Neuroscience

Methods

in

Drug Abuse

Research

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Neuroscience Methods in

Drug Abuse Research

Editors:

Roger M. Brown, Ph.D.

David P. Friedman, Ph.D.

Yuth Nimit, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 62

1985

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C 20400
NIDA Research Monographs are prepared by the research divisions of the

National Institute on Drug Abuse and published by its Office of Science. The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and integrative research reviews. Its dual publication emphasis is rapid and targeted dissemination to the scientific and professional community.

Editorial Advisors

Martin W. Adler, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

Sydney Archer, Ph.D.

Rensselaer Polytechnic Institute

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Richard E. Belleville, Ph.D.

NB Ascciates. Health Sciences

Rockville, Maryland

Gilbert J. Botvin, Ph.D.

Cornell University Medical College

New York, New York

Joseph V. Brady, Ph.D.

The Johns Hopkins University School of Medicine

Baltimore, Maryland

Theodore J. Cicero, Ph.D.

Washington University School of Medicine

St. Louis, Missouri

Sidney Cohen, M.D.

Los Angeles, California

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric Institute

San Francisco, California

Denise Kandel, Ph.D.

College of Physicians and Surgeons of Columbia University

NewYork, NewYork

Herbert Kleber, M.D.

Yale University School of Medicine

New Haven, Connecticut

NIDA Research Monograph Series

Jerome Jaffe, M.D.

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Neuroscience Methods in

Drug Abuse Research
ACKNOWLEDGMENT

This monograph is based upon papers presented at a technical review

which took place on September 10-11, 1984, at Bethesda, Maryland.

The conference was sponsored by the Division of Preclinical

Research, National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from

the copyright holders to reproduce certain previously published

material as noted in the text. Further reproduction of this

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Citation of the source is appreciated.

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appearing in this publication are used only because they are

considered essential in the context of the studies reported herein.

DHHS publication number (ADM)85-1415

Printed 1985

NIDA Research Monographs are indexed in the Index Medicus. They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Preface

The explosive growth in neuroscience research over the last decade

is paralleled by the impressive array of new approaches now being

applied in drug abuse research. In particular, techniques in

neuroanatomy, neurophysiology, and molecular biology are now coming

into prominence along with the more classical pharmacological and

biochemical techniques that once dominated the drug abuse field.

More importantly, however, it has been the creative use of

multidisciplinary tactics, as illustrated by the papers in this

review, that has substantially increased the impact of technological

advances in the drug abuse field. The papers in this monograph are

based on a technical review held on September 9 and 10, 1984, and

are presented in three sections. We hope these papers will

stimulate multidisciplinary research on drug abuse issues by serving

as examples, as well as information sources, to the field.

In the first section, the application of in vitro autoradiography to

brain and drug abuse research is described by Michael Kuhar and

Miles Herkenham. This method, which has supplied us with a high

resolution technique for localizing receptors to drugs and neurotransmitters in the central nervous system, is described and

analyzed. This analysis is timely now, because after an exciting

decade of use as a qualitative tool, autoradiography is about to

become even more productive as a quantitative technique as well.

The authors concentrate on the promises and problems associated with

quantitative autoradiography and supply some of the best background

material available on the subject.

In the second section, approaches integrating electrophyslology with

behavioral analysis are applied to the study of drug actions on the

nervous system. This section begins with a description of a unique

application of a now classical technique in drug abuse research,

electrical self-stimulation of the brain's reward system. These

studies, by Conan Kornetsky, use the subjects' willingness to work

for intracranial electrical stimulation to define what is and is not

rewarding and illustrate how a drug's interaction with this

behavioral model is a useful predictor of abuse liability. John

Chapin and and his colleagues and Sam Deadwyler show how to relate

neural activity to complex behaviors in experimental animals. In

their analyses, they show how drugs alter the activity of both

single neurons and ensembles of neurons, and that the drug-induced

changes in neural activity are correlated with and may predict overt

behavioral effects of drugs. Chapin uses a paradigm that explores

sensorimotor integration, while Deadwyler's model focuses on sensory

processing as it relates to higher cognitive functions. Finally,

v
Henri Begleiter and Bernice Porjesz describe the use of evoked brain

potentials to study the effects of drugs on humans. Their work with

alcoholics, using a computer-assisted analysis of the electrical

brain activity associated with both simple and complex cognitive

functions, has revealed alterations in CNS activity after acute and

chronic alcohol exposure, after abstinence from alcohol, and even in

the offspring of the alcoholics. These fascinating studies suggest

that it may be possible to find markers for individuals who are

susceptible to drug abuse. Clearly, electrophysiology is undergoing

a renaissance in neuroscience research. This revival has been aided

by the integration of the computer into the laboratory, but would

not have progressed as it has without the creative drive of

investigators such as those who have contributed to this section.

In the final section, some of the consequences of chronic drug use

are explored. Lewis Seiden describes, by a variety of neuropharmacological, neurochemical. and histological studies, the methamphetamine-induced loss of dopamine and dopamine-containing neural

elements in the striatum. This toxic effect may occur after a

single high dose or a more prolonged, lower dose regimen. Interestingly, the damage caused by methamphetamine results from the brain's

own dopamine. This startling finding suggests that neurotoxic

consequences may follow the abuse of other drugs that act to block

monoamine reuptake mechanisms. Horace Loh and his colleagues

summarize their work on opiate mechanisms in which they have used a

neuroblastoma x glioma hybrid cell line that contains delta opiate

receptors. They have used this cell line as a model to study the

molecular mechanisms of tolerance and dependence. In this model

system, tolerance is manifested by the loss of the opiate-induced

inhibition of adenylate cyclase upon prolonged opiate administration. This loss appears to be caused by an initial decrease in

opiate receptor affinity followed by an actual loss of opiate

receptors in the external membrane of the cell. Dependence is

manifested by a naloxone-induced increase in adenylate cyclase

activity after chronic opiate exposure. This is related not to

changes in the opiate receptor, but to the accumulation of two

cytosolic components: adenosine and (tentatively) calmodulin. These

substances activate adenylate cyclase, although the relationship of

this activation to naloxone occupation of opiate receptors is not

understood. Finally, Sandra Moon describes the effects of in utero

drug exposure on developing neurochemical systems. This work, which

utilizes both receptor autoradiography and fluorescence to reveal

changes in opiate receptors and dopamine in the striatum caused by

haloperidol administration, shows how alterations in one neurochemical system may alter other systems. It also illustrates the

kind of creative use of anatomical techniques now occurring in drug

abuse research.

R.M. Brown. Ph.D.

D.P. Friedman, Ph.D.

Y. Nimit. Ph.D.

Neurosciences Research Branch

Division of Preclinical Research

National Institute on Drug Abuse

vi
Contents

Preface

THE AUTORADIOGRAPHIC ANALYSIS OF

DRUG EFFECTS ON THE NERVOUS SYSTEM

Receptor Mapping in Neuropharmacology by Autoradiography:

Some Technical Problems

Michael J. Kuhar, James R. Unnerstall, and

Errol B. De Souza

v

1

Levels of Quantitative Analysis of Receptor Autoradiography:

Technical and Theoretical Issues

Miles Herkenham

ELECTROPHYSIOLOGICAL APPROACHES TO THE STUDY

OF DRUG EFFECTS ON THE NERVOUS SYSTEM

Brain-Stimulation Reward: A Model for the Neuronal Bases

for Drug-Induced Euphoria

13

Conan Kornetsky 3 0

The Neural Systems Approach to Examining the Actions of

Drugs on the Central Nervous System

John K. Chapin, Stephen M. Sorensen, and

Donald J. Woodward 51

Correlating Behavior with Neural Activity: An Approach

to Study the Action of Drugs in the Behaving Animal

Sam A. Deadwyler 63

The Use of Event-Related Potentials in the Study of Alcoholism:

Implications for the Study of Drugs of Abuse

B. Porjesz and H. Begleiter 7 7

CONSEQUENCES OF CHRONIC DRUG USE

Methamphetamine: Toxicity to Dopaminergic Neurons

Lewis S. Seiden 100

Molecular Mechanisms of Opiate Tolerance and Dependence

in a Clonal Cell Line

Alan K. Louie, Ping-Yee Law, and Horace H. Lob 117

Consequences of Perinatal Drug Administration on Brain

Development: Striatal Opiate Receptors and Dopamine

Sandra Lynn Moon 129

List of NIDA Research Monographs 146

vii
Receptor Mapping in Neuropharmacology by Autoradiography:

Some Technical Problems

Michael J. Kuhar, James R. Unnerstall, and

Errol B. De Souza

INTRODUCTION

Techniques for receptor mapping have been steadily improving

for the past decade. Microscopic receptor mapping is now as

feasible and popular as neurotranamitter and enzyme

histochemiatry has been for many years. A main reason for this

has been the development and adaptation of microscopic

autoradiographic methods in parallel with the biochemical

binding techniques which have so dramatically advanced the

study of receptors at the molecular level. The goals of this

chapter are to summarize the main issues in receptor mapping by

autoradiography, to discuss some important current technical

problems which profoundly affect the methods that one should

select to do experiments, and also to discuss the

interpretation of autoradiograma.

The fundamental advantages of microscopic receptor mapping are

increased anatomical resolution and greatly increased

sensitivity of measurement (at least when compared to

biochemical approaches). The increase in anatomical resolution

is obvious because these are microacropic techniques which

always have greater resolution than microdissection techniques.

The great sensitivity in measurement derives from the fact that

one can measure receptor densities in very small microscopic

regions. This increase in sensitivity can be orders of

magnitude greater than that found in biochemical studies.

Thus, in general, receptor mapping will be useful in any

situation requiring receptor measurement in small regions or in

situations where overall quantities (but not necessarily

densities) of receptors are low. An example of such a

situation is an experiment on the axonal flow of receptors;

while it can be done biochemically, light microscopic

autoradiography provides a sensitive method of measurement and

anatomical resolution not available by other means (Kuhar and

Zarbin 1984; Zarbin et al. 1983). Additional information on

other more general uses of receptor mapping is presented below.

1
In general, there are two important principles that one must

keep in mind when carrying out receptor mapping experiments.

In our experience, these principles are critical in guiding the

investigator to carrying out a useful experiment. The first

principle is selective or preferential labeling of receptors.

This has been facilitated by the development of receptor

binding techniques over the past decade. The development of

large numbers of radioactive liganda and the availability of

extensive pharmacological studies on many receptors has

provided the ability to define quite precisely the type of

receptor to which one is binding. A second principle is the

selection of appropriate methods of visualization. This is

important because one does not visualize the receptor directly,

but rather the ligand which binds to it. Thus, one must

produce an autoradiogram or image in such a way that one

prevents or minimizes the diffusion of ligand away from the

receptor. It does no good or is even misleading if a receptor

is appropriately labeled but the ligand is lost from the tissue

during a preparation process that obtains the image by any

means. This is obviously not an important issue where

irreversibly binding ligands are used. However, the vast

majority of available liganda are reversible binding agents and

the autoradiography must be done in a way which takes into

account the diffusible nature of the signal (Stumpf and Roth

1966; Roth and Stumpf 1969; Young and Kuhar 1979; Barnard

1979). Perhaps a third principle should be added, i.e., a

principle of appropriate interpretation. As will be discussed

below, the use of tritium in producing autoradiograma has some

special pitfalls because of the low energy of the beta

particles emitted by tritium. The efficiency with which beta

rays are converted into autoradiographic grains varies from

region to region because of this low energy which results in a

differential absorption of beta rays by different tissue

regions. The absorption varies depending on the dry mass

content of the region. This is discussed in more detail below.

There are two main experimental approaches to receptor mapping

experiments. They are in vitro labeling procedures and in vivo

labeling procedures. - The first is the more popular and more

versatile one. In in vitro labeling procedures, slide-mounted

tissue sections are incubated with radioactive ligands so that

the receptors are labeled under very controlled conditions.

Following the labeling, the slide-mounted tissue sections can

be rinsed to remove nonreceptor-bound drug and improve specific

to nonspecific binding ratios. In in vivo labeling procedures,

the receptors are labeled in intact tissues in vivo after

systemic administration of the drug (ligand). If the ligand

has a very high affinity for the receptor, tracer quantities of

the drug can be injected into the animal, and after a

relatively short time interval, the drug is carried to the

brain by the blood, diffuses into the brain, and binds to the

receptors. The nonreceptorbound drug is then removed from the

brain and other tissues by various excretory processes. The

high affinity of the drug for the receptor causes a retention

of the drug on or in the vicinity of the receptor molecule.

2
Detailed strategies for carrying out both in vivo and in vitro

labeling experiments have been described (Kuhar 1982a, 1982b.

1983). In vitro labeling procedures have-many advantages over

in vivo labeling procedures. Since the experiments are carried

out in vitro, one can control the labeling conditions so that

there is greater specificity and efficiency in the labeling.

One can add inhibitors to prevent metabolism of the labeling

ligand and one can also use ligands which would not cross the

blood-brain barrier in in vivo labeling procedures. One can

also quite easily compare various receptor distributions

because it is possible to use different ligands with

consecutive sections of tissue. Also, this approach has a

relatively low cost since an entire animal does not have to be

labeled with drug and it is possible to carry out studies with

human postmortem tissues. Yet, an additional advantage of in

vitro labeling is that one can quite precisely define the

receptor type that one is studying because of parallel

biochemical experiments that can be carried out with the

slide-mounted tissue sections. Nevertheless, in vivo labeling

experiments are still quite important. Recently, PET scanning

images of receptors have been utilize because of the ability to

label neurotransmitter receptors in vivo. These PET scanning

procedures are noninvasive and provide an extraordinary

opportunity to examine receptors in clinical populations

(Wagner et al. 1983; Eckelman et al. 1984).

The Uses of Receptor Mapping

Historically at least, the study of receptors has been within

the discipline of pharmacology. Perhaps this is the reason

that receptor mapping has most often been considered a

pharmacological tool. Receptor maps provide powerful insights

into the mechanism of drug action by identifing those brain

regions which have the receptors and, therefore, those areas

which are affected by drug administration. There are many

examples of this. One of the first applications of

this approach may have involved the opiate drugs.

Administration of opiate drugs causes a variety of

physiological affects, including analgesia, pupillary

constriction, respiratory depression, and suppression of

various visceral reflexes. Perhaps the only way this wide

range of physiological action can be fully understood is to

associate the various physiological effects with receptors in

various neuronal circuits which mediate the effects (Kuhar

1978, 1981). In other words, identifying the neuronal circuits

which have the receptors and which will be affected by drug

administration may provide an understanding of how the drug

produces its effect. Moreover, receptor mapping studies can be

an impetus for developing new drugs. For example, several

studies have indicated that there are high densities of

neurotensin receptors on dopamine neurons in the midbrain.

Recent studies by Uhl and Kuhar (1984) show that these

neurotensin receptors up regulate after chronic administration

of antipsychotic drugs, indicating the involvement of

3
neurotensin in the nigrostriatal system and possibly in systems

involved in schizophrenia. These studies suggest that drugs

aimed at the neurotensin receptor might be useful in treating

movement disorders or emotional disorders associated with

dopaminergic systems.

Receptor mapping is also a valuable compliment to

neurotransmitter mapping. Receptor maps help complete our view

of the biochemical organization of the brain. Recent studies

on the corticotropin-releasing factor (CRF) receptors (De Souza

et al. 1984) have shown that these receptors are found in many

regions of the brain where endogenous CRF-containing neurons

have been identified. A part of the brain containing very high

levels of CRF has been the median eminence, and this region

also has very high levels of CRF receptors (De Souza et al.

1984). In addition, CRF receptors were found in many other

areas of the brain indicating that CRF may be a

neurotransmitter in the brain as well as a hypothalamicreleasing factor.

Other uses of receptor maps include identifying changes in

neuropathology and also providing needed data for a variety of

other types of experiments, such as locating targets for

experiments involving direct drug injections.

TECHNICAL PROBLEMS

Problems With Diffusion

As has been discussed briefly above, diffusion of ligand in

receptor mapping procedures can distort the image such that it

provides wrong or misleading information. Thus, the

development of methods for dealing with diffusible molecules by

Lloyd Roth and Walter Stumpf has been very helpful (Stumpf and

Roth 1966; Roth and Stumpf 1969). Several approaches have been

developed to prevent diffusion of radioactive ligands. Perhaps

the safest and most widely used are the “dry” techniques. In

these procedures, the radiolabeled tissues are kept dry and not

exposed to any solvents which might provide an opportunity for

diffusion or leeching of drug until after the autoradiographic

image is formed. Other approaches involve barriers, such as

plastic or carbon films placed over the radiolabeled tissues

prior to dipping in warm emulsion. The barrier would at least

theoretically prevent the wet emulsion from promoting diffusion

while still allowing the close apposition of emulsion for the

creation of the image.

Young and Kuhar (1979) developed a procedure for labeling

receptors in slide-mounted tissue sections. Autoradiograms

were generated by apposing dry emulsion on flexible coverslips.

Diffusion was minimized at every step in this procedure by

keeping samples cold and dry whenever possible.

4
Yet another approach involve? fixation of ligands into tissues.

Irreversibly binding ligands can be said to be “fixed.”

Another way to fix radioactive ligands in tissue sites is to

expose the radiolabeled tissues to fixatives. Fixation by

formaldehyde vapors has been used and is obviously advantageous

to fixation with liquids where diffusion may occur before

fixation. Herkenham and Pert (1982) have proposed the use of a

method which includes the vapor fixation of radioactive ligands

in slide-mounted tissue sections followed by wet defatting

procedures prior to the dipping into emulsion for production of

autoradiograms. Recently, it has been found that the defatting

procedures subsequent to the vapor fixation are especially

useful in that they eliminate the different absorption of

tritium-emitted B particles that are found in gray and white

matter. This issue is discussed in more detail below.

Autoradiograms prepared by these procedures appear to be quite

FIGURE 1

Time Course of [3H]Ligand Loss in Ethanol Solution

Slide-mounted tissue sections were prepared and labeled with

[

3

H]dihydromorphine (DHM), [3H]naloxone (NAL), or

[

3

H]N-methylscopolamine (NMS). Some slides were not

incubated (controls) and others were incubated for varying

amounts of time in 70% ethanol. Specific binding was then

measured. See text for discussion. From Kuhar and Unnerstall

(1982). Copyright 1982, Elsevier Scientific Publishers, B.V.

5
similar to those prepared by "dry" techniques where opportunity

for diffusion is minimal. However, Kuhar and Unnerstall (1982)

have found that the labeling ligand was at least partially lost

from the tissue during the ethanol dehydration and xylene

defatting treatment. At least one ligand, tritiated

N-methylscopolamine (3H-NMS), was totally lost from the

tissue (figure 1). These results indicate that the vapor

fixation by formaldehyde does not result in complete attachment

of radioactive ligand to tissue elements. Also, the degree of

attachment appears to vary with the ligand employed. Although

it has not been shown, it is possible that the degree of

attachment may vary also with the region of brain examined or

possibly even with the subtype of receptor that the ligand is

binding to. Thus, "diffusion" might not be the best

description of the problem in this case, since the ligand which

is fixed by the formaldehyde will not diffuse. However,

"leeching" of ligand from tissue is a problem with this

approach because not all of the ligand is covalently linked and

there appears to be significant loss of ligand from the tissue

during the wet dehydrating and defatting procedures. Stumpf

and Roth (1966) examined the effect of gaseous formaldehyde

fixation on steroid receptors labeling with 3H-estradiol.

These authors found that some degree of diffusion and loss of

ligand from tissue definitely occurred, and they stated that

one should not assume that fixation of tissue includes

immobilization of the labeled compound. Studies by Kuhar and

Unnerstall (1982) indicate that only partial fixation occurs

for most ligand and, in some cases, at least for 3H-NMS, no

fixation of ligand occurs.

Thus, the introduction of dehydration, defatting, and wet

emulsion application is not without problems, even with fixed

tissues. The loss of label found by Kuhar and Unnerstall

varies with the ligand chosen and opens the possibility that

leeching of ligand will occur in the section to the extent that

anatomical resolution will be reduced. Even if this were not a

difficulty, the loss of label would require that

proportionately longer exposure times be used. Also, the

practically total loss of at least one ligand, 3H-NMS,

indicates that these procedures are not generally applicable

for receptor autoradiography. Further, while it has not been

shown, it is important to rule out the possibility that

preferential leeching will occur from some brain regions or

from subtypes of receptors so that the resultant image may

mislead investigators as to receptor distributions. Defatting

does seem to have the significant advantage with tritium in

that it reduces or eliminates the grey/white differences in

efficiency described below (Herkenham and Sokoloff 1984).

Gray/White Differences with Tritium

Autoradiographic studies with tritium-labeled substances in

brain have revealed differences in autoradiographic efficiency

between gray and white matter (Alexander et al. 1981; Herkenham

and Sokoloff 1984; Taylor et al. 1984). Basically, the finding

6
matter (figure 2).

is that tritium in white matter did not produce

autoradiographic grains as efficiently as trituim in gray

FIGURE 2

Double in Plots of Standard Data Indicating Different Autoradiographic Efficiency for Tritium in Grey and White Matter

Tissue sections from bovine caudate (grey) and internal capsule

(white) were incubated in varying concentrations of tritiated

isoleucine. DPM/section are not corrected for the dry mass of

the tissue. Optical densities were determined by a video-based

image analysis system. Further details are presented in the

text. Reproduced from J.R. Unnerstall’s Ph.D. dissertation.

The problem appears to be that white matter has a higher dry

mass content than gray matter and, therefore, absorbs more beta

rays than does the gray matter. Significantly, it has been

found that the more energetic emissions from carbon-14 and

iodine-125 (I125) do not show these gray/white differences.

7
These regional differences in efficiency with tritium are

troublesome, particularly since most available ligands for

receptor mapping are tritium labeled. If one attempts to

quantify the receptor content in autoradiographic experiments,

one must understand the relative “quenching” caused by

different regions. Moreover, this is a difficult problem

because the relative content of gray and white matter varies

throughout the brain. Hence, investigators have sought some

way to cope with these regional differences in efficiency.

Kuhar, Unnerstall, and coworkers have attempted to develop a

method for determining the relative amount of quenching in each

brain region. These methods involve soaking slide-mounted

tissue sections in tritium-labeled substances such that equal

quantities of tritium are found in all regions of the tissue

section. Autoradiograms produced from these sections showed

varying grain densities and shoved the expected gray/white

differences. Since the quantity of radioactivity in all brain

regions was known, the relative efficiency of or a “quenching

factor” for each brain region can be calculated. Another

approach utilized by Kuhar and coworkers involved thaw mounting

thin sections of brain on slides coated with tritium and other

radioactive substances. As the beta rays passed through the

thin sections of brain to form an image on film, the absorption

of beta rays was proportional to the dry mass in the various

regions. The image produced in these experiments also provided

a means of directly calculating the relative quenching of a

given brain region.

While these approaches have provided a way to cope with these

differences in efficiency, they are obviously tedious and must

be applied to every brain region studied. Also, it is possible

that various experimental treatments will cause a difference in

the tissue density of various brain regions and, hence, these

techniques must be carried out for experimental animals as well

as for controls.

Herkenham and Sokoloff (1984) found that defatting of

slide-mounted tissue sections following formaldehyde vapor

fixation resulted in a reduction of the gray/white matter

difference in efficiency. It appears that the defatting

procedures remove significant matter from the tissue sections

so as to make the relative amount of quenching from gray and

white matter regions the same. Thus, there is no need to

correct optical densities for regional differences in

efficiency. While these conclusions have not been supported

with quantitative data from several brain regions, the

qualitative information shown in autoradiograms from Herkenham

and coworkers supports this idea quite clearly. Thus,

defatting of the tissues after vapor fixation provides a

substantial advantage in quantftation of autoradiograms with

8
tritium. However, as pointed out above, defatting procedures

result in a significant loss of ligand from the tissues. Also,

as stated above, this loss indicates that proportionately

longer exposure times will be needed and also places one in

danger that significant other artifacts may be occurring, as

described above. Thus, it appears that neither approach with

tritium is completely satisfactory.

Because of these problems, autoradiography with tritium can be

undertaken only if attempts are made to understand the relative

problems involved. Perhaps, the best solution will be to use

I

125-labeled ligands. Not only do I125-labeled ligands not

have a regional efficiency problem, the specific activity of

these ligands is substantially higher than those with tritium

and much shorter autoradiographic exposure times are required.

Qusntitation of Autoradiograms

Aside from the above problem, the quantitation of

autoradiograms is still not a trivial issue because the

response of emulsions to radioactivity is not linear. In other

words, increasing the radioactivity content of a tissue does

not result in a linearly related increase in grain density or

optical density. Several laboratories have explored this under

conditions used for receptor mapping (Geary and Wooten 1983;

Penney et al. 1981; Rainbow et al. 1982; Unnerstall et al.

1981, 1982) and found that appropriate translation of grain

densities into femtomoles of ligand bound requires the use of

standards to calibrate the experimental system used. While

this is a difficult problem, it can be solved with a reasonable

effort.

SUMMARY

In the past decade, substantial advances in receptor mapping

have been made. It is now as feasible to carry out receptor

localizations as it is to carry out neurotransmitter

localizations. A great deal of data has accumulated on various

technical procedures and it is clear that significant problems

occur with tritiated ligands. These problems involve different

autoradiographic efficiencies in tissue regions, and steps must

be taken to either correct for these different efficiencies or

to eliminate them. It seems that I125-labeled ligands will

become more and more important in the field.

9
REFERENCES

Alexander, G.M.; Schwartzman, R.J.; Bell, R.D.; Yu, J.; and

Renthal, A. Quantitative measurement of local cerebral

metabolic rate glucose utilizing tritiated 2-deoxyglucose.

Brain Res 223:59-67, 1981.

Barnard, E.A. Visualization and counting of receptors at the

light and electron microscopic levels. In: O'Brien. R.D.,

ed. The Receptor. Vol. I. General Principles and

Procedures. New York: Plenum Press. 1979. pp. 247-310.

De Souza, E.B.; Perrin, M.H.; Insel, T.R.; Rivier, J.;

Wylie, W.V.; and Kuhar, M.J. Corticotropin-releasing factor

receptors in rat forebrain: autoradiographic identification.

Science 224:1449-1451, 1984.

Eckelman, W.C.; Reba, R.C.; Rzesyotarski, W.J.; Gibson, R.E.;

Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.;

Eng, R.; and Grissom, M.P. External imaging of cerebal

muscarinic acetylcholine receptors. Science 223:291-292,

1984.

Geary, W.A., II, and Wooten, G.F. Quantitative film

of opiate agonists and antagonist binding in rat brain.

J Pharmacol Acol Exp Ther 225:234-240, 1983.

Herkenham. M., and Pert, C.B. Light microscopic localization

of brain opiate receptors: A general autoradiographic method

which preserves tissue quality. J Neurosci 2:1129-1149,

1982.

Herkenham, M., and Sokoloff, L. Quantitative receptor

autoradiography: Tissue defatting eliminates differential

self-absorption of tritium radiation in gray and white

matter of brain. Brain Res, in press.

Kuhar, M.J. Histochemical localization of opiate receptors and

opioid peptides. Fed Proc 37:153-157, 1978.

Kuhar. M.J. Autoradiographic localization of drug and

neurotransmitter receptors in the brain. Trends Neurosci

4:6O-64, 1981.

Kuhar, M.J. Localization of drug and neurotransmitter

receptors in brain by light microscopic autoradiography. In:

Inverson, S.D.; Iverson, L.L.; and Snyder, S.H., eds.

Handbook in Psychopharmacology. Vol. I. New York: Plenum

Publishing, 1982a. pp. 299-320.

Kuhar, M.J. Localizing drug and neurotransmitter receptors in

vivo with tritium-labeled tracers. In: Eckelman, W.C., ed.

Receptor-binding Radiotracers. Vol. I. Boca Raton: CRC

Press, 1982b. pp. 37-50.

Kuhar, M.J. Autoradiographic localization of drug and

neurotransmitter receptors. In: Bjorklund, A., and

Hokfelt, T., eds. Methods in Chemical Neuroanatomy,

Handbook of Chemical Neuroanatomy. Vol. I. Amsterdam:

Elsevier, 1983. pp. 398-415.

10
Kuhar, M.J., and Unnerstall, J.R. In vitro receptor

autoradiography: Loss of label during ethanol dehydration and preparation procedures. Brain Res 244:178-181,

1982.

Kuhar, M.J., and Zarbin, M.A. Axonal transport of muscarinic

cholinergic receptors and its implications. Trends Pharmacol

Sci supplement, Jan. 1984.

Penney, J.B.; Pan, H.S.; Young, A.B.; Frey, K.A.; and Douth,

G.W. Quantitative autoradiography of 3H-muscimol binding

in rat brain. Science 214:1036-1038, 1981.

Rainbow, T.C.; Bleisch, W.V.; Biegon, A.; and McEwen, B.S.

Quantitative densitometry of neurotransmitter receptors.

J Neurosci Methods 5:127-138, 1982.

Roth, L.J., and Stumpf, W.E. Autoradiography of Diffusible

Substances. New York: Academic Press, 1969:

Stumpf, W.E., and Roth, L.G. High resolution autoradiography

with dry-mounted, freeze-dried frozen sections. Comparative

study of six methods using two diffusible compounds,

3

H-estradiol and 3H-mesobilirubinogen. J Histochem

Cytochem 14:274-287, 1966.

Taylor, N.R.; Unnerstall, J.R.; Mashal, R.D.; De Souza, E.B.;

and Kuhar, M.J. Receptor autoradiography: Coping with

regional differences in autoradiographic efficiency with

tritium. Soc Neurosci 10:557, 1984.

Uhl, G.R., and Kuhar, M.J. Chronic neuroleptic treatment

enhances neurotensin receptor binding in human and rat

substantia nigra. Nature 309(5967):350-352, 1984.

Unnerstall, J.R.; Kuhar, M.J.; Niehoff, D.L.; and Palacios,

J.M. Benzodiazepine receptors are coupled to a subpopulation

of GABA receptors: Evidence from a quantitative

autoradiographic study. J Pharmacol Exp Ther 218:797-804,

1981.

Unnerstall, J.R.; Niehoff, D.L.; Kuhar, M.J.; and Palacios,

J.M. Quantitative receptor autoradiography using

3

H-ultrofilm: Application to multiple benzodiazepine

receptors. J Neurosci Methods 6:59-73, 1982.

Wagner, H.N.; Burns, H.D.; Dannals, R.F.; Wong, D.F.;

Langstrom, B.; Duelfer, T.; Frost, J.J.; Ravert, H.T.;

Links, J.M.; Rosenbloom, S.B.; Lukas, S.E.; Kramer, A.V.; and

Kuhar, M.J. Imagining dopamine receptors in the human brain

by positron tomography. Science 221:1264-1266, 1983.

Young,W.S., III, and Kuhar, M.J. A new method for receptor

autoradiography 3H-opioid receptor labeling in mounted

tissue sections. Brain Res 179:255-270, 1979.

Zarbin, M.A.; Palacios, J.M.; Wamsley, J.K.; and Kuhar, M.J.

Axonal transport of beta-adrenergic receptors. Mol

Pharmacol 24:341-348, 1983.

11
AUTHORS

Michael J. Kuhar, Ph.D.

Professor of Neuroscience, Pharmacology, and Psychiatry

Department of Neuroscience

The Johns Hopkins University

School of Medicine

Baltimore, Maryland 21205

James R. Unnerstall, Ph.D.

Predoctoral Fellow

Department of Neuroscience

The Johns Hopkins University

School of Medicine

Baltimore, Maryland 21205

Errol B. De Souza, Ph.D.

Instructor

Department of Neuroscience

The Johns Hopkins University

School of Medicine

Baltimore, Maryland 21205

12
Levels of Quantitative Analysis of

Receptor Autoradiography:

Technical and Theoretical Issues

Miles Herkenham

Autoradiography is a tool used to localize radiolabeled substances

contained within slide-mounted tissue sections. Labeled sections

are apposed to a radiation-sensitive emulsion. When developed

like a photograph, the exposed emulsion provides qualitative information about location of labeled material in different areas of a

heterogeneous structure such as the brain. While anatomical localization can be highly informative (Pert and Herkenham 1981), often

additional information about the amount of material incorporated

is useful or necessary.

The autoradiographic film image results from the interaction of radiation ( particles) with silver halide crystals in the emulsion

layer of the film, producing clusters of reduced silver atoms (Rogers

1979). The size of the clusters (grains) is a feature of the emulsion that affects the resolution and sensitivity of the film. The

number of these grains produced is proportional to the level of radiation exposure. When developed, the silver grains are opaque,

and the visible image is the result of different levels of light

transmittance in different portions of the film. Quantifying the

relative differences between different transmittance levels by densitometry seems to be a relatively easy matter, but assigning

"absolute" values is considerably more difficult since, for example,

the time of exposure would be a critical variable. In practice,

the use of an independent measuring device, e.g., a liquid scintillation spectrofluorimeter, provides a means of assessing baseline

values. However, for accurate assessment of radioactivity levels

by either relative or absolute means, the response characteristics

of the film over the range of radiation exposure must be known.

The quantitative densitcmetric analysis of autoradiographs can be

carried out at four distinct levels which are described Bela.

LEVELS OF QUANTITATIVE ANALYSIS

Rank Order of Density

Regardless of the amount of exposure (radioactive disintegrations

x time), film transmittance levels can be assigned a rank order

13
which would be invariant for a given image. With autoradiography

of brain tissue sections containing receptor-bound radiolabeled

ligands, differential light transmittance through different parts

of the film will reflect differential amounts of ligand binding

sites across brain structures. Even this most basic form of quantification is subject to several qualifications. First, since tritium emits only weak particles, these emissions are differentially

self-absorbed ("quenched") in brain regions depending on the myelin

content in each region (Alexander et al. 1981; Herkenham and

Sokoloff 1984). As a result of differential quenching, two structures containing the same amount of radioactivity but different

amounts of white matter will show different transmittance levels

(figure 1). Removing the lipids in the myelin by the histological

procedure of defatting after aldehyde fixation (Herkenham and Pert

1982) eliminates the differential quenching and "corrects" the

autoradiograph (Herkenham and Sokoloff 1984).

A second factor is that rank ordering is reliable only within the

same film image and, hence, only among areas in the same brain section or among several sections exposed identically to the same film.

If rank ordering of structures contained in different brain sections

is required, the sections must also have been treated identically

(identically prepared, co-incubated in vitro, washed and dried together).

Comparing the transmittance levels in different parts of the film is

usually accomplished by densitanetric analysis using some form of

photocell. The transmittance is translated to a measure of optical

density (OD), which will be discussed in greater detail below.

Magnitude of Relative Differences

In order to describe "how much" different the radioactivity levels

are in different structures, or in the same structure after various

manipulations, assignment of numerical values to levels of film

darkness is needed. At this level of quantification, the absolute

magnitude of each value is not known, but relative differences among

structures can be expressed as a ratio, a proportion, or a percent

difference. ODs are used in this way when the relationship between

OD and ligand bound has not been determined.

A densitometric analysis serves to quantify relative differences in

OD, but OD differences will not accurately reflect differences in ligand bound if the film response to radioactivity is not linear over

the range of values measured. For example, densitometry of two

structures with different amounts of radioactivity will show one

value of OD differences at a brief exposure and a smaller value at

a longer exposure if the film saturates to produce a "ceiling effect." The prudent investigator must determine the range over which

the film response is linear for the exposre levels encountered

and/or convert densitmtric data to values of radioactivity by the

use of standards prepared with known amounts of radioactivity and

tissue content. The remaininy parayraphs of this section describe

the theoretical and practical issues pertinant to the relationship

14
FIGURE 1

Regional Tritium Quenching

Silver stain of myelin (a) and autoradiographs of [3H]succinimidyl

propionate binding (b-d) at level of rat cerebellum. Succinimidyl

propionate is a protein acylating agent that binds homogeneously

and covalently to all brain regions. The autoradiogaphs are all

of the same section exposed before defatting (b and d) and again

after defatting (c), both times for 10 days. The actual film images

are photographed in b and c; d shows a computer-generated image in

which ODs are coded in five shades of gray (as in the scale bar).

The dotted lines in b and c show where computer-averaged densities

were plotted as a function of horizontal position in a narrow rectangle containing cerebellar gray and white matter and the film.

The histograms show relative ODs in these regions before and after

defatting. From Herkenham and Sokoloff (1984).

15
between OD and radioactivity. The third section describes how

standards can be calibrated to achieve units of absolute value.

When light is passed through an exposed and developed film, the

amount transmitted is inversely proportional to the number of reduced silver grains in the emulsion. The situation is analogous

to absorption spectrometry, in which, according to Beer's Law, the

absorption of light and the concentration (or density) of the medium through which the light passes can be expressed as:

I = Io e

where I = intensity of transmitted light

Io = intensity of incident light

= absorption coefficient

1 = length of light path

C = concentration (density) of light absorbing

compound.

From this equation OD has traditionally been defined (converting

to base 10).

Io/I = 10

OD = log (IO/I) =

In practice, OD is determined by measuring Io and I and solving the

above equation. For our densitometry system (Goochee et al. 1980),

Io is arbitrarily set at a value of 100, and I is determined as a

proportion of IO, so OD = 2 - log I. In the latter instance, OD

units would range between a value of zero and two. Using the first

equation, an OD value of two would indicate a 100/1 ratio of incident

to transmitted light; values above two represent nearly total

absorption by the medium.

Since a and 1 are constants, OD should be linearly related to c,

the concentration of radioactivity (in units of exposure), but only

if the film response is linear. A plot of OD versus exposure should

reveal the response characteristics of the film.

Quantitative densitometry of receptors has thus far been done with

only one kind of film: a tritium-sensitive film called 3H-Ultrofilm

(LKB, Gaithersburg, MD). However, despite its wide use for both

3H and 125I I, the response characteristics of this film are not generally appreciated. A prerequisite for determining the response

characteristics has been the preparation of tritium standards. Several method papers describing standards preparation and their application to film autoradiography have been published (Penney et al.

1981; Unnerstall et al. 1982; Altar et al. 1984; Rainbow et al. 1984;

Geary et al., in press), but only a few have shown a plot of OD versus exposure. In one study such a plot revealed a curve best fit by

a logarithmic equation (Rainbow et al. 1984), while in another it

was fit by a rectangular hyperbola (Unnerstall et al. 1982). Using

a drum scanner system (Goochee et al. 1980), the plot of OD versus

radioactivity concentration in a set of commercially made standards

16
(tritium in plastic, from American Radiochemicals Corporation,

St. Louis, MO) resembles both these functions, but a power function

provides a closer fit (figure 2). Figure 2 shows that a linear

relationship between OD and expsure does not exist and is approximated only at very low OD values (Bela about 0.6). That is, LKB

film begins to saturate even with low exposures to tritium; complete

saturation occurs at or around an OD value of two.

FIGURE 2

LKB Film Response

Eight different concentrations of tritium in plastic (commercial

standards) were apposed to film for 1 month, and their ODs were

measured as described. The relationship that best fits the resulting curve was computer derived. The deviation from linearity

represents the saturation of the film.

The LKB film response to radiation seems to be independent of the

nature of the source, as the saturation curve for 125I is very similar to that for tritium (McLean et al., in press). This indicates

that saturation is a property of the film itself. The large grain

size of LKB film must play a major role in the shape of the saturation curve. Of course, the higher energy 125I emissions expose the

film in hours or days instead of weeks or months for tritium.

When standards are used to convert ODs to units of radioactivity,

some aspects of film response to radiation should be considered. One

17
is that the rates of image generation are different for different

isotopes; because of this noncovariance, the isotope in the standard should be the same as in the tissue being masured (Geary et al.,

in press). Another consideration arises when comparing ODs of images

generated by the same isotope in two different media, such as plastic

and brain tissue. Though there is covariance of 3H-tissue and 3Hplastic sources (Geary et al., in press), plastic quenches tritium

muchg more than does brain (Alexander et al. 1981). Therefore, equivalent amounts of radioactivity in each would produce different ODs.

The values, therefore, can only be used in a relative fashion.

By the equation that describes the relationship between OD and tritium exposure, a conversion from OD to units of radioactivity can be

made, permitting valid comparisons of relative differences in radioactivity levels in different brain regions. Similarly, relative

differences in tritium quenching among different brain regions can

be quantified. For example, the quenching shwn in figure 1 was

quantified in table I of Herkenham and Sokoloff (1984). Cerebellar

gray and white matter had OD values of .35 and .09, respectively.

Using a power function that relates OD to radioactivity in plastic

at 2 weeks exposure, these values become 27.8 and 5.1 nCi/mg, respectively. Thus, a 4-fold OD difference converts to a 5.4-fold

quench difference. In general, because of film saturation, OD differences underestimate the magnitude of radioactivity differences;

furthermore, the underestimate increases with increasing ODs (as

emphasized by the changing slope in figure 2). Thus, conversion to

units of radioactivity is required for determining the relative differences at any level of density.

In many studies the nonlinear response of the film is "corrected" by

logarithmic or log-log transformations. Traditionally, in photography, OD has been plotted as a function of the logarithm of exposure

(Farnell 1966), producing the "H and D" curve, named after Hurter

and Driffield who defined OD in 1890 (Rogers 1979). The H and D

curve is S-shaped, with a long central straight portion (e.g., figure

2b of Unnerstall et al. 1982). This curve is instructive for several

reasons. First, since our eyes transduce light in a logarithmic

fashion, the log relationship approximates our perception of relative

"darkness." Second, the flattening at the low end is said to be an

index of film background, or "fog," whereas the high end shows some

practical limits of film saturation. Finally, the slope of the linear

portion is a measure of film "contrast" as we perceive it.

A linear plot of OD versus log exposure would be expected if the relationship between OD and exposure is a logarithmic one. Hwever, in

our hands a pwer function was a better fit, so a log-log transformation should reveal a straight line. Figure 3 shws that this is the

case. The flattening at the low end seen with the log transformation

is nearly eliminated by the log-log transformation. This flattening

is more pronounced at low exposures (i.e., the same standards for one

week, not shown) but disapears if fog (OD of the film itself) is

subtracted. The flattening at the high end, for our densitometry

system at least, is mainly a consequence of our definition of OD as

2 - log I.

18
FIGURE 3

Log-Log Transformation of Film Response Data

The same data used for figure 2 are plotted on a log-log scale

here. A straight line best fits the data points. The deviations

from linearity at high OD values result from an OD ceiling of 2.0

and at low values from the contribution of film fog, or background,

which typically has an OD of 0.05 to 0.08.

Straight lines are often convenient for transforming and comparing

data. Hwever, logarithmically transformed data pose difficulties

for statistical analysis, since the magnitude of error of estimated

concentration, interpolated from the OD measurement, will be constant

only over the same range for which OD is linearly related to exposure.

As the slope of that relationship changes (figure 2), the error size

of estimated concentration increases with increasing ODs. Because

3

H-Ultrofilm shows saturation over virtually the entire OD range,

statistics based on the assumption of constant error will be invalid.

Although there are statistical procedures for varying

error size (weighted linear regression, Snedecor and Cochran 1980),

for simplicity, a constant error might be approximated for OD values

on the "linear" part of the OD versus exposure curve, i.e., for OD

values below 0.6 (figure 2).

19
These issues are raised here not only because they affect how densitometry data are analyzed, but because previous descriptions of LKB

film response to standards have used logarithmic data transformations

to empasize the "usable" or "workable" part of the OD range. Although the "workable" range of film response might be considered as

that for which OD is linearly related to exposure, or that for which

a curve can be closely fit to relate the two, in other studies the

workable range is defined as that for which a straight line is found

after logarithmic (Unnerstall et al. 1982; both transformations in

figure 3 of Raibow et al. 1984) or log-log transformations (Geary

et al., in press). Since the data for the plastic standards are closely fit with the densitometry system by a power function (figure 3),

the log-log transformation is linear over nearly the entire range of

the film. This result is much different than reported by others

(Unnerstall et al. 1982; Geary et al., in press), who saw deviation

fran linearity at OD values above 1.0 (the OD values reported by

Unnerstall et al. are x 100). Interestingly, in both studies

the reported deviation at ODs above 1.0 was based on only one data

point. Some studies also describe deviations at very lcw values of

OD, where signal detection becomes a major problem.

Thus, deviations from linearity can reflect several variables, including errors in determining both ODs and the radioactivity in the standards, as well as a failure of the equation to closely fit the data.

Since film is not an "ideal" medium, we should not expect its response

to fit same "ideal" equation. In practice, whatever equation that most

closely describes the relationship between OD and exposure in the

standards is preferred--this may be one of the equations already described, or it might be another, such as a polynomial (Reivich et al.

1969). When a cubic equation is used, for example, straight lines

after log or log-log transformations would not necessarily be expected.

In conclusion, LKB film response to tritium exposure is nonlinear.

Film saturation is noticeable at the lowest OD values, and so conversion of OD units to units of radioactivity is necessary before comparisons of relative differences are valid. Conversion is achieved

by curve-fitting procedures: much of the OD range can be described

by a power function, but different equations may better fit narrow

intervals, especially at both low and high ends of the OD range.

Nearly all of the OD range is usable for quantification of relative

differences in radioactivity levels, whose accuracy is determined by

the closeness of fit provided by the equation that relates OD to exposure and by the quality of the standards and the densitometer.

However, very little of the OD range can be used for statistics based

on the assumption of constant error size of estimated radioactivity

over the range of measured OD values. A log or log-log transformaation of the data obscures the fact of changing error size, but it is

of value for comparing different sets of data , as in differential film

response to different isotopes, or to the same isotope in different

media (e.g., Alexander et al. 1981; Geary et al., in press).

20
Expression of Amount of Ligand Bound per Amount of Tissue

The third level of quantification can be achieved if the standards

are calibrated to enable determination of the actual amount of radioactivity (hence the amount of ligand) present in the tissue. When

the radioactivity per weight of tissue is calculated, the amount of

ligand bound can be easily determined by a formula (Unnerstall et al.

1982) if the specific activity of the labeled compound is known.

Calibration can be achieved in several ways. By sure methods, brain

homogenates with known amounts of radioactivity are both assayed by

liquid scintillation counting and by application to slides for autoradiography (Unnerstall et al. 1982; Rainbow et al. 1984). By another

method (Reivich et al. 1969), a brain containing a homogeneously

distributed labeled compound, such as methylglucose or antipyrene,

is sectioned at the midline--half the brain is homogenized for scintillation counting of radioactivity, and the other half is sectioned

and exposed to film with a set of standards covering a wide range of

radioactivity levels. After the counting efficiency is determined

for the machine and fluor used, the OD of the brain sections of known

radioactivity calibrates the standards against brain.

If the isotope is 14C or 125I, all brain regions will show approximately equivalent ODs if the labeled compound is homogeneously distributed (Orzi et al. 1983; Herkenham and Sokoloff 1984). If the

isotope is 3H, however, the differential quenching across brain regions will produce a heterogeneous image (figure 1). "Efficiency"

factors can be assigned for each structure based on a comparison with

a weighted average (Unnerstall et al. 1984). Alternatively, the

tissue can be defatted to eliminate the differential quenching (Herkenham and Sokoloff 1984; Geary et al., in press), but then two opposing factors must be assessed: the increased autoradiographic efficiency and the loss of label that occurs by defatting (Kuhar and

Unnerstall 1982). Unfortunately, it is quite difficult to measure

each factor independently. For example, a defatted tissue standard

cannot be prepared without some loss of label in the preparation.

Furthermore, it is difficult to measure radioactivity that is fixed

in tissue. We can estimate it by comparing scintillation counts of

radioactivity in tissue that is unfixed, fixed, or fixed and defatted.

To best extract counts from radiolabeled ligand that is fixed in the

tissue, 1 ml tissue solubilizer (e.g., Protosol, New England Nuclear)

is added to sections in scintillation vials, and these are shaken

overnight. Then 250 μl methanol are added, to reduce chemiluminescence, followed by 10 ml of a nondetergent fluor (e.g., Econofluor,

New England Nuclear).

Determination of Number of Receptors per Weight or Volume of

Tissue

The "ultimate" in quantification is the determination of the actual

number of receptors per volume of tissue, per cell, or even per synapse.

A number of issues pertain to this level of quantification, including

interpretation of binding data and the rather elusive definition of

of what is meant by a "receptor."

21
The previous section discusses the third level of quantification

and establishes the requirements for determining the amount of ligand bound per amount of tissue. In order to relate the amount of

ligand bound to number of receptors, one must determine the percent

occupation of the receptors under the in vitro binding conditions

used. Using slide-mounted sections which bound ligand is

quantified by scintillation counting, the duration of incubation required to reach an equilibruim state should be determined. Next, an

analysis of binding saturation, by incubating in different concentrations of ligand, will reveal both the affinity of the ligand for

the binding site (Kd) and the number of available binding sites (Bmax).

The qualification that must be kept in mind is that Kd and Bmax are

numbers that can change with binding conditions. Often binding is

increased by pre-incubation, changes in incubation temperature, or

the addition of ions, allosteric effectors and protease inhibitors.

Sane binding increases take the form of Kd reductions and others, Bmax

elevations. A change in Bmax is not always easy to interpret. Unmasking binding sites by removing endegenous ligand is commonly suggested, but "cryptic" binding sites may be made available by some of

the treatments (e.g., Rouot et al. 1980; Chang et al. 1983).

Since most ligands bind to more than one site, altered binding conditions may raise the affinity for a low-affinity site, resulting

in complicated Scatchard plots requiring sophisticated analysis.

When the binding site is coupled to other molecules, such as ion

channels, second messengers or enzymes, binding data can be altered by incubation conditions. In vitro binding conditions used to

bring about these effects become far removed from the in vivo environment in which the receptor normally exists. There large chasm

between what is known about the action of the receptor in vitro and

what it might be doing in vivo.

Some sobering observations about receptor localization and function

are already emerging. These take us the full circle, from “absolute”

quantification of receptor number, back to the mere localization of

receptors to some brain areas and not to others. A striking finding

in nearly all neurotransmitter/receptor systems examined to date is

that the locations of high-affinity binding sites, which are the easiest to identify autoradiographically, do not necessarily correspond

to the areas of the brain that contain the terminations of pathways

that release the transmitter for that receptor: there may be no correlation at all between receptor distribution and transmitter distribution for any given putative transmitter-receptor system (see Herkenham and McLean, in press,for review); and, in some cases, there may

even be a negative correlation (e.g., Goedert et al. 1984). This

puzzling situation has emerged for receptors to transmitters including

peptides, catecholamines, indolamines, and the amino acids.

Specific examples of the mismatches are abundant. In the opiate system, it is classic knowledge that opioid peptides have their highest

levels in the globus pallidus, a structure which contains only modest

numbers of all of the opiate receptor subtypes. Likewise, in the

substance P system, the substantia nigra pars reticulata contains

22
the highest levels of substance P terminal immunoreactivity in the

brain (see Mclean et al. 1985); but, in the pars reticulata, substance P receptors are not detectable, and binding for a putative

substance P receptor subtype, marked by the analog eledoisin, is

barely detectable (figure 4). Conversely, areas that contain little

or no demonstrable substance P immunoreactivity, like the hippocampus

and cerebral cortex, have very dense concentrations of receptors.

One doesn't need quantitative autoradiography to appreciate such profound mismatches!

In our enthusiasm to be quantitative we may miss some of the truly

remarkable uses of "quantitative" autoradiography. For example, by

examining just the patterns of substance P and eledoisin binding,

one might be at a loss to explain why these were ever considered to

be related peptides. Pattern recognition is one of the most powerful

uses of autoradiography. In a recent

the similar patterns of [3H]MPTP and [3Hlpargyline binding supported

tudy by Rainbow et al. (1985),

the suggestion that MPTP produces parkinsonian effects after conversion to a toxic ion by monoamine oxidase (Markey et al. 1984).

Another, perhaps more striking, mismatch can be found in the deep

cerebellar nuclei, which are known to be the main sites for relaying

cerebellar outputs. For many years physiologists have known that a

main feature of cerebellar outflow is an inhibitory connection between Purkinje cell axons and cells of the deep cerebellar nuclei.

Subsequently, it was shown that this postsynaptic inhibition is produced by the actions of the neurotransmitter, GABA (Obata et al.

1967), and that Purkinje cells (Pibak et al. 1978) and their terminals (McLaughlin et al. 1974) are immunoreactive for the GABA-synthesizing enzyme, GAL Finally, deep cerebellar nuclei are known to

contain moderately high levels of GABA (Fahn and Coté 1968). Given

these facts, one would expect to see high levels of GABA receptors

in the deep cerebellar nuclei. However, autoradiography of [3H]muscimol binding, produced by in vitro conditions that selectively label

"synaptic GABA receptors" (Penney et al. 1981), does not reveal any

detectable binding in the deep cerebellar nuclei of the rhesus monkey (figure 5a).

Recently a class of low-affinity GABA receptors has received much

interest because it appears to be the class that interacts with benzodiazepines and barbiturates and to mediate physiological responses

to GABA (Olsen et al. 1984b; Tallman and Gallager 1985). This class

can be marked for autoradiography by the GABA antagonist [3H]bicuculline methochloride (Olsen et al. 1984a). Its autoradiographic distribution matches that of benzodiazepine receptors (Wamsley et al.

1984) and differs somewhat from that of [3H]muscimol. Autoradiography of [3

H]flunitrazepam binding, under conditions which would

label the benzodiazepine/low-affinity GABA receptor complex (O'Neill

et al. 1984), reveals detectable binding in the deep cerebellar nuclei (figure 5b), though it is still quite low in relation to binding

in the cerebellar and cerebral cortices. Interestingly, the presence

of [3H]flunitrazepam binding in a region where there is no detectable

23
FIGURE 4

Substance P

Autoradiographic film images at the level of rat substantia nigra.

a) Substance P immunoreactivity marked by iodinated antibodies

(McLean et al. 1985) is extremely dense in the nigra and represents

terminations of substance P containing striatonigral projections.

Autoradiograph courtesy of S. McLean. b) Substance P receptors

marked by iodinated substance P (Rothman et al. 1984b) are undetectable in the nigra. c) Similarly, eledoisin, a substance P analog

which may bind to a subtype of the substance P receptor (the SP-E

site), has only barely detectable binding in the nigra (Rothman et

al. 1984a). Mismatches of substance P terminals and receptors

are apparent in the cortex as well.

24
FIGURE 5

GABA

Autoradiographs at level of monkey deep cerebellar nuclei (arrows

point to Location of dentate nucleus) a: The binding of 50 nM

[

3

H]muscimol, under conditions that Label the high-affinity GABA

receptor (Penney et al. 1981), is undetectable in the deep cerebellar

nuclei, which are known to receive dense and functional GABA-ergic

inputs. Autoradiograph courtesy of R.P. Hammer, Jr. b: The

binding of 1 nM [3H]flunitrazepam, under conditions that label

the benzodiazepine/low-affinity GABA receptor complex (see text),

is low but detectable in the deep cerebellar nuclei. Autoradiograph

courtesy of J.B. O'Neill, D.P. Friedman, and J.M. Crawley.

25

484-443 O - 85 - 2
[

3

H]muscimol binding appears to contradict the idea that the lowaffinity site is the same receptor as the high-affinity site, but

in a conformationally different form (Tallman and Gallager 1985).

For this to be the case, the low-affinity sites should comprise a

subset of the high-affinity sites. Clearly more work needs to be

done on this exciting problem. Recent indication that the sites are

different comes fran their dissociation in mutant mouse cerebellum

(Fry et al. 1985).

Such examples of the gross mismatch between receptor localization

and neurotransmitter localization suggest that functional receptors

may exist with such low affinity for available ligands that they

would be undetectable by autoradiographic anaylsis. While this

may indeed be the case, especially for synaptic transmission where

endogenous ligand concentrations are high, we must still consider

the roles of the high-affinity sites in mediating pharmacological

effects. We often known that the high-affinity receptors are the

ones that mediate drug actions, since structure-activity studies

show good correlations between the potencies of analogs to inhibit

ligand binding to the receptor and to produce physiological or behavioral effects. Other observations suggest possible nonsynaptic

functions for the apparently mislocalized high-affinity sites. A

recent ultrastructure study showed that in rat striatal patches,

which contain very high densities of high-affinity μ opiate receptors,

the large majority of binding sites are not located at synapses as

classically defined (Hamel and Beaudet 1984). Thus, nonsynaptic

nerve transmission can be suggested. The possibility that transmitters can diffuse through extracellular spaces to reach distant receptors has been discussed (Herkenham and McLean, in press). Another

recent study shows that high-affinity benzodiazepine receptors have a

very rapid turnover rate--their half-life is on the order of hours

(Borden et al. 1984). Receptor turnover may be amenable to regulation

by transmitters and drugs. In support of this concept, studies have

shown upregulation of high-affinity [3H]muscimol binding after removal

of GABA-ergic afferents (Penny et al. 1981). While receptor affinity

and specificity help determine drug potency in structure-activity

tests, under normal physiological conditions, many factors intervene

between binding kinetics and function. The ultimate value of guantitative autoradiography may not be to discover how many receptors there

are in a given area, but rather to help elucidate the mechanisms by

which drugs drugs and transmitters use and regulate the receptors,

and thus reveal dynamic brain processes.

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28
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ACKNOWLEDGMENTS

Dr. Louis Sokoloff, NIMH, provided guidance on the theoretical

basis of autoradiography. Dr. Peter Munson, NICHHD, provided

statistical expertise.

AUTHOR

Miles Herkenham, Ph.D.

Laboratory of Neurophysiology

National Institute of Mental Health

Building 36, Room 2D10

Bethesda, Maryland 20205

29
Brain-Stimulation Reward: A

Model for the Neuronal Bases

for Drug-Induced Euphoria

Conan Kornetsky

Not in short supply in the field of drug abuse are theories. A

National Institute on Drug Abuse (NIDA) monograph (Lettieri et

al. 1980) entitled “Theories on Drug Abuse” presented 43 separate

essays, each describing a theory attempting to explain the

nonmedical use of drugs. For the most part, with some

exceptions, the theories explicitly stated or implied that use is

engendered only in those individuals who are in sane way deviant

This deviation may be in the realm of personality, biology, or

social systems. Thus, the drug is used to restore one to a

normal state. I believe that this point of view was eloquently

stated by Beckett (1974), ". . . that heroin addiction in some is

symptormatic of an underlying chronic depression in a wounded

personality . . ." Khantzian (1980) argued ". . . that opiates

counteracted regressed, disorganized, and dysphoric ego states

associated with overwhelming feelings of rage, anger and related

depression." Dole and Nyswander (1967, 1980), in introducing the

use of methadone as a treatment modality, argued for the

existence of a metabolic disturbance in the narcotic addict and

that the drug use is restorative.

An argument could be made that these theories are probably all

correct to sane degree. Certainly depressed individuals might

find opiates or cocaine helpful in relieving their depression.

Opiates will decrease aggressive behavior so that we can assume

it decreases feelings of aggression. Methadone certainly will

restore an individual who, as Dole and Nyswander (1980) have

argued, developed a long lasting metabolic deficiency from use of

narcotic drugs. Certainly a depressed existence can become more

tolerable by the use of drugs that elevate one’s mood. However,

drug seeking behavior would not exist and none of these putative

restorative states would happen if in some way the abused substances did not cause some feeling of well-being or euphoria.

Despite these multiple causative factors, this cheer will present evidence from my laboratory, as well as others, that there

may exist a common underlying neuronal action of many abuse

substances. At the level of the CNS, it is manifest as an

activation of those areas of the brain for which electrical

3 0
stimulation is rewarding, and for the user it is translated into

some pleasurable feelings that have been described as the “high’

In an animal the effect is sufficiently reinforcing that the

animal will work to receive the drug or it will make the animal

more sensitive to rewarding brain stimulation.

The. possibility that abuse substances achieve their reinforcing

value by activating a reward system in the brain was suggested

not too many years after the original discovery of Olds and

Milner (1954) that animals will work in order to receive such

brain stimulation. The phenomenon has been called intracranial

self-stimulation (ICSS) as well as brain-stimulation reward, For

the most part, but not exclusively, the former term has been used

when the animals are in an operant paradigm in which the

dependent variable is rate of response. Early investigators,

including Olds, saw in the technique a method for the study of

the effects of drugs on brain function. The first report of the

effects of drugs on rewarding brain stimulation was a 1956 paper

published in Science by Olds et al. describing the effects of

chlorpromazine and reserpine. In 1957, Killam and coworkers

reported at a meeting of the American Society for pharmacology

that 10 mg/kg of pentobarbital slightly increased the rate of

responding in rats with electrodes in some areas, but not all, of

the hypothalamus. Also, they reported that amphetamine caused an

increase in response rate as well as a possible lowering of the

reward threshold. The inference was that rate of response

increased because the brain stimulation became more rewarding.

Olds and Travis (1960) described a series of experinmts in which

they studied the effects of morphine as well as a number of other

drugs. The most common effect they observed with morphine was a

decrease in response rate. Of interest is that the first

published paper I could find on the effects of opiates after the

Olds and Travis report was published in 1972 (Adams et al.). A

more detailed description by this group was given by Lorens and

Mitchell (1973). They, as well as other investigators using rate

of response as the dependent variable, found that after low doses

of morphine rate of response was first decreased followed by

facilitation 2 to 3 hours after drug administration. They also

reported that after a relatively short period of chronic

treatment there was no decrease in rate of response, and

significant facilitation in self-stimulation was observed during

the second hour after morphine administration.

A further indication that moraine caused an activation of the

brain pathways involved in intracranial self-stimulation was

given in the early 1970s (Nelsen 1970; Nelsen and Kornetsky

1972). Rats prepared with two bipolar electrodes, one in the

medial forebrain bundle-lateral hypothalanus area and one in the

brainstem, showed a dissociation in the EEG recorded simultaneously from these two brain sites after morphine administration. In the former, a positive reinforcing site, there was an

activation as reflected in a shift from high to low amplitude

waves. At the same time, the EEG recorded from the brainstem, an

31
aversive site, showed the opposite -- a shift from low to high

amplitude waves. An example of this effect is shown in figure 1.

FIGURE 1

Effects of morphine in a single animal on the relative high

amplitude waves (percent total waves) in the EEG; recorded from

depth electrodes. The top panel shows data from a positively

reinforcing (+) brain site (lateral hypothalamus). The bottom

panel shows data recorded from a negatively reinforcing (-) brain

site (mesencephalic reticular formation). Epochs of EEG; were

simultaneously recorded from both sites. Adapted from Nelsen

(1970). (Fran Kornetsky and Wheeling 1982M

Earlier, Stein and Ray (1960) reported that d-amphetamine lowered

the threshold for ICSS using a two-lever reset method. In this

procedure the animal has two response levers available. A

response on one lever results in rewarding brain stimulation, but

each response on this lever results in a fixed decrease of

intensity of stimulation in a step-wise manner. A response on

the second lever resets the intensity of stimulation available

from the first lever back to its original intensity. Threshold

is defined as the average intensity at which the animal will

press the reset lever. Stein and Ray clearly replicated the

earlier study by Killam et al. (1957).

32
Throughout the 1960s and into the 1970s, ICSS was not used to

study to any great degree the effects of abuse substances and, as

mentioned, opiates were not studied at all during this period.

This failure to evaluate the action of abuse s&stances on ICSS

was probably not surprising, considering the early equivocal

results with morphine and the emergence in the 1960s of the drug

self-administration procedure (Weeks 1962) and the use of drugs

as discriminative stimuli (Overton 1964). The drug self-&ministration procedure clearly had face validity for the studv of

abuse substances, and the drugs as discriminative stimuli procedures, in many ways, is an animal analogue of the Drug

Addiction Research Center's (Lexington, KY) method of determining

the abuse potential of new compounds in man. Although both of

these methods are useful, they do not directly tell us anything

about the neuronal bases for the reinforcing effect of a drugonly whether or not it is reinforcing.

As previously stated, there was a resurgence in the 1970s of the

use of brain-stimulation reward as a model for the study of the

euphoria caused by abuse substances. In 1974, Marcus and

Kornetsky demonstrated the functional significance of our earlier

EEG; findings (Nelsen and Kornetsky 1972); see figure 2. In this

experiment, it was found that morphine lowered the threshold for

FIGURE 2

Effects of morphine on rewarding (+) and aversive (-) stimulation. Ordinate is percent change in threshold from pre- to

post-injection (POST-PRE/PRE x 100). Each line represents the

mean threshold chanqe for three subjects each on escape and

reward thresholds. Adapted from Marcus and Kornetsky (1974).

(From Kornetsky and Wheeling l982b)

33
rewarding stimulation and raised the threshold to aversive stimulation. Of importance was that it was the first classical

psychophysical study of ICSS that measured the threshold in a

rate (of response) free paradigm Threshold was defined as that

intensity of stimulation which half the time the animal would

find reinforcing and half the time it would be nonreinforcing.

Also, it was the first clear demonstration that morphine lowers

the threshold independent of any stimulating motor effects.

Although a "double staircase" (Cornsweet tracking method of

intensities was used in this experiment, subsequently we have

used the psychophysical method of limits in our studies.

The behavioral paradigm that we use is schematically presented in

figure 3. The procedure allows us, in a manner of speaking, to

ask the animal after the first stimulus (S1), "if you wish you

may have another stimulus exactly the same as the first stimulus

by turning the cylindrical manipulandum." By varying the

intensity of the stimuli according to a psychophysical schedule

of stimuli presentations we can determine the intensity at which

half the time the animal says "yes" to our question and half the

time it says "no."

Figure 4 illustrates a pre- and post-drug power-function after

the administration of 4 mg/kg of morphine. Plotted is the

percent of times the animal decided to respond at each intensity

of stimulation. As can be seen, a shift to the left indicates

increased sensitivity with the threshold defined as the intensity

at which the animal responded 50 percent of the time.

Thus far in our studies we have yet to find a false positive,

that is, a drug that lowers the threshold for rewarding brain

Stimulation that is neither an abuse substance nor a drug that

has been demonstrated to cause euphoria in humans. However, we

have found false negatives, that is drugs that are abused but do

not lower the threshold for reward. As might be expected, the

opiates that are abused lower the threshold, as do amphetamine

(Esposito et al. 19801, cocaine Esposito et al. 1978), and

phencyclidine (Kornetsky et al. 1980). Abuse substances we have

studied that have no threshold lowering effect are alcohol

(Unterwald et al. 1984), pentobarbital (unpublished), and

lysergic acid diethylamide (unpublished). If a complete dose

response curve is generated for a drug that lowers the threshold

for reward, the curve is U-shaped. This is illustrated in figure

5, which shows the dose-effect relationship after the administration of d-arqhetamine. Since the curve is U-shaped, the maximum

effective dose for an individual animal or animals can be

determined.

After our initial success with demonstrating that a number of

drugs lower the threshold when abused, we expanded our studies to

mixed agonist-antagonist opioids. We tested threeofthese:

cyclazocine, nalorphine and pentazocine (Kornetsky et al. 1979).

Of these three drugs, only pentazocine consistently lowered the

34
threshold. However, the extent of this lowering (maximum

efficacy) did not reach the level observed after morphine,

cocaine, or d-amphetamine. The relative threshold lowering

effect of these mixed agonist-antagonist drugs as well as

morphine, cocaine, and d-amphetamine is shown in figure 6.

FIGURE 3

A schematic representation of the method for determining brainstimulation reward thresholds. (I) A sequence of a single trial

when an animal doss not respond. (II) The sequence when the

animal makes a response. A trial begins with the delivery of a

non-contingent stimulus train (S1); if the animal fails to

respond by turning the wheel within 7.5 seconds from the onset of

the S1, the trial is terminated and 15 seconds later the next

trial is begun. If the wheel is turned as indicated in example

II, the rat receives a contingent stimulus (S2). Intensity of

stimulation is varied according to a modification of the method

of limits. A response after the 7.5 second available response

time postpones the presentation of the next stimulus for 15

seconds. (Fran Kornetsky and Bain 1982)

3 5
FIGURE 4

The effects of 4 mg/kg of morphine (subcutaneous) on the

threshold for brain-stimulation reward. The percent of trials in

which the subject responded at each current intensity are

presented. PRE and POST data were obtained immediately prior and

subsequent to drug injection. Thresholds are indicated as the

median rewarding intensities at which the subject responded.

(From Kornetsky and Wheeling 1982a)

Also shown in figure 6 is the effect of phencyclidine (PCP). Of

the five animals studied, only in one did PCP not lower the

threshold. However, as can be seen, the extent of this threshold

lowering effect is slight and approximates the same level

established by pentazocine.

In order to test the model further, we decided to examine a

common street combination of drugs, tripelennamine and

pentazocine (called “T’s and Blues” on the streets). As

described above, we had already demonstrated that pentazocine had

significant but relatively low efficacy in lowering the threshold. Before studying the combination, we Qtermined the effect

of tripelennamine alone on the threshold for rewarding intracranial stimulation. To our surprise we found that

tripelennamine significantly lowered the reward threshold

(Unterwald et al. 1984b). However, this finding could have been

predicted from the experiments of Jasinski et al. (1983) who

found that human subjects given tripelennamine alone report an

increase in euphoria as measured by the Addiction Research Center

Inventory. The effect of tripelennmine alone is shown in the

36
FIGURE 5

Mean Z-score changes in threshold after various doses of damphetamine. Z-scores are based on the post-pre changes in

threshold after drug as a function of the and standard deviation of post- pre-changes in threshold after saline for each

animal. Z-scores are conputed as follows:

Saline (post - pre) - treatment threshold (post - pre)

Standard deviation of saline

Thus changes of 2 indicate the 95% confidence limits. Error

bars indicate the standard error of the Z-score.

FIGURE 6

Median effects of various drugs on brain-stimulation reward (POSTPRE/PRE x 100) based on the optimal lowering of threshold,

independent of dose. Negative scores indicate the extent of

effect below the maximal decreases obtained with vehicle. CYC:

cyclazocine, N=4: NAL: nalorphine, N=4; PTZ: pentazocine, N=4;

MOR: morphine, N=10; CCC: cocaine, N=7; d-A: d-amphetamine, N=4;

PCP: phencyclidine, N=8. (From Kornetsky and Wheeling 1982a)

37
the upper graph, and the lower graph shows the effects of

pentaxocine alone and these same doses in combination with an

ineffective dose of tripelennamine (0.625 mg/kg).

As can be seen, not only is the dose effect curve moved to the

left, but the maxinum effect is clearly greater with the combination than with any dose of pentazocine alone. Also, the effect

of the combination is greater than the effect of any dose of

tripelennamine alone.

We tested one other mixed agonist-antagonist alone and with

tripelennamine. This drug, nalbuphine, is currently only

available in injectable form for clinical use as an analgesic.

Jasinski and Mansky (1971) reported that it has significantly

less abuse potential than morphine. An example of the type of

effect of this combination in one animal is given in figure 8.

As can be seen, not only does nalbuphine lower the threshold but

the interaction with tripelennamine is similar to that seen with

the tripelennamine and pentazocine combination

The results of this combination suggest that if nalbuphine

becomes generally available, it may be used in combination with

tripelennamine as pentazocine currently is. However, it has been

reported that nalbuphine has greater efficacy as an opiate

antagonist than does pentazocine Schmidt 1983). Thus, in the

morphine or heroin dependent individual, the use of nalbuphine

may precipitate abstinence which would preclude its use.

However, in the nordependent individual the combination clearly

has abuse potential.

The question that these experiments raise is whether or not there

is a common neuronal substrate for these threshold lowering

effects. Most investigators believe that the underlying neurochemical substrate is catecholaminergic with heavy emphasis on

the dopamine system (e.g., Fibiger 1978; Wise and Bozarth 1982;

Gallistel et al. 1981; Olds and Forbes 1981). However, recently

Prado-Alcala and Wise (1984) and Prado-Alcala et al. (1984) have

found that there is not a close correspondence between the

boundaries of the brain reward system and those of the dopamine

terminal fields. These findings suggest that a direct activation

of a dopaminergic system does not account for the rewarding

effects of stimulation to a variety of brain sites.

The facilitation of the threshold lowering effect of the mixed

agonist-antagonists by the antihistamine tripelennamine suggests

that a central histamine system may also play a role in the reinforcement effects of opiate drugs. Belluzzi and Stein (1977)

have suggested a role for an endorphin system in brain-stimulation reward. This is based, for the most part, on their findings

that naloxone will block or attenuate brain-Stimulation reward.

Although some investigators also have found that naloxone will

attenuate ICSS behavior (Stapleton et al. 1979; Schaeffer and

Michael 1981), other investigators using similar procedures with

38
FIGURE 7

Mean Z-score effects of various doses of tripeleunamine (top

panel) and pentazocine (bottompanel). The bottom panel also

shows the effects of 2.5 mg/kg of tripelennamine with various

doses of pentazocine. Error bars indicate the standard error of

the Z-score. A Z-score of ± 2 indicates the 95% confidence

limits. (Fran Unterwald and Kornetsky 1984)

electrodes in similar brain sites have failed to find an effect

of naloxone Wauquier et al. 1974; Holtzman 1976; van der Kooy et

al. 1977; Lorens and Sainati 1978).

Stapleton et al. (1979) reported that relatively large doses (10

mg/kg) of naloxone did not abolish self-stimulation but caused

relatively small reductions in lever-press rate. They concluded

that endorphinergic processes my modify reward but did not seen

to be critical for reward We have found that doses of naloxone

up to 16.0 mg/kg, both acutely (Esposito et al. 1980) and chroniically (Berry et al. 1981) administered, had no effect on the

reward threshold in animals with electrodes in the medial forebrain bundle (MFB) or ventral tegmntal area (VTA) except to in39
FIGURE 8

Mean effect of various doses of nalbuphine alone and in

combination with an ineffective dose of tripelennamine.

crease within subject variability. We have, however, found that

naloxone at doses of 2.0 to 4.0 mg/kg will reverse the effects

of 0.5 or 1.0 mg/kg of d-amphetmine. Higher doses of naloxone

often did not block as well as the 2.0 to 4.0 q/kg. The effects

of naloxone on the threshold lowering effect of d-amphetamine, as

well as cocaine and morphine, are shown in figure 9. Note that

FIGURE 9

Effects of various combinations of naloxone with cocaine, damphetamine, or moprphine on brain-stimulation reward threshold.

Z-scores are based on the mean and standard deviation of threshold changes (POST-PRE) after respective vehicle injections. Zscores beyond ± are considered significantly different Data are

based on the man Z-score for 6 subjects with cocaine, 4 subjects

with d-amphetamine, and a single morphine subject. The naloxone

block or attenuation of the respective drug effect was observed

in all animals. (Fran Kornetsky and Wheeling 1982a)

40
there is a tremendous difference in potency between naloxone’s

effect compared to its ability to block cocaine or d-amphetmine.

These results support the hypothesis that there may be endorphinergic receptors on the presynaptic dopamine neuron which

serve as modulators of the dopamine system and thus may be part

of a common neuronal substrate involved in the euphoria caused by

the narcotic analgesics as well as cocaine and d-amphetamine.

Contrary evidence, however, is given by Pettit et al. (1984).

They found that destruction of dopamine terminals in the nucleus

accumbens of the rat would selectively attenuate cocaine but not

heroin self-administration. This discrepancy in interpretation

of our findings using brain-stimulation reward and the Pettit et

al. findings using self-administration suggests that the two

models may not be measuring the same phenamenon. Only future

research will solve this apparent contradiction in findings.

A major question is whether or not drugs cause differential

effects as a function of where the stimulating electrodes are

implanted. Ever since the first drug study of the effects of

drugs on ICSS by Olds et al. (1956), there have been attempts to

use this technique to determine site of action of the drugs.

These attempts have been criticized on procedural grounds

primarily because there has been a confounding between rate of

response and threshold. Most of the site specificity studies

have used rate as the dependent variable or a rate dependent

threshold procedure. Many investigators have found differential

effects of drugs as a function of site of stimulation. This has

been especially true for studies with d-amphetmine (e.g., Carey

et al. 1975; Franklin and Robertson 1982; Goodall and Carey 1981)

and opioids (e.g., Liebman and Segal 1977; Lorens 1976; Nazzaro

et al. 1981). The major difficulty with these studies is that

the sites themelves seem to engender different response rates.

Even if response rates were equated, as Esposito and Kornetsky

(1978) and Liebman (1983) point out, response rates may be at a

ceiling at one placement and not at another placement. Also, if

baseline response rates are not equated there is still the

problem of “rate-dependency.” Further, Schenk et al. (1981) did

a within subject comparison of the effects of morphine on lateral

hypothalamic and central gray self-stimulation In addition,

they were also able to compare between subject differences. They

found that the time course and magnitude of drug-caused changes

in effects in the two placements was a function of individual

animal differences, not electrode placement differences.

As mentioned previously, the baseline rates of responding will

differ as a function of site of electrode placement It is not

even clear whether or not these rate differences directly reflect

the reward value of the stimulation Over 20 years ago Hobos and

Valenstein (1962) found, in a paradigm involving two electrodes

associated with two levers respectively, that animls did not

necessarily choose the lever that resulted in stimulation to a

site that generated the highest rates. Although the number of

subjects was small, Esposito et al. (1979) compared the effects

41
of morphine on the threshold for rewarding stimulation in animals

with electrodes in the VTA, the ventrolateral portion of the

central gray, or the anterior tip of the locus coeruleus. They

concluded that, at least from this data, it would be premature to

specify a precise neuroanatomical and/or neurochemical substrate

for the observed effects.

We directly compared rate of response and threshold in the same

animals in order to test the hypothesis that rate was inversely

related to thresholds (Payton et al. 1983). Figure 10 presents

the scatter plot from this experiment. As can be seen, there is

no significant correlation. Complete power functions were done

on each animal and the greatest rate of response was the datum

used for an individual animl. Since electrodes were aimed for a

number of structures, a comparison of threshold as well as rate

of response as a function of site of stimulation could be

compared. This is shown in table 1. Although significant

differences in threshold as a function of site were found, no

such differences were found in rate of response. Also, when

rank, from lowest to highest in both threshold and rate is

compared, there is no relashionship between threshold and rate.

FIGURE 10

Threshold/rate scatter plot, Each point represents a separate

animal. POA: preoptic area: MFB: medical forebrain bundle; VTA:

ventral tegmental area; DMH: dorsal medial hypothalamus; AHA:

anterior hypothalamus; VTHA: ventral thalmic area; PAHA: preoptic-anterior hypothalamic area,

42
Somewhat contrary data are given in the recent reports of Gratton

and Wise (1983) and Prado-Alcala et al. (1984). They did mapping

studies in which they used a dorsal-ventral movable electrode.

They measured rate of response and a threshold which they defined

as the maximum current intensity that sustained 10 or less

responses per minute. They classified the rate data from each

animal as either low, medium, or high. Although they did not

publish the mean threshold obtained at each of the response rate

levels, enough of the data were presented that I was able to

calculate the mean and SD for each level. Going from low rate to

high rate, the mean threshold ± SD was 36.4 ± 10.9, 39.9 ± 10.5,

and 22.8 ± 8.7 μA, respectively. There was a clear difference in

threshold between the high rate sites and either the law or

medium rate sites. Although we found that the highest rate of

response was from the MFB, as did Gratton and Wise, cur finding

that the MFB also yielded the highest absolute threshold was

contrary to Gratton and Wise. This would indicate that their

threshold measure was not independent of rate of response.

TABLE 1

Comparison of Threshold and Response Pates

from Stimulation to 5 Brain Sites

Brain Sites Threshold* (Rank)** Rate of Response* (Rank)**

(μA) (Response/Min)

Dorsal Medial

Hypothalamus 37.7 ± 6.7 (1) 55.6 ± 8.4 (4)

Anterior Hypothalamic Area 68.8±.2 (2) 52.5±2.6 (2)

Preoptic Area 68.9±10.2 (3) 41.9±2.6 (1)

Ventral Tegmental Area 81.3±4.1 (4) 53.7±3.9 (3)

Medial Forebrain

Bundle 86.5±6.0 (5) 62.8±7.9 (5)

ANOVA F ratio 9.32 (P<.001) 1.89 (P<.10)

* Mean ± SE.

** From lowest to highest. (If rate reflects threshold then the

lower the rate the higher the threshold).

The threshold measure we use is in classical psychophysical terms

the absolute threshold. That is, the intensity of stimulation

that is recognized half the time by the subject. The threshold

used in the previously described studies by Wise and his

43
collaborators, as well as others, is defined as the intensity of

stimulation that gives a particular rate of response. These

levels are clearly above the intensity at which the animal will

respond only half the tine. Thus, the type of threshold may be

important.

Interpretation of site of action based on thus locus of the tip of

the stimulating electrode can be misleading. We determined the

minimum level of stimulation that an animal could detect when the

minimum level of stimulation that the animal found rewarding had

already been determined (Kornetsky and Esposito 1981). In both

types of threshold determination the same electrodes in the same

animals were used; thus, the site of stimulation was identical.

As might be expected, the detection threshold is well below the

reward threshold. We then compared the effects of cocaine on

both thresholds. Figure 11 shows the results of this experiment.

At doses at which cocaine lowered the brain-stimulation reward

threshold it raised the threshold for detection. This

dissociation clearly points to the difficulty in attributing a

drug effect as being at the tip of the stimulating electrode.

FIGURE 11

Men effect of various doses of cocaine on the threshold for

brain-stimulation reward (reinforcement) and on the threshold

for brain-stimulation detection, Data are expressed as Z-scores

based on the respective mean and standard deviation of the

effects of saline N=4). Adapted from Kornetsky and Esposito

(1981) (Fron Kornetsky and Rain 1982)

44
More important, it suggests that the task and the integrated

systems involved in the demands of the task are more important in

understanding a particular drug effect than is the site of

Stimulation per se.

CONCLUSION

Our work has stemmed from the hypothesis that the threshold

lowering effect of abuse substances reflects the drugs’ ability

to cause euphoria in man (Marcus and Kornetsky 1974). This

position has been substantiated by many studies by a variety of

investigators. Further, it has been established (Esposito and

Kornetsky 1977) that tolerance does not develop to the threshold

lowering effects of morphine. This suggests that physical

dependence is not necessary for the rewarding effects of drugs

(Kornetsky and Esposito 1979; Kornetsky et al. 1979). Recently,

Bozarth and Wise (1984), in an experiment in which animals were

trained to press a lever for microinjections of morphine into the

ventral tegmental area, found no signs of physical dependence,

but physical depedence was seen after similar infusions into the

periventricular gray region

Finally, strong support for the role of the reward system in the

action of abuse substances is found in the experiment by Seeger

et al. (1984). This group used the quantitative 2-Deoxyglucose

method of Sokoloff (1982) to study the effects of amphetamine on

ICSS. Animals were prepared with electrodes in the VTA. They

compared four groups of animals: high current, low current plus

0.5 mg/kg of d-amphetamine, 0.5 mg/kg d-amphetanine but not

allowed to self-stimulate, and a no treatment group In the high

current and low current plus amphetamine groups, response rates

for rewarding stimulation was approximately the same. Rates of

local cerebral glucose utilization (LCGU) was lower at stimulation site and pathway in the low current plus d-amphetamine group

than the high current group. However, in a number of the projection areas of the VTA, LCGU's were equivalent, i.e., the nucleus

accumbens, the medial frontal cortex, the basolateral amygdala

and the locus coeruleus. In the olfactory tubercle and the

sulcal cortex, LCGU was greater in the low stimulation plus damphetamine group than the high Stimulation group.

Although these are sane of the first drug-brain stimulation

studies done by this group of investigators, these findings do

support the contention that abuse substances activate functional

reward systems and a reasonable assumption is that this may be

the basis for the euphoria and reinforcing properties of abuse

substances.

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ACKNOWLEDGMENTS

This work was supported in part by grant DA02326 from the

National Institute on Drug Abuse and Research Scientist Award

DA00099 from the National Institute on Drug Abuse.

AUTHOR

Conan Kornetsky, Ph.D.

Division of Psychiatry

Boston University school of Medicine

80 East Concord Street

Boston, MA 02118

50
The Neural Systems Approach to

Examining the Actions of Drugs

on the Central Nervous System

John K. Chapin, Stephen M. Sorenson, and

Donald J. Woodward

INTRODUCTION

One of the goals of the study of drug actions is to create a

continuity of understanding between different levels of

experimental analysis. Current approaches to the problem

range from the quantum chemistry of receptor binding to the

social behavior of human groups. The "neural systems"

approach involves study of the activity of neuronal circuits

within the intact, functioning brain, with the aim of

determining the substrates of relevant behaviors, In terms of

a hierarchy of "levels" of functionality of the central

nervous system, this approach might be considered to occupy

the niche between approaches at the cellular level and the

purely behavioral level, As such, investigations of the

effects of iontophoretic or systemic administration of drugs

on single cells on well-understood brain circuits, and in the

context of well-defined behaviors, are commonly carried out

with the hope of forming a bridge between cellular and

behavioral pharmacology.

Why be optimistic about the ability of the neural systems

approach to bridge these disciplines? Rapid advances are now

being made in describing the anatomy and physiology of the

neural circuits and their roles in the control of behavior.

Furthermore, investigations into the roles of various

neurotransmitters in these circuits are progressing rapidly.

Each advance in these traditional disciplines opens new

possibilities for parallel studies on the mechanisms of action

of drugs of abuse. Our conviction is that the most rapid

advances occur when these studies are planned and executed

together.

Drug investigations at the neural systems level seek to shed

light on the "circuit mechanisms" of their action on the

51
brain, as distinct from "cellular mechanisms." While it is

theoretically possible that an understanding of drug effects

on cellular mechanisms alone could lead to a prediction of

their effects on overall brain circuits, the circuits'

complexity appears to preclude such an outcome. Several

factors could contribute to this difficulty in predicting the

behavior of neural circuits. For example, a circuit may

respond strongly to a drug at the biophysical level, but cause

no behavioral change because of internal compensation

mechanisms that counteract the drug effect. Alternatively, a

drug may have little detectable effect at the biophysical

level, but produce a marked behavioral change. This could

result from a multiplication of subliminal effects at each

relay in a multisynaptic system. In view of these

possibilities, it appears that a proper search for the

mechanism of drug action must be conducted at the level of the

emergent properties resulting from the circuit architecture,

in addition to interactions at the biophysical level.

To illustrate how such an approach might be implemented we

will briefly summarize some work which has recently been

carried out in this laboratory. This involved

characterization of the effects of ethanol and other

anesthetic substances on single unit discharges in the awake,

behaving rat.

ETHANOL AND SENSORY TRANSMISSION

These studies were initiated in an effort to provide

information about the circuit mechanisms underlying the potent

effects of alcohol and other drugs on sensori-motor and

cognitive functions. Human performance studies have shown

that some of the lowest dose effects of ethanol involve

reductions in the ability to quickly move in reaction to a

particular sensory stimulus during simultaneous presentation

of distracting stimuli (Moskowitz and Sharma 1974; Levine et

al. 1975; Barry 1979). Such a deficit may be a major factor

in alcohol-related traffic accidents.

What are the circuit mechanisms underlying these drug

effects? It is reasonable to suspect that some components of

such "sensorimotor filtering" are normally expressed as part

of the feature extractive and/or sensory modulating

capabilities of the cerebral cortex. We have approached this

problem, therefore, by developing a neurophysiological model

in the rat of behavior-linked sensory modulation in the

sensorimotor cortex.

It has been shown in several mammalian species (Nelson 1984

[monkey]; Coquery 1978a, 1978b [cat]) that cutaneous sensory

transmission through the afferent pathways to, and within, the

somatosensory cortex is modulated just before onset and during

active movement. This modulation may be caused by direct or

indirect actions of motor cortical projections to the primary

52
somatosensory (SI) cortex (Jones et al. 1978 [monkey]; Chapin

et al. 1981 [rat]), or afferent systems to the SI cortex (see

Towe 1973). Such higher-order control of afferent

transmission may play a role in sensori-motor coordination.

We have investigated such sensory modulation phenomena in

experiments involving single unit recording in the forepaw

area of the SI cortex of awake, moving rats (Chapin and

Woodward 1981, 1982b, 1982c). These experiments (described

below) demonstrated that sensory transmission from the skin of

the paw to certain cortical neurons may be momentarily "gated

out” during reaching movements of the forelimb. This appeared

to result in a division of cortical cells into categories

which selectively respond either to “expected” sensory input

(type A cells) or to “unexpected” input (type B cells). An

example of a type B cell is shown in figure 1. This shows

responses of a single neuron to touch stimuli during rest and

running behavior. Figures IA and 1B show poststimulus time

FIGURE 1

Responses of a single SI cortical neuron to passive

and active touch of forepaw. A and B: poststimulus histograms

of unit responses (composed of an early excitatory peak, a

postexcitatory inhibition, and a late peak) to passive touch

of palm and wrist (at dotted line) with a probe (1 msec bins).

C: perievent histogram showing same unit’s sensory response

to sudden start of the treadmill (10 msec bins; same time

scale as in B). D: Unit’s response to touching on the wrist

with a hand held probe during treadmill locomotion. E: Unit’s

response to contact of the same forepaw on the ground during

treadmill locomotion.

53
histograms of the unit response to probe touch on palm and

wrist respectively (1 msec/bin). The three perievent

histograms below show the response of the same neuron to the

mechanical stimulation of the paw experienced when the

treadmill started (C), to touching the wrist with a hand-held

probe during the swing phase of locomotion (D), and to contact

of that paw on the ground during another locomotor sequence

(E). It can be seen that though the unit did not respond to

footfall (E), it did respond to probe touch of the forepaw

during swing phase (D), and to the mechanical stimulation of

the forepaw caused by the sudden onset of treadmill movement

(C). In type A cells, a roughly opposite pattern was

evident, i.e., they responded to footfall, but not to natural

or electrical stimulation during swing phase.

In order to examine such responses during drug exposure, a

quantitative stimulation paradigm was developed. In these

studies, a small stimulating electrode, chronically implanted

under the skin of the forepaw, was used to quantitatively test

afferent transmission to single units in the SI forepaw area

during different behaviors or during various phases of limb

movement. Using this test, such transmission was found to be

subject to a time-varying, selective inhibition during

treadmill locomotion. Thus, in type B cells, this had the

effect of "gating out" sensory responses to touching the skin

of the paw to the ground at footfall (whose timing is

predictable based on the trajectory of forelimb movement), but

"gating in" responses to stimuli delivered during the swing

phase (when unpredicted obstacles in the running path might be

encountered), Conversely, in type A cells, sensory responses

to skin stimulation during swing phase were gated out, while

responses to footfall were gated in.

Figure 2 illustrates a schematic diagram of the

neurophysiological findings relevant to these two types of

cells, as well as hypothetical mechanisms which may explain

these findings. Schematic perievent histograms (figure 2:A,Bright, in black) averaged around the footfall (FF) in the step

cycle show the typical responses of these cells to footfall

and to probe stimuli delivered during swing phase. "Perievent

afferent modulation histograms," shown below the perievent

histograms in A and B (labeled "time course of inhibition")

show typical time courses of inhibition (expressed as a

percentage of total possible inhibition) of sensory

transmission over the step cycle for these two types of cells.

Dotted lines above the perievent histograms show how they

would theoretically look in the absence of this inhibitory

gating. Thus, in this model, two different patterns of phasic

afferent inhibition appear able to allow sensory inputs from

the paw to different groups of cells according to whether the

predictable footfall event is imminent or not.

54
FIGURE 2

Schematic diagrams of hypothetical mechanism of sensory

gating. Perifootfall histogram (C) shows hypothetical

responses of cutaneous afferents (from forepaw) to: 1)

footfall, and 2) probe touch delivered during the swing phase

of the step cycle. In the SI cortex, type A cells are gated

to respond only to the footfall stimulus (see perifootfall

histogram A: c). Type B cells only respond to the touch during

swing phase (B:c). The "perievent gating histograms" (A:d and

B:d) show time course of inhibition of cutaneous afferent

transmission which produces the observed gating effect.

Hypothetical circuit mechanism for the gating (A:b and B:b)

involves axonal projections from neurons in adjacent motor

cortex (dark cells at left), which, through interneurons (open

circles) may inhibit SI cortical neurons (dark cells at

right). Perifootfall histograms in A:a and B:a show observed

activity in different classes of motor cortical neurons.

One hypothesis to explain the observed inhibition is that

this inhibition may result from the activity in the rostrally

adjacent motor cortex of movement correlated units, which have

been shown to project to the somatosensory‘cortex. (In figure

2, hypothetical collaterals of motor cortical neurons are

shown inhibiting SI cells through interneurons.) In our

experiments, most such motor cortical cells discharged

strongly either during the middle swing phase or just before

footfall. These time courses corresponded closely with those

of afferent inhibition previously shown in the somatosensory

cortex.

To the extent that this "selective gating” phenomenon

constitutes an example of higher order sensoriwtor processing

55
in the cortex, it may be of value as a model for investigation

of the effects of alcohol and other drugs which affect

sensorimotor coordinative abilities. Figure 3 shows results

of an experiment which assessed the effects of a low dose of

ethanol (0.3 g/kg body weight, intra-peritoneal) on responses

of a single neuron in the cortex of an awake, freely moving

rat to constant stimulation through an electrode chronically

implanted under the skin of the forepaw. (According to

benchmark experiments, such an injected dose should reach 0.55

to 0.6 g/kg blood concentration between 5 and 10 minutes

postinjection.) The histogram in figure 3A shows the response

of this unit to such stimulation during normal awake, resting

behavior. The initial excitatory responses of such neurons

are often divided into two separate early excitatory peaks

(E1a'and Elb, figure 3A).

FIGURE 3

Responses of a single SI cortical neuron to paw stimulation

during "resting" (A,B) and "walkingw (C,D) behavior before

(A,C) and after (B,D) ethanol administration. Bin width: 1

msec; X-axis ticks: 25 msec; Y-axis ticks: 5 spikes/sec/bin

(normalized for all histograms).

The histogram in figure 3C, which was constructed by using

the same stimulus parameters during treadmill running, shows

that both components of this neuron's response to the same

stimulation during locomotion behavior were markedly reduced.

Five to fifteen minutes after ethanol injection the following

changes occurred: 1) the Elb (late) component of the initial

response was selectively reduced (see figure 3B), and at

higher doses replaced with an inhibition. 2) Though the Ela

(early) response was itself reduced somewhat, it became much

less variable across behaviors. In particular, the level of

56
this response during movement in ethanol intoxication was

actually increased relative to similar locomotor behaviors

before the drug injection (compare figures 3C and 3D). Thus,

ethanol reduced an inhibitory process which is normally

associated with selective gating of sensory transmission in

the normal state. Similar, though more profound, effects have

been found at higher doses (Chapin and Woodward 1983).

What might be the circuit mechanism of this effect of ethanol

on sensory gating? The hypothesis set forth in figure 2

suggests that the gating may result from the activity of

movement correlated cells in the rostrally adjacent motor

cortex. Figure 4 illustrates the effect of a low dose of

ethanol (0.5g/kg) on a neuron isolated in the forelimb area of

the motor cortex. As is typical of neurons in this cortical

area, this neuron discharged most actively during active

movement of the forelimb, specifically the forepaw-projection

movement just preceding footfall during locomotion. The

strength of this movement correlate was quantitated through

the use of a computer synchronized videotape recording of the

rat’s movements. Each video field-frame (with 17 msec

resolution) was labeled with an automatic video frame counter

so that subsequent frame-by-frame analysis could be used to

correlate movement and unit activity.

The perievent histograms in figure 4 were generated by

averaging unit activity around the footfall event (which

marked the termination of the movement relevant to this cell).

These were constructed for each of 28 l-minute running

periods (each containing from 30 to 40 steps) before and after

ethanol administration. The intensity of unit firing (in

spikes/sec) during the movement-related discharge (marked by

"E" in the bottom-left histogram, and the line plot in figure

4), as well as the inhibitory period (I), were measured with

the computer and plotted as a percent change from the

discharge rate during a control epoch (C) in the histogram.

Time of ethanol administration is marked by an arrow at the

left of the time axis. Before ethanol administration (see

bottom-left histogram), the discharge rate (spikes/set) during

the reaching movement averaged about 80% above that measured

in the late stance phase (considered as a control period). No

inhibition was seen after this excitatory phase. Starting at

3 minutes postethanol, a steady decline in the motor response

was measured, and after 11 minutes It was only about 20%

above the control level (top-middle histogram). In addition,

57

484-443 0 - 85 - 3
FIGURE 4

Time course of ethanol's effect on forelimb movement

correlated discharge in the rat motor cortex, Perifootfall

histograms: pre-ethanol (bottom-left); 11 mins postethanol

(top-center); 24 mins postethanol (bottom-right). Bin width:

2 msec; X-axis ticks: 50 msec; Y-axis ticks: 10 spikes/set/bin.

See text for further explanation.

an inhibitory period appeared in the postexcitatory phase

(corresponding to early stance phase). This had a time course

similar to that of the reduction in the excitatory response.

Even while these motor cortical responses were drastically

changed only a moderate disturbance of the locomotory gait was

observed. After 20 to 25 minutes (bottom-right histogram),

both excitatory and inhibitory responses recovered to prealcohol levels. From 4 to 20 minutes after ethanol injection,

the normal movement correlated activity of this cell was

markedly reduced or almost abolished. Nearly full recovery

was subsequently achieved. We have not observed effects of

this magnitude at such low doses in recordings elsewhere in

the brain (especially the cerebellum).

This powerful effect on motor cortical unit activity could be

involved in some aspects of ethanol's disruption of motor

coordination. In addition, in the simplified model of this

circuit brought out in figure 2, such an action on motor

cortex could also bring about ethanol's observed disruption of

sensory gating in the somatosensory cortex. Figure 5

illustrates, in the same schematic format used in figure 2,

one possible explanation which is consistent with our findings

58
FIGURE 5

Schematic diagram showing hypothetical mechanism of ethanol's

effect of sensory eating. "Pre-ethanol" schematic (top;

I:a-e) is similar to that for type B cell in figure 2. I:e

shows perievent histogram of hypothetical response to footfall

in cutaneous afferent system; II:e shows response after

ethanol. Normal type B pattern of cortical gating shown in

I:c,d. Dotted line in I:c shows how perifootfall histogram

would look in absence of gating; I:d shows perifootfall gating

histogram. In the postethanol condition (II:c,d), there is

some tonic inhibition, but no selective gating. Perievent

histograms I:a and II:a show observed activity of motor

cortical units over the step cycle in the pre- and post-ethanol

conditions.

in both cortices. The "pre-ethanol" condition of a

typical type B cell is illustrated at the top of figure 5.

The "postethanol" condition (bottom) schematizes the observed

reduction in the selective "gating-out" of the footfall

response. This might be attributable, in this simple model,

to a reduction in activity of motor cortical units which

normally subserve such inhibition. While the real situation is

undoubtably much more complex, this model appears consistent

with all currently available evidence. If other regions of

frontal or prefrontal cortex are similarly affected by such

low ethanol doses, a wide range of perceptual, cognitive, and

behavioral control deficits could result.

It is instructive to compare these actions of ethanol to the

those of other sedating or anesthetic drugs, such as halothane,

59
barbiturate, and ketamine. We have found that each of these

drugs produces a selective reduction in the longer latency

(E1b) sensory responses of single somatosensory cortical

neurons (Chapin et al. 1981a). However, the behavioral

concomitants of the phenomenon vary considerably according to

the drug. For example, a dose of ethanol capable of producing

coma results in a virtual block of all somatosensory input to

the cortex, whereas after barbiturate-induced coma, the

short-latency response remains strong.

The "dissociative anesthetic" ketamine, on the other hand,

produces markedly different effects than ethanol. First, it

does not selectively reduce the longer latency component of

the excitatory sensory response in the cortex, but instead

reduces both early excitatory responses (E1a and E1b; see

figure 3) and amplifies a much longer latency (>100 msec)

component. Further, it does not disinhibit responses to

sensory input during movement as does ethanol. Finally,

whereas ethanol reduces both spontaneous and movement

correlated discharge in the motor cortex, ketamine increases

both categories of firing. Thus, while moderate doses of both

drugs produce ataxic "staggering" behavior, ethanol is

correlated with a reduction in motor and "associative"

cortical activity, and ketamine is correlated with an increase

in such activity. This is consistent with the results of

Hammer and Herkenham (1983) who showed that metabolic activity

(as indicated by uptake of 2-deoxyglucose) of associative

regions of the rat cerebral cortex was markedly increased

relative to primary sensory regions after ketamine

administration.

DISCUSSION

The question to be considered is how results of experiments

at the cellular level may relate to results of experiments at

the systems level such as those described here. Our view is

that the emergent properties of intact brain circuits make it

difficult to account for drug-induced behavioral changes based

on the results of information obtained from experiments at the

cellular or membrane level. For example, in the awake,

behaving animal we found that ethanol (and other sedative

hypnotics such as barbiturate, or anesthetics such as

halothane) markedly increased the postexcitatory inhibitory

response to sensory stimulation. Initially, this appears to

be consistent with a hypothesis suggested by Hunt (1983) that

ethanol selectively enhances pre-synaptic release of

gamma-aminobutyric acid (GABA).

On the other hand, our finding that the powerful inhibition

of sensory input normally observed during movement was

reduced, rather than increased, by ethanol seemed to

contradict the notion that such phenomena are all attributable

to increased GABA release. A possible explanation may lie in

the suggestion (see figure 5) that this reduction in sensory

60
gating may be caused by a reduction in activity of motor

cortex cells, which may normally provide the gating bias

signal.

It is not clear at this point where or how ethanol acts to

produce this effect on the motor cortex, and this is the crux

of the matter. It is not unreasonable to suspect, for

example, that frontal cortical excitability may be controlled

by afferents rising through multisynaptic pathways from the

basal ganglia, cerebellum, or brain stem, which themselves may

be compromised by more direct physical effects of ethanol.

This loss of excitatory input may decrease the ability of

motor cortical neurons to respond appropriately during

movement, thereby reducing the inhibitory input to SI.

Thus, the possibility that this drug effect may be caused by

changes in the activity of neurons which may be several

synapses removed from the recorded neuron reduces the

predictive ability of a model based on information derived

from reduced systems. At the very least, this reinforces the

argument that an understanding of drug effects on behavior

will require further knowledge not only of molecular,

membrane, and synaptic mechanisms, but also of the circuit

mechanisms. Overall, it will be necessary to build a

continuity of understanding that serves to connect information

gained at all these levels. The type of study described here

is one way to address this issue.

REFERENCES

Barry, H. Behavioral manifestations of ethanol in intoxication

and physiological dependence. In: Biochemistry and

Pharmacoloy of Ethanol. New York: Plenum, 1979. pp. 511-529.

Chapin, J.k., and Woodward, D.J. Modulation of sensory

responsiveness of single somatosensory cortical cells during

movement and arousal behaviors. Exp Neurol 72:164-178, 1981.

Chapin, J.K., and Woodward, D.J. Cortico-cortical connections

between physiologically and histologically defined zones in

the rat SI and MI cortices. Abstr in Soc for Neurosci Ann

Meeting, 1982a.

Chapin, J.K., and Woodward, D. J. Somatic sensory transmission

to the cortex during movement: I. Gating of single cell

responses to touch. Exp Neurol 78:654-669, 1982b.

Chapin, J.K., and Woodward, D.J. Somatic sensory transmission

to the cortex during movement: II. Phasic modulation over

the locomotor step cycle. Exp Neuronal 78:670-684, 1982c.

Chapin, J.K., and Woodward, D.J. Ethanol’s effect on selective

gating of somatic sensory inputs to single cortical neurons.

Pharm Bioch Behav 18:489-493, 1983.

Chapin, J.K.; Waterhouse, B.D.; and Woodward, D.J., Differences

in cutaneous sensory response properties of single

somatosensory cortical neurons in awake and halothane

anesthetized rats. Brain Res Bull 6:63-70, 1981.

61
Coquery, J.H. Selective attention as a motor program. In:

Requin, J., ed. Attention and Performance. Hillsdale:

Lawrence Erlbaum, 1978a.

Coquery, J.M. Role of active movement in oontrol of afferent

input from skin the cat and man, In: Gordon, G., ed. Active

Touch. Oxford: Pergammon Press, 1978b. pp. 161-179.

Hammer, R.P., and Herkenham, M. Altered metabolic activity in

the cerebral cortex of rats exposed to ketamine. J

Neurol 220:396-404, 1983.

Hunt, W.A. The effect of ethanol on GABAergic transmission.

Neurosci Biobhav Rev 7:87-95, 1983.

Jones, E.G.; Coulter, J.D.; and Hendry, S.H. Intracortical

connectivity of architectonic fields in the somatic sensory,

motor and parietal cortex of monkeys. J Comp Neurol 181:

291-348, 1978.

Levine, J.H.; Kramer, G.G.; and Levine, E.N. Effects of alcohol

on human performance: An integration of research findings

based on abilities classification. J Appl Psych 60:

285-293, 1975.

Moskowitz, H., and Sharma, S. Effects of alcohol on peripheral

vision as a function of attention. Hum Factors 16:174-180,

1974.

Nelson, R.J. Responsiveness of monkey primary somatosensory

cortical neurons to peripheral stimulation depends on “motor

set”. Brain Res 304:143-148, 1984.

Towe, A.L. Somatosensory cortex: descending influences on

ascending systems. In: Iggo, A., ed. Handbook of Sensory

Physiology. Vol II. Somatosensory System. New York:

Springer-Verlag, 1973. pp. 701-718.

ACKNOWLEDGMENTS:

This work was supported by USPHS grants NS-1804, AA-3901, DA-02338,

AA-30533, and an award from the Biological Humanics Foundation.

AUTHORS

John K. Chapin

Stephen M. Sorensen

Donald J. Woodward

Department of Cell Biology

University of Texas Health Science Denter at Dallas

5323 Harry Hines Blvd.

Dallas, Texas 75235

62
Correlating Behavior with Neural

Activity: An Approach to Study

the Action of Drugs in the

Behaving Animal

Sam A. Deadwyler

INTRODUCTION

The assessment of drug action in the central nervous system (CNS) often requires

three different but related approaches. A logical first step is the delineation of

where in the brain or spinal cord the drug is likely to act. More specifically, it

is necessary to have anatomical evidence that a given drug acts at a particular

location in the nervous system. This type of evidence is supplied by the vast

array of histochemical, biochemical, and more recently, molecular genetic

techniques which provide information regarding the presence of receptors (Atweh

and Kuhar 1977), endogenous ligands to bind to those receptors (Elde et al.

1976), and which neural structures incorporate those ligands (Cuello 1983; Sar et

al. 1978; Walmsley et al. 1980).

The second approach is likely to be some assessment of the physiological action

of the particular drug. Traditionally, examination of the neurophysiological

effects of a drug in acutely anesthetized animals and observation of the

neurological signs following injections in the unanesthetized animal have been the

methods of choice. More recently, advances in brain slice technology

(Dingledine 1984) have made it possible to study drug actions at the cellular and

synaptic levels using intracellular and, most recently, single channel recording

techniques (Sakman and Neher 1983). However, while slice electrophysiology--

which reveals the actions of drugs at single membrane loci and receptor

elements--has supplied a great deal of new information regarding drug actions,

there are obvious limitations in terms of extrapolating the findings to the intact

nervous system.

A related approach to describing the functional consequences of drug action in

the CNS has been to study the effects such influences might have on behavior.

Psychopharmacological approaches have characterized various behavioral assays,

and highly specific drug influences can now be detected and related closely to

known pharmacological indices of drug action (such as rank-order potency,

dissociation constants, PA2 analyses, etc. See Iverson et al. 1984). However,

even in the latter case, once a drug-dependent behavior has been isolated and

characterized, the linkage of the physiological substrate(s) to the behavioral

changes produced by the drug are frequently little more than theoretical

assumptions.

63
The third stage, therefore, in the complete analysis of the actions of abused

drugs is to identify specifically those neural mechanisms altered by the drug

which define the drug-dependent behavior. Historically, this has been the most

difficult approach because it requires examination of neurophysiological

mechanisms in behaving, unanesthetized animals. In the past, recording from

freely moving animals required a high degree of technological sophistication.

Now, however, the recording of concomitant neural activity and behavior has

been perfected and is available to most neuroscience investigators. In addition,

interpretation of such data has become more meaningful due to the use of

identified cell populations with known I) neurotransmitter and neuromodular

affiliations (cf. Jacobs et al. 1984; Foote et al. l983), or 2) identified inputs and

outputs (cf. Evarts et al. 1971; Fetz and Finocchio 1979; Chapin et al., this

volume).

OPIATE DRUGS IN THE BRAIN

Several important features of opioid action on neural tissue have been described

in recent years (Bradley et al. 1978; Duggan and North 1984). Extensive

investigations have shown that opiates and opioid peptides alter the physiological

activity of nerve cells through specific receptor coupled membrane processes (cf.

Miller 1984). The most commonly reported postsynaptic action of the alkaloid

opiate compounds (morphine, levorphanol, etorphine, and normorphine) is a

naloxone reversible depression in firing (Duggan and North 1984). The

decreased firing results from an opioid receptor-mediated increase in potassium

conductance. It is not yet clear whether this increase in potassium conductance is

produced by an increase in intracellular calcium (North and Williams 1983b). It

has become clear in recent years, however, that opiates probably have the same

actions in many different cell types within the CNS. North and Williams (1983a)

report that morphine and the opioid peptides leucine, methionine enkephalin, and

appropriate analogs produce an increase in potassium conductance in the

neurones of the locus coeruleus, substantia gelatinosa, and myenteric plexus.

Although the consequences of this increased potassium conductance and

subsequent decreased excitability may differ with respect to location and cell

population within the CNS (Dingledine 1981; Nicoll et al. 1980), there is little

evidence to suggest that opiates behave differently with respect to physiological

actions on those cell types that possess opiate receptors. However, there are

other effects of opiates on neural tissue that have been reported. These range

from direct transmitter-like influences to humoral-like modulation of both preand post-synaptic mechanisms (Barker 1978; West and Miller 1983).

The discovery and subsequent confirmation by many investigators of multiple

opiate receptors (Martin et al. 1976; Lord et al. 1977) have added a new

dimension of complexity to the understanding of opioid action in central neural

processes (cf. Martin 1984). In addition, a variety of opioid peptides have been

described in the brain, spinal cord, and periphery (Imura et al. 1983; Hughes

1983). It is clear from these investigations that in the future a consideration of

the specific nature of opioid action on central neural mechanisms will require a

detailed analysis of the physiology and pharmacology of specific opiate receptor

subtypes as well as a thorough investigation of their possible endogenous ligands

(Watson et al. 1982; McGinty et al. 1984; Goodman et al. 1980; Atweh and Kuhar

1983). As has been recently reported (Walker et al. 1982; Dunwiddie et al. 1980).

reversibility by the mu-receptor antagonist naloxone may no longer be a criterion

for the unequivocal establishment of a neurophysiological effect mediated by

opioid peptides.

64
EFFECTS OF OPIATE DRUGS IN HIPPOCAMPUS

One of the striking divergences from the consensus of reports regarding opiate

and opioid peptide neural effects in the brain is the well-characterized excitatory

influence of these substances in the hippocampus. Since Nicoll et al. (1977)

initially reported the excitatory effects of both morphine and methionine

enkephalin following microiontophoresis of these substances within hippocampus,

there have been many detailed studies confirming the excitatory nature of opiates

in this structure (cf. Corrigall 1983). The basis of the excitatory influence of

opioids has been suggested to reside in the inhibitory action of opiates and opioid

peptides on a select population of hippocampal inhibitory interneurones. The

inhibition of these interneurones produces a disinhibition of pyramidal cells

which receive tonic and/or phasic inputs from the opiate-sensitive inhibitory

cells (Nicoll et al. 1980; Duggan and North 1984). This hypothesis was originally

proposed by Zieglgansberger et al. (1979) on the basis of differential extracellular

firing patterns from two distinct cell types following microiontophoresis of

enkephalin in the CA1 region of hippocampus. Subsequent reports both in

support of (Lee et al. 1980) and in conflict with (Haas and Ryall 1980;

Dingledine 1981) the disinhibitory hypothesis have since appeared in the

literature.

The analysis of the direct effects of opiate and opioid peptides on hippocampal

cellular activity and synaptic mechanisms in CA1 has been provided by

Dingledine (Dingledine 1981; Valentino and Dingledine 1982). These investigations have shown that mu- and delta-receptor agonists. but not kappa-receptor

agonists, have dose-dependent effects on the synaptic and cellular excitability of

pyramidal cells in the CA1 field of hippocampus. Concentrations of opiates

(morphine) and opioid peptide analogs (D-ala-dleucine enkephalin) in the

nanomolar range were shown to increase the excitability of hippocampal cells.

These findings are consistent with the mechanism proposed recently by Newberry

and Nicoll (1984) that opiates and opioid peptides decrease a dendritically located

inhibitory influence which may be tonically or phasically activated by excitatory

afferent terminals.

The results of in vivo experiments are consistent with the above in vitro

hippocampal slice findings and have shown that apparent endogenous as well as

exogenous opioid substances produce epileptiform discharges in hippocampus

(Urca and Liebeskind 1979; Frenk et al. 1978, 1983; Henricksen et al. 1978).

Behaviorally convulsant influences appear to be mediated by delta receptors,

while epileptic discharges which do not produce behavioral convulsions are

mediated specifically by mu receptors. Effective routes for producing the

increased excitability of hippocampal tissue in vivo are intraperitoneal,

intravenous, and intracerebroventricular (Frenk et al. 1983). The in vivo

epileptiform effect, but not the convulsant aspects of opioid substances, can be

reversed by systemic administration of naloxone.

In summary, it can be said that opiates with mu-receptor agonist properties and

certain opioid peptides with delta-receptor agonist properties increase the

excitability of hippocampal pyramidal cells. Kappa receptors are not implicated

since opiate substances with high affinity for this receptor do not produce either

epileptiform activity or convulsions at equivalent effective concentrations of muand delta-receptor agonists. The nature of hippocampal process activated by

opioid receptor binding is not as yet identified. The most likely candidates for

such a mechanism appear to be 1) an opiate receptor-mediated inhibition of the

release of gamma-aminobutyric acid (GABA) from a specific population of

65
inhibitory interneurones (Zieglgansberger et al. 1979; Robinson and Deadwyler

1981; Nicoll et al. 1980). or 2) a phasically activated convergent inhibitory

afferent fiber system with opioid receptors located on GABA axon terminals

(Newberry and Nicoll 1984).

FUNCTIONAL SIGNIFICANCE OF OPIOID SUBSTANCES IN HIPPOCAMPUS

The presence of endogenous opioid peptides in hippocampus has been described

in detail by Gall et al. (1981) and McGinty et al. (1983). These investigators

have shown an enkephalin-like immunoreactivity in the perforant path region of

the dentate gyrus and in the cells of origin of this pathway in the entorhinal

cortex. These findings have importance for the eventual understanding of opioid

peptide action in the hippocampus and possibly other limbic areas because of the

considerable knowledge regarding the functional nature of this synaptic

connection. The perforant path connection in the dentate gyrus exhibits several

features which indicate a memory-like function (Lynch and Baudry 1984). In

this context, the presence of an endogenous opioid peptide in the perforant

pathway suggests a possible neurotransmitter or neuromodulator role of opiates in

the establishment of long-term representations of sensory events. Haas and Ryall

(1980) have demonstrated excitatory actions of enkephalin analogs on the granule

cells of the dentate gyrus, the targets of the perforant path projections.

For several years, we have been developing a model of hippocampal function that

has as its goal the identification of the behavioral circumstances which provoke

sensory activation of synaptic pathways in the hippocampus of the

unanesthetized, freely moving rat. In so doing. we have devised a means of

studying the functional properties of the perforant path in the behaving rat

(figure 1). Our past research has shown that an auditory evoked potential (AEP)

recorded in the outer molecular layer (OM) of the dentate gyrus (the region of

termination of the perforant path fibers) during performance of a sensory

discrimination represents the transmission of sensory information into the dentate

gyrus from the entorhinal and perirhinal cortex (Deadwyler et al. 1979a, 1979b,

1981). We have termed this evoked potential the OM AEP.

Prior investigations have documented several factors which control the amplitude

changes in the two negative peaks (N1 and N2) (figure 2) of the OM AEP which

appear to be specific to the stimulus context in which the animal performs the

behavioral task. The Nl peak is large during acquisition of the behavior and

decreases in amplitude as the tone stimulus gains significance through

instrumental conditioning (figure 2). The N2 peak is nonexistent prior to

conditioning and following extinction, but it increases and is maintained at

maximal amplitude during acquisition and achievement of criterion behavioral

performance. During extinction, the amplitude of Nl is increased and N2 is

markedly decreased (figure 2). Recent evidence suggests that the N1 peak is a

reflection of event-specific information regarding the nature of occurrence of

positive-reinforced and negative-nonreinforced stimuli in a two-tone auditory

discrimination task (West et al. 1982). Depth profile analyses of perforant path

elicited extracellular field potentials and the N1 component of the OM AEP, as

well as lesion studies which eliminated the entorhinal cortex or severed the

perforant path connections to the hippocampus, strongly implicate the perforant

path as the generator of the N1 component of the OM AEP (Deadwyler et al.

1981, 1982). The generator of N2 has not been identified; however, the integrity

of the septohippocampal system is required for the behavioral modulation of this

component (Deadwyler et al. 1981).

66
RECORDING PROCEDURE

FIGURE 1

Recording procedures for chronic animal preparation. #A. Schematic drawing of

entire rat brain showing orientation of stimulating (S1 and S2) and recording (R)

electrodes. Stimulating electrodes are placed in the perforant path fibers (S1)

originating from the entorhinal cortex (Entor. denoted by crosshatching in A) and

the dentate gyrus commissural (Comm.) system (S2) originating in the

contralateral hippocampus. The recording electrode (R) is placed in the dorsal

hippocampus via phyiological criteria (i.e., electrically elicited monosynaptic field

potentials) in each animal prior to behavioral testing. #B. An exploded drawing

of the hippocampal region illustrated in A. A granule cell within the dentate

gyrus is shown receiving convergent inputs from the two main fiber systems

shown in A. These cells, in turn, project to hippocampal pyramidal cells, shown

as triangles. #C. A further three dimensional expansion of this same dentate

gyrus region showing a dentate granule cell with laminated afferent inputs from

perforant path and commissural system with recording electrode positioned in the

outer molecular layer (region of granule cell dendrites and afferent fiber systems)

as during recording of the OM AEP. The perforant path region of the molecular

layer is the location of enkephalinergic axons, as described by Gall et al. (1981).

67
The fact that the N1 component of the OM AEP seems to accurately reflect the

perforant path synaptic input to the dentate gyrus indicates that it can be utilized

as a “physiological marker” for the functional involvement of this pathway in

behaviorally relevant neurophysiological processes. Because of our interests in

the functional significance of the opioid peptides in the perforant path. we

examined whether or not morphine and the potent mu-receptor agonist peptide

FK 33-824 (Roemer et al. 1977) would alter the OM AEP in behaving rats

(Christian et al., in press). Both intraperitoneal (5.0 to 7.0 mg/kg, morphine) and

intracerebroventricular (36.0 ng/ul, FK 33-824) injections of these compounds

increased the amplitude of the perforant path component (NI) of the OM AEP

(figure 3). Intraperitoneal (i.p.) injections of naloxone (1.0 to 2.0 mg/kg)

following morphine or FK 33-824 administration reversed the opioid-induced

increase in the N1 and decrease in the N2 component (figure 3).

Examination of the extracellular synaptic current generated by monosynaptic

electrical activation of perforant path fibers in these same animals showed

naloxone reversible increases at all stimulus intensities, but were more marked at

the higher voltages (figure 4). While these treatments did not have deleterious

effects on behavioral responding at the indicated doses, increased doses (7.0 to

10.0 mg/kg) usually resulted in termination of behavioral responding even though

the reinforcement contingency remained in effect.

Thus, opiates increased the N1 component of the OM AEP and the efficacy of

the perforant path-to-dentate granule cell synapses simultaneously, while

provoking an extinction-like correlate of the electrophysiological changes

previously reported in nondrugged animals (see figure 2). In other studies,

opioid substances have recently been shown to abolish both the behavior and the

hippocampal neural correlate of the classically conditioned nictitating membrane

response in the rabbit (Mauk et al. 1982. 1983). These findings suggest a

function for the recently identified enkephalin-like opioid peptides within the

perforant path and possibly other systems in the hippocampus. Whether or not

these effects reflect a direct transmitter-type action of opiates and opioid

peptides on dentate granule cells or modulation of other nonopioid transmitters in

this region is not known. Gall et al. (1981) and McGinty et al. (1983) reported

immunohistochemical labeling of opioid peptides associated with other regions of

the dentate gyrus in addition to the perforant path. Enkephalin-like

immunoreactivity was present in the mossy fiber axons and cell bodies of the

dentate granule cells, which also showed positive reactivity for dynorphin

(McGinty et al. 1983). The perforant path region is devoid of dynorphin

immunoreactivity (McGinty et al. 1983). These results provide an anatomical

basis for the above demonstrated electrophysiological effects of opioid substances

on the OM AEP.

The results of our studies in the chronic animal demonstrate that exogenously

administered opiates and opioid peptides alter the amplitudes of sensory evoked

potentials in the hippocampus. A corresponding excitatory influence was

obtained in recordings of monosynaptic field potentials elicited by electrical

stimulation of the perforant path. Since the entorhinal cortex receives input

from the primary cortical sensory areas (Van Hoesen and Pandya l975), our

results reveal a possible selective capacity for endogenous opioid peptides to

modulate and/or regulate the transmission of sensory information into specific

limbic brain regions (Lewis et al. 1981). Such a presumed action of endogenous

opioid peptides would not be unlike that demonstrated in pain pathways in the

68
FIGURE 2

Examples of OM AEP’s illustrating relevance to behavioral performance in

auditory discrimintion task. Early: Top trace shows a composite (mean) of

several OM AEPs (solid trace) recorded early in training prior to establishment of

criterion performance. The N1 and N2 components are denoted underneath their

respective peaks in this record. Each tracing is a composite normalized average

of several individual OM AEPs recorded within different 50 trial training

sessions. Dotted trace indicates session-to-session variance (standard deviation)

of individual OM AEPs across 14 sessions. Late: Composite OM AEP recorded

after achievement of criterion performance in the auditory discrimination task.

Extinction: Lower trace illustrates changes in composite OM AEP during

extinction of the auditory discrimination behavior. Calibration: 33 uV.

69
FIGURE 3

Effects of opioids on the OM AEP. Top trace (NOR): The effect of morphine

administered i.p. on N1 and N2 components of the OM AEP. Solid curve is the

control vehicle injection record. Dotted curve is the record obtained following a

5-7 mg/kg dose of morphine sulfate. Note increase in N1 and decrease in N2

caused by morphine. FK 33-824: Middle tracing illustrates similar but more

dramatic effect of the mu-type receptor agonist FK 33-824 (Sandoz)

administered intracerebroventricularly at a dose of 36 ng in 10 microliters of

vehicle (saline). Large increase in N1 and reduction in N2 amplitude are similar

to that seen with morphine (IP) and during extinction of the behavior (as

illustrated in figure 2). NAL REV: Reversal of FK 33-824 effect (solid curve)

by subsequent injection of 1-2 mg/kg of naloxone i.p. (dotted curve). Each trace

is the normalized average of all sessions and all animals exposed to the indicated

treatment.

70
STIMULUS VOLTS STIMULUS VOLTAGE

FIGURE 4

Effects of morphine on perforant path extracellular synaptic potential (ESP). Left: Input/output curve of ESP (see

inset) prior to and after i.p. administration of 5.0 mg/kg dose of morphine. Calibration 1.0 mv and 5.0 msec. Arrow

indicates voltages at which inset records were obtained on the curve. Right: Group data for three different animals

showing percent change in ESP at different stimulus intensities following morphine injection (7 mg/kg). Bars indicate

standard errors. Stimulus voltages were normalized across animals from lowest to highest intensities (100-400 uA).
spinal cord (Zieglgansberger 1980; Yaksh 1981; Cuello 1983). An opioidregulated synaptic mechanism could, in this context, modify the responsiveness

of hippocampal cells to sensory inputs arriving via the perforant path from the

entorhinal cortex.

EFFECTS OF CANNABINOIDS IN HIPPOCAMPUS

Studies similar to those described above have recently been instituted to examine

the psychoactive effects of cannabinoids on the OM AEP and unit discharges in

the dentate gyrus. Animals trained in the same auditory discrimination task as

described previously were subjected to low and high doses of delta-9-

tetrahydrocannabinol (THC), the major psychoactive derivative of marijuana.

These investigations showed that all doses of THC altered behavior in a dosedependent manner. At doses between 0.5 mg/kg and 1.5 mg/kg there was a

tendency to selectively increase responding (above control levels) to the negative

(nonreinforced) tone in a two-tone discrimination task. These behavioral effects

were not observed even with very high doses of opiates (>10.0 mg/kg). In

contrast, i.p. doses of THC above 2.0 mg/kg caused the animals to cease

responding altogether.

The above doses of delta-9-THC produced dose-dependent changes in OM AEPs.

Control OM AEPs were stable across nondrug days. The 0.5 mg/kg dose slightly

decreased N1 but not N2 in the OM AEP, however, doses of 1.0 to 2.0 mg/kg

produced a substantial decrease in N1 recorded during both single and

differential tone discrimination performance. N2 amplitude was increased when

N1 was decreased and vice versa. Like opiates, these changes were observed for

up to 2 hours after drug administration, at which point they tended to recover to

control levels.

The systematic effects of dosage indicate that the changes in the OM AEP may

be functionally related to corresponding changes in the behavior observed at the

same dose levels. The data indicate that 1) dentate gyrus neural activity is

affected by pharmacological doses of delta-9-THC, and 2) dose-dependent

changes can be expected from usage of this substance. The above findings

confirm the fact that the effective dose range for observing alterations in

hippocampal activity in behaving rats is similar to the dose range reported

previously for other, unanesthetized preparations (Turkanis and Karler 1981).

The biphasic action of this substance previously documented in in vitro

preparations (Vardaris et al. 1977; Weisz et al. 1982), however, was not observed

in this study. Furthermore, our results suggest that gross disruptions in

behavioral performance probably occur within the same dose ranges in which

hippocampal effects were observed. It has recently been shown that cannabinoids

exert a profound effect on memory in humans and it has been suggested that the

effects are mediated via alteration in the cholinergic projections to the

hippocampus (Miller and Branconnier 1983; Cheney et al. 1981). Our results are

compatible with these assumptions.

CONCLUSION

The arsenal of techniques available to examine the effects of drugs of abuse on

neural processes now encompasses recordings made at the single cell and

subcellular levels, as well as the detection of changes in neural events related to

“associative” or “cognitive” processes in behaving animals. These techniques,

coupled with equally sophisticated biochemical and anatomical analyses of drug

72
dispersion, site of action, and relation to endogenous neurotransmitter and

neuropeptide systems, make it possible to characterize the continuum of neural

effects necessary for determining the abuse potential of untested drugs, while

providing new insights into the neural effects of more traditionally abused

substances. The use of chronic animal preparations will allow determination of

not only the consequences of drug action on behavior but also the effects on

identified neural events following acute, as well as repeated, drug exposure.

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AUTHOR

Sam A. Deadwyler, Ph.D.

Bowman Gray School of Medicine

Wake Forest University

Winston-Salem, North Carolina 27103

76
The Use of Event-Related Potentials

In the Study of Alcoholism:

Implications for the Study of Drugs

of Abuse

B. Porjesz and H. Begleiter

While it has long been apparent that the human brain is

susceptible to both the acute and chronic effects of alcohol, it

is only recently, with the development of sophisticated

computer-assisted technology, that these effects can be studied

in vivo. With the use of this technology, evoked brain

potentials (EPs) can be studied to examine more subtle forms of

brain damage and/or dysfunction by examining the relation of an

EP to a specific sensory or behavioral event. Event-related

potentials (ERPs) offer a unique approach for assessing level of

brain functioning, as they permit the simultaneous observation of

electrophysiology and cognition.

An ERP is obtained by recording with a non-invasive scalp

electrode the time-locked brain electrical activity that follows

the delivery of a discrete stimulus to any sensory modality

(e.g., auditory, visual). Signal averaging techniques make it

possible to extract these time-locked neuroelectric signals

(i.e., the ERPs) from the background random "noise," which is

canceled out with these procedures. Depending on stimulation

parameters and recording sites, these time-locked signals

represent activity at neural generators from the peripheral end

organ to higher integrative centers of the brain. The

quantitative measurement of salient features extracted from ERP

recordings provide objective neurophysiological data reflecting

various aspects of brain function and integrity.

ERP techniques have proven to be especially valuable in indexing

electrophysiological concomitants of complex cognitive tasks

(Hillyard et al. 1978; Donchin 1979; Donchin et al. 1978). They

can be recorded in conjunction with behavior, or even when no

behavioral response is required, to both attended and unattended

stimuli. Thus, the ERP techniques are very sensitive indices of

the functional integrity of the brain. They differ from

computerized axial tomography (CT-scan) or nuclear magnetic

resonance (NMR) in that they reflect subtle, dynamic,

moment-to-moment changes in brain functioning that are elicited

77
while the brain is being challenged, rather than the static gross

brain damage that is apparent on the scans. As a result, ERP

aberrations are often observed in the absence of brain damage as

visualized on CT-scan.

In order to consider how EP techniques may be used to study

problems of drug abuse, it may be useful to examine how they have

been used to study the effects of alcohol on the brain. EP

techniques have provided indices sensitive to the various alcohol

effects, namely: alcoholization, tolerance, withdrawal, and

long-term brain dysfunction resulting from chronic alcohol use.

Alcoholization is characterized by marked decreases in EP

amplitude (Bierley et al. 1980) and prolongations in conduction

velocities of the brain stem potential (BSP) (Squires et al.

1978a, 1978b; Chu et al. 1978). Chronic alcohol intake is also

accompanied by EP amplitude reductions (Porjesz et al. 1976;

Begleiter and Porjesz 1977) and by BSP delays that are less

pronounced once tolerance develops Squires et al. 1978a, 1978b;

Chu et al. 1978; Zilm et al. 1981). Withdrawal is characterized

by increased EP voltages and extremely shortened BSP latencies,

suggestive of underlying CNS hyperexcitability (Begleiter and

Porjesz 1977, 1979; Begleiter et al. 1980a; Squires et al. 1978a,

1978b; Chu et al. 1978; Hunter and Walker 1980). Finally,

long-term abstinence is marked by decreased EP amplitudes

(hyporeactivityj and prolonged BSP latencies and slower

conduction velocities (Begleiter et al. 1981; Porjesz and

Begleiter 1983). The duration of these prolonged central nervous

system (CNS) disturbances and their potential recovery are not

yet known.

We have recently recorded auditory BSP's from hospitalized

alcoholics who were abstinent from alcohol for 1 month (Begleiter

et al. 1981). This technique allows investigation of subcortical

brain functioning with a noninvasive scalp electrode (Sohmer and

Feinmesser 1967; Jewett 1970; Jewett and Williston 1971). These

"far-field" potentials consist of 7 time-locked positive waves,

(designated I to VII), each presumed to reflect activity at

different sites along the auditory pathway from the auditory

nerve through the brain stem (Jewett 1970; Lev and Sohmer 1972;

Buchwald and Huang 1975; Starr and Achor 1975; Starr and Hamilton

1976; Stockard and Rossiter 1977). The latencies of each of

these peaks, as well as "central conduction time" (the latency of

each peak with respect to peak I), are accurate in localizing

sites of pathology from the peripheral end organ to the brain

stem; the time interval between the first peak and peak V of the

inferior colliculus is most often taken as a measure of brain

stem transmission time (Fabiani et al. 1979).

In our study, we found that alcoholic patients manifested

significant delays in latencies and central conduction velocities

of peaks II to V. These findings are remarkably similar to those

reported with acute doses of alcohol in animals (Squires et al.

1978a) and man (Squires et al. 1978b). Our study provided the

78
first systematic electrophysiological evidence of brain

dysfunction at levels other than neocortex in alcoholics without

overt clinical signs of neurological damage. The increase in

neural transmission time may reflect the process of

demyelination, which has long been suspected in alcoholics (Adams

et al. 1959) and has been observed in rats chronically exposed to

alcohol (Moscatelli and Demediuk 1980). Similar results have

recently been reported in neurologically impaired alcoholics

(Rosenhamer and Silfverskiold 1980; Chu and Squires 1980; Nickel

and Ludewig 1981; Haas and Nickel 1981; Chu et al. 1982), and in

neurologically intact alcoholics (Cassvan et al. 1984). The

etiology of these auditory BSP delays and the drinking history

factor(s) (e.g., length of drinking history, amount consumed per

sitting, number of withdrawals, and nutritional factors) that

result in brain stem aberrations have not yet been definitively

determined. It is even possible that nutritional deficiencies

alone produce demyelination and hence the BSP delays because

nutritional deficits are known to lead to demyelinating diseases

such as polyneuropathy (Hillman 1974). At present, we are

investigating the relationship between drinking history,

nutritional factors, and the magnitude of BSP aberration. Our

preliminary data suggest that alcoholics with signs of

nutritional deficits and/or polyneuropathy display different BSP

waveforms than those alcoholics without nutritional deficits.

Furthermore, length of drinking history does not seem to

correlate with BSP delay; in fact, alcoholics with relatively

short heavy drinking histories (< 8 years) and evidence of

nutritional deficits manifested greater BSP aberrations than

alcoholics with long drinking histories (> 20 years) and no

signs of nutritional deficits. The results of animal studies

(Chu et al. 1978) suggest that other factors besides chronic

alcohol exposure are necessary to produce BSP abnormalities;

chronic alcohol ingestion was not sufficient to cause BSP delays

after withdrawal in laboratory animals that did not also have

nutritional deficits. Taken together, these findings suggest

that BSP aberrations in alcoholics may be the result of alcohol

and/or nutritional factors.

For the past several years, we have also systematically examined

ERPs in medication-free alcoholics who are abstinent for

approximately 1 month. These ERP techniques require the subject

to be engaged in a task (usually information processing). Each

task is designed to examine deficits of a particular ERP

component which has been well documented to vary predictably

under specific conditions in normal subjects (Hillyard et al.

1978; Donchin et al. 1978).

In one bimodal (visual and auditory) study, we investigated the

ability of alcoholics to focus on a relevant stimulus modality

and inhibit responding to an irrelevant modality by examining

the Nl component of the ERP (Porjesz and Begleiter 1979), which

occurs at around 100 msec. The Nl component is sensitive to

attention to a relevant stimulus modality; it is enhanced to

79
all stimuli in a relevant stimulus modality, and reduced to

stimuli in irrelevant modalities (Hillyard et al. 1973, 1978;

Picton and Hillyard 1974). The patient was presented with a

sequence of randomized single flashes and clicks with rarely

occurring double flashes and clicks interspersed among them.

The patient was required to "shift attentional sets," in order to

count either the double flashes or double clicks, or ignore all

stimuli, in an otherwise identical stimulus sequence.

Consistent with the ERP literature (Hillyard et al. 1973, 1978),

control subjects in our study manifested significantly enhanced

Nl components to stimuli in the relevant as compared to the

irrelevant modality; however, alcoholics maintained the same low

amplitude of N1 regardless of the degree of task relevance.

Often it is the differential voltage between ERPs recorded to

stimuli in relevant and irrelevant channels that is more

revealing about the nature of brain functioning than absolute

voltages to either relevant or irrelevant stimuli. These results

suggest that alcoholics may be incapable of appropriate "sensory

filtering," as they do not differentiate neurophysiologically

between relevant and irrelevant channels.

In addition, the results indicated that abstinent alcoholics

manifested abnormally reduced late component amplitudes ( > 100

msec), but not early component amplitudes. These findings in

abstinent alcoholics are remarkably similar to results obtained

when healthy subjects ingest single doses of alcohol (Lewis et

al. 1969; Porjesz and Begleiter 1975; Rhodes et al. 1975). This

suggests that the neurophysiological brain dysfunction observed

in abstinent alcoholics may resemble brain functioning detected

in normal persons under the influence of alcohol.

We have also used the P3 component of the ERP in many different

experimental paradigms to examine brain dysfunction in alcoholics

(figure 1). The P3 or P300 component is a large, positive

deflection that occurs approximately 300 msec to 500 msec after

the stimulus. It can only be elicited under certain rather

specific conditions related to the "subjective significance" of a

stimulus, namely: task relevance (Sutton et al. 1967),

unpredictability (Donchin et al. 1978) and infrequency (Tueting

et al. 1971). as well as by motivational factors (Begleiter et

al. 1983). The characteristics of P3 are unrelated to stimulus

parameters, and can even be elicited in the absence of an

expected stimulus (emitted potentials) (Klinke et al. 1968). In

terms of scalp topography, P3 has been found to be maximum over

parietal areas; it is bilaterally distributed without apparent

hemispheric asymmetry, with similar distributions regardless of

the sensory modality of the stimulus (Simson et al. 1976, 1977a,

1977b).

In one of our laboratory studies, we investigated the P3

component with a visual target-selection paradigm (Porjesz et al.

1980). The target-selection paradigm is most frequently used to

80
FIGURE 1

Grand mean ERP wave forms recorded at parietal electrode (Pz) to

the target stimulus (solid linej and nontarget stimulus (dashed

line) in healthy subjects. Notice the prominent P3 component

(large positive deflection occurring between 300 and 450 msec) to

the target stimulus.

81
elicit a P3 component; it requires the subject to detect a

designated rarely occurring target stimulus embedded in a series

of frequently occurring nontarget stimuli. ERPs recorded to

frequently occurring nontarget stimuli elicit N1 components, but

no P3, while rare target stimuli elicit both N1 and P3

components (figure 1). In our study (Porjesz et al. 198O), the

stimuli were geometric shapes. One rare geometric shape (e.g.,

triangle) was designated target, and the subject was required to

press a button only in response to that stimulus. Target and

nontarget stimuli were alternated every other block enabling the

recording of ERPs to the same stimulus when it was target or

nontarget. ERPs were recorded to targets (rarely occurring,

task-relevant geometric shapes), nontargets (frequently occurring

task irrelevant geometric shapes), and novel stimuli (rarely

occurring task irrelevant random shapes).

We found that P3 amplitudes were significantly reduced or absent

in alcoholic patients to rare target stimuli under conditions

optimal for eliciting large P3s (Donchin et al. 1978) (figure

2). This finding was most pronounced over parietal areas, where

P3 amplitude is maximal at scalp (Simson et al. 1577a. 1977b;

Ritter et al. 1968). Furthermore, while controls manifested

differentially enhanced P3 components to target stimuli,

alcoholics manifested identical low amplitude P3 waves with the

same P3 latencies, regardless of whether a stimulus was a target

or nontarget. Moreover, despite the fact that all stimuli were

in the relevant channel, we found that the Nl amplitudes were

significantly reduced in alcoholics to all stimuli, to levels

comparable to an irrelevant stimulus modality. As in our bimodal

study (Porjesz and Begleiter 1979), this suggests that

sensory-filtering mechanisms are impaired in chronic alcoholics.

Thus, the major ERP aberrations manifested by alcoholics are the

lack of differentiation between their responses to relevant and

irrelevant inputs, and the low voltages of their event-related

activity. This seems to suggest underlying brain dysfunction

that impairs sensory-filtering and "probability-matching"

processes.

Recent evidence implicates the amygdala and hippocampus as

possible neural yenerators of P3. One recent study investigating

the neural origins of P3 with implanted electrodes in humans

reported that P3 was maximum at subcortical loci (Wood et al.

1980). Similarly, Halgren et al. (1980) have recently recorded

large late potentials from limbic system with implanted

electrodes in humans. They postulate that P3 may be generated in

hippocampus or amygdala. Magnetoencephalographic studies have

also suggested the hippocampus as a possible neural generator of

the P3 component (Okada et al. 1983).

Thus, our results that alcoholics manifest low-voltage or even

absent P3 components under conditions designed to elicit maximum

P3 component amplitudes may be indicative of hippocampal

82
FIGURE 2

Grand mean ERP wave forms recordea at parietal (Pz) to the target

(solid line) and nontarget (dashed line) stimuli in the alcoholic

group. Compare the P3 component of the target stimulus to that

of the control group (figure 1). and notice how reduced it is in

amplitude. Also notice the lack of difference between P3

amplitudes to target and nontarget stimuli in the alcoholic group

in this figure.

83
deficits. While these results do not rule out the contributions

of neocortical sites, they emphasize the important role of limbic

structures in generating the P3 component. The involvement of

the hippocampus in chronic alcohol intake in the absence of

malnutrition has been recently demonstrated in neuropathological

and neurophysiological studies in animals (Begleiter et al.

1980a; Walker et al. 1980, 1981; Riley and Walker et al. 1978).

Long-term ethanol consumption has been found to result in the

loss of dendritic spines in the hippocampus of both the mouse

(Riley and Walker 1978) and rat (Walker et al. 1980). In our

laboratory, we have also demonstrated a hippocampal

susceptibility to both acute and chronic alcohol effects with EPs

recorded from monkey hippocampus (Begleiter et al. 1980a).

We were interested in determining the relationship between

electrophysiological deficits and structural deficits assessed

with CT-scan in alcoholics (Begleiter et al. 1980b). We selected

two groups of alcoholics who had been subjected to CT-scans

following 1 month of abstinence: namely, those manifesting a

high degree of widened cortical sulci (Pos-CT) and those without

any evidence of widened cortical sulci (Neg-CT). Patients in the

two groups did not differ with regard to age, education, and

drinking history (duration and amount). ERPs were recorded on

the same day as the CT-scan and involved the same P3 paradigm

previously described (Porjesz et al. 1980). Alcoholics with

enlarged cortical sulci (Pos-CT) had significantly reduced (or

absent) P3 amplitudes to target stimuli, as compared to

alcoholics without signs of enlarged cortical sulci (Neg-CT);

however, both groups manifested smaller P3s to targets than did

control subjects. Furthermore, both groups of alcoholics

displayed similar P3 components to all categories of stimuli,

regardless of task relevance.

Because Neg-CT alcoholics also manifested diminished P3

amplitudes when compared to healthy nonalcoholics, neocortical

shrinkage alone cannot explain these P3 reductions. These

findings suggest that chronic alcohol abuse not only results in

changes in the neocortex, but may also involve

electrophysiological aberrations indicative of other brain (e.g.,

limbic system) deficits. Often the neocortical deficits in

chronic alcoholics are emphasized while subcortical aberrations

are overlooked. Our results suggest that alcoholics manifesting

observable widened cortical sulci on CT-scan are more likely to

also manifest hippocampal deficits. This, perhaps, lends support

to the hypothesis that alcohol produces diffuse brain damage not

solely circumscribed to neocortical areas.

We have recently completed a study examining the N2 or N200

component of the ERP in abstinent alcoholics (Porjesz and

Begleiter 1981a, 1981b; Porjesz and Begleiter, in press). The N2

component is a modality-specific negative deflection with a

maximum amplitude at occipitoparietal scalp for the visual

modality and at central regions for the auditory modality.

84
Recent evidence suggests that the latency of N2 can be taken as

an early index of stimulus evaluation time (Renault and Lesevre

1979); the more difficult the discrimination, the longer the

latency of N2 (Ritter et al. 1979; Towey et al. 1980; Gaillard

and Lawson 1980). This component is thus a better index of

stimulus evaluation time than the reaction time (RT), because it

is not confounded by the motor response. The RT is a complex

measure of speed of information processing, as it depends on the

end product of stimulus evaluation, response selection and

organization, and motor response. Therefore, although there are

some reports of delayed RTs in chronic alcoholics (Talland 1963;

Vivian et al. 1973; Bertera and Parsons 1973),

cannot determine which aspect(s) of information

(are) slower in alcoholics.

these studies

processing is

In order to use the N2 component of the ERP as an

of stimulus evaluation, we designed an RT study

index of speed

involving easy

and difficult line orientation discriminations. This

visuospatial RT design enabled us to investigate the relationship

between difficulty of discrimination, N2 latency, P3

characteristics, and RT in abstinent alcoholics. ERPs were

obtained to frequent nontarget (vertical line), and infrequent

easy (90 degree deviant from vertical) and difficult (3 degree

deviant) line orientations.

Our results indicated that the latency of N2 reflected difficulty

of discrimination in the control subjects, being significantly

delayed to the difficult when compared to the easy

discrimination. By contrast, it failed to do so in the

alcoholics, where N2 latencies were similar regardless of

difficulty of discrimination. Furthermore, the N2 latency

occurred significantly later in the alcoholic group than in the

control group for both easy and difficult discriminations,

suggesting that alcoholics need more time for stimulus

evaluation and may therefore find the discrimination task more

difficult. The latency difference between groups was even more

apparent for the easy discrimination than for the difficult

discrimination. This suggests that alcoholics need

proportionately more time to make an easy discrimination

(vertical from horizontal) when compared to controls (who can

process this information more quickly), than to make a

difficult discrimination (which both groups presumably find

difficult).

In addition, alcoholics manifested delayed P3 latencies to easy

discriminations when compared to controls; these P3 latencies

were comparable to those expected for a difficult task. These

results suggest that alcoholics adopt an undifferentiated mode of

responding regardless of task requirements, finding all tasks

difficult. While the amplitude of N2 was larger for easy

discriminations than difficult discriminations in the control

group, the amplitude of N2 was the same in the alcoholics

regardless of task difficulty. The amplitude of N2 has been

85
shown to be related to the degree of stimulus deviance in normal

subjects (Naatanen 1981).

There were no significant differences in RTs between the two

groups of subjects, although the alcoholics tended to have faster

RTs and make more errors than controls. This response pattern

implies that alcoholics adopt different response strategies from

controls, stressing speed over. accuracy (Kutas et al. 1977).

Their apparent inability to withhold responding until certainty

of accuracy or correctness has been established suggests a lack

of inhibition in alcoholics.

In addition to these latency results, we found that alcoholics

had significantly decreased P3 amplitudes. This low amplitude P3

was even more apparent for the easy discrimination, where

controls exhibited very high P3 voltages. The P3 voltage was

significantly larger for the 90 degree target when compared to

the 3 degree target in the control but not the alcoholic

subjects. This result is predicted by many ERP studies which

have demonstrated that the more deviant a rare stimulus is from

the background (the more easily discriminable it is) the larger

the P3 amplitude (Towey et al. 1986; Ritter et al. 1972; Ford et

al. 1979; Johnson and Donchin 1978; Ruchkin and Sutton 1978).

Perhaps the lack of P3 amplitude difference in the alcoholic

group indicates that they are more uncertain of the correctness

of their decision than are controls, as they stress speed over

accuracy.

Thus, on the basis of both the N2 and P3 ERP components, it was

concluded that alcoholics have difficulty evaluating the

potential significance of a stimulus. They do not

electrophysiologically differentiate between relevant and

irrelevant or easy and difficult discriminations, but rather

maintain the same ERP characteristics (both amplitude and

latency) regardless of the task requirements. This perhaps

indicates that their template for match/mismatch decisions is

lost or not readily available. In either case, it suggests a

memory deficit where each incoming stimulus must be evaluated

anew. Our data suggest that alcoholics manifest both types of

brain deficits: the delay in N2 latency suggests that the

template for comparison is not as readily accessible in

alcoholics, while the low P3 voltages suggest that once

retrieved, the match/mismatch processes themselves are impaired

in alcoholics.

We are currently examining the reversibility of the BSP and ERP

deficits observed at 1 month of abstinence and following 4 months

of continued abstinence in the same hospitalized alcoholics

(Porjesz 1983; Porjesz and Begleiter, in preparation).

Preliminary data following 4 months of abstinence indicate

improved morphology of BSP waveforms, shortening of latencies,

and improved conduction times.

86
The relative roles of abstinence from alcohol and nutritional

factors in so-called "recovery" still remain to be determined.

Throughout the long-term abstinence program in our hospital,

patients receive extensive vitamin therapy and may be manifesting

improvements in nutritional status. Furthermore, the role of

withdrawal cannot be overlooked; CNS hyperexcitability may be

followed by a period of subacute hypoexcitability. We might

speculate that this hypoexcitability is manifested by a

prolongation of brain stem latencies caused by aberrant

fluidizing effects on the neuronal membranes which may result in

edema. Edema resulting from osmotic stress can lead to

demyelination (Lewis 1976; Yates 1976; Feigen and Budzilovich

1978, 1980; Kleinschmidt-DeMasters and Norenberg 1981).

However, it should be noted that those alcoholics who remained in

treatment for the full 4 months had less impaired BSPs at initial

testing (3 to 4 weeks) when the data were analyzed

retrospectively. As we are only able to examine reversibility in

alcoholics who remained in long-term treatment, and these

alcoholics tend to be less impaired initially, we cannot be

certain that recovery occurs in all alcoholics regardless of

degree of impairment. It remains to be determined whether

recovery occurs as a function of the initial degree of

impairment, whether greater impairment requires longer time

periods for reversibility, or whether recovery ceases beyond a

certain critical level of impairment.

We are currently investigating electrophysiological aberrations

in another group of nonhospitalized alcoholics sober from 3 to 10

years, and thus far we have found that they manifest normal BSPs.

This suggests that perhaps 4 months is not a long enough time

interval to investigate reversibility of brain dysfunction

following years of heavy drinking. However, it should be noted

that these long-term abstinent alcoholics (> 3 years) were not

tested initially; therefore the extent of BSP aberration they may

have manifested immediately following alcohol abuse is not known.

It is possible that these alcoholics never exhibited BSP delays,

as we do not see BSP delays in all alcoholics tested. While the

issue of reversibility is still unresolved, the data seem to

indicate slow reversibility of BSP deficits with prolonged

abstinence.

Although the BSP delays seem to improve with prolonged

abstinence, the decreased voltages in the P3 component of the ERP

do not seem to change with prolonged abstinence (Porjesz 1983;

Porjesz and Begleiter, in preparation). We examined the

possibility of reversibility of late component P3 deficits in

abstinent alcoholics following 3 weeks and 4 months of

abstinence. Interestingly, no improvement in ERP morphology or

late component amplitude was noted following 4 months of

abstinence in the same alcoholics; in fact, the waveforms were

strikingly similar at initial test and retest. Furthermore,

there was no improvement in the differential enhancement of P3

87
amplitudes on the basis of task relevance to target stimuli in

these abstinent alcoholics. In addition, P3 aeficits were still

observed in our group of nonhospitalized alcoholics sober from

3 to 10 years. Thus, even following 3 to 10 years of abstinence,

alcoholics still manifest abnormally low P3 amplitudes. We have

observed these P3 decrements in response to both auditory and

visual target stimuli in a bimodal target-selection study. These

results suggest that the P3 deficits may not be reversible, or

perhaps reverse much more slowly following very long abstinence

periods.

Thus, it seems that some electrophysiological aberrations

observed in alcoholics improve with prolonged abstinence while

other electrophysiological aberrations do not change with

prolonged sobriety. Caution is suggested in interpreting the

results as they are based on small samples. We are currently

examining this issue with larger sample sizes in an effort to

determine the important factors of susceptibility and

reversibility of brain dysfunction in alcoholism.

As these P3 deficits do not seem to recover following prolonged

sobriety (> 3 years), it is even possible that these deficits

may precede the development of alcoholism. While it has

generally been assumed that the brain abnormalities observed in

alcoholics are due to the toxic effects of alcohol on the brain,

nutritional deficits, or an interaction of alcohol and

nutritional-related factors, these brain deficits may represent a

predisposing factor differentiating those individuals with a

susceptibility to alcoholism. There is increasing evidence that

certain individuals are at high risk for developing alcoholism.

Specifically, sons of alcoholic fathers are four times more

likely to develop alcoholism than sons of nonalcoholics (Goodwin

1979; Goodwin et al. 1973), even when they are separated from

their biological parents soon after birth. Studies of male

adoptees indicate that the biological rather than the adoptive

parent is predictive of later drinking problems (Goodwin et al.

1973; Bohman 1978; Cadoret and Gath 1978; Cadoret et al. 1980;

Goodwin and Guze 1974; Schuckit et al. 1972). Furthermore, the

concordance rate for alcohol abuse among identical twins is

almost double the rate for fraternal twins (Kaij 1960); patterns

of alcohol consumption have also been found to be highly

concordant among identical twins (Partanen et al. 1966; Jonsson

and Nilsson 1968; Loehlin 1972). Taken together, these studies

suggest that a genetic factor predisposing sons of alcoholics to

alcoholism may be involved.

An exciting use of EP techniques is in identifying possible

biological marker(s) for those at risk for developing alcoholism.

It is likely that brain function is involved in the genetic

predisposition for alcoholism. There is good evidence to

indicate that brain EP waveforms are genetically determined.

Monozygotic twins, for example, manifest EP waveforms that are as

concordant with each other as EPs obtained from the same

88
indiviaual tested twice (Dustman and Beck 1965).

he have recently undertaken a major project which has already

demonstrated the possibility that sons of male alcoholics

manifest differences in EP that antedate any exposure to alcohol.

In order to study this problem, we are recording ERPs in boys

between the ages of 6 to 18 and comparing electrophysiological

responses from sons of alcoholics (high risk) and from age and

education-matched sons of nonalcoholics (low risk). Children

whose mothers abused alcohol are excluded to rule out the

contribution of the fetal alcohol syndrome (FAS).

In one ERP study (Begleiter et al. 1984), we examined the P3

component in boys between the ages of 7 to 13 who had no prior

experience with alcohol. The high-risk (HR) group consisted of

25 sons of alcoholic fathers with a mean age of 11.9 (S.D. =

2.1). In each case, the father had received the exclusive

diagnosis of alcoholism (DSM III) and had been in treatment for

alcoholism at some time. Boys whose mothers had ingested alcohol

during pregnancy or who drank excessively after giving birth were

excluded. Only boys without medical problems and without

exposure to alcohol or other substances of abuse were included in

this study. The 25 normal control subjects (NC) were boys who

were matched for socioeconomic status (SES) and age to the HR

subjects. The NC group had a mean age of 12.5 years (S.D. =

2.4). They were included only if they had no exposure to alcohol

or other substances of abuse, and had no history of alcoholism or

other psychiatric disorder in first or second degree relatives.

Except for alcohol history, the same exclusion criteria were used

as for the HR group.

The experimental design consisted of a visual head orientation

task (figure 3). Subjects pressed one of two microswitches as

quickly and accurately as possible (reaction time) with either

the right or left index finger to indicate whether the right or

left ear was present on the display, respectively. They

performed this task under randomized "easy" and "difficult"

conditions.

Reaction times for easy stimuli were significantly shorter than

for difficult stimuli (p <.0001); there were no significant

reaction time differences between groups. However, the number of

correct behavioral responses was significantly less for the HR

group for easy (p < .001) and difficult stimuli (p < .001).

The entire raw data set was subjected to a principal component

analysis with varimax rotation using the covariance matrix

(PCAV). Basis waveforms were extracted, and the component scores

for each of the four factors were then subjected to an analysis

of variance (ANOVA). Our results indicated that only the factor

representing the P3 component was significantly different between

the high- and low-risk groups; the P3 amplitudes were found to

be significantly smaller in the HR group as compared to the NC

group. This group difference was found to be significant at the

89

484-443 O - 85 - 4
FIGURE 3

Experimental paradigm for head-orientation task. The target

stimulus is a rarely occurring aerial view of the head with the

nose and one ear drawn in, rotated in four possible positions:

nose up and right ear, nose up and left ear, nose down and right

ear, nose down and left ear drawn in. The nontarget stimulus is

a frequently occurring oval presented in the center of a

computer-generated display. In the "easy" condition, the head

was facing forward (nose up on the screen) and the left ear or

right ear appeared on the same side that corresponded to the

appropriate button, in the "difficult" condition, the head was

facing back (nose down on the screen) and the left or right ear

appeared on the side opposite the corresponding button. The

subject's task was to press the button corresponding to the ear

present in the display as quickly as possible.

90
parietal electrode (where P3 is maximum) for both the easy

condition (p < -01) and the difficult condition (p < .002).

These findings are the first to indicate a significant difference

in P3 amplitude between boys, not exposed to alcohol, who are at

high risk for alcoholism and normal controls.

Differences in electrophysiological recordings in response to

challenge doses of alcohol have recently been reported between

males with some family history of alcoholism and control

subjects. This has been reported for EEG (Pollock et al. 1983)

and ERP (Elmasian et al. 1982) recordings. In the ERP study,

Elmasian et al. found that male college students with family

histories of alcoholism manifested different ERPs to challenge

doses of both placebo and alcohol, when compared to matched

controls without a family history of alcoholism; these

differences between the two groups were apparent in the P3

component.

Our findings are particularly interesting as they were obtained

without the use of alcohol. We found that approximately 36% of

the sons of alcoholics manifested this P3 difference. However,

whether these low amplitude ERPs are in fact markers for a

preaisposition to alcoholism remains to be tested. Studies are

under way in our laboratory to retest these children each year to

determine whether those manifesting ERP differences are in fact

those who actually develop problems with alcohol.

Because ot these findings, we were interested in determining

whether other electrophysiological deficits observed in alcoholic

patients would be apparent in boys at risk for alcoholism.

Therefore, we decided to record BSPs in high-risk boys. In this

study (Begleiter et al., in preparation), we examined another

sample of 23 sons of alcoholic fathers between the ages of 7 and

13 with a mean age of 12.2 (S.D. = 2.1). The 23 NCs were boys

who were matched for SES, age and education to the HR boys. The

NC group had a mean age of 12.4 (S.D. = 2.3). The inclusion and

exclusion criteria were identical to those previously described

for the P3 study. Again, we only included boys without prior

exposure to alcohol or other illicit drugs. As in our study with

alcoholics, the latency of the tirst five positive peaks and the

interpeak latencies between peak 1 and each successive peak were

measured.

We did not find any differences in the auditory BSPs obtained

trom sons of alcoholic fathers and those obtained from matched

control subjects. The individual peak latencies and the brain

stem transmission times were found to be similar in the two

groups. The lack of significant differences in BSPs between HR

and NC subjects is interesting in light of our observed

difference in P3 between HR and NC subjects. These findings

indicate that, while P3 deficits may antecede the development of

alcoholism in some high-risk individuals, the brain stem deficits

which we have observed in abstinent alcoholics are most probably

91
alcohol-related changes. Further evidence for this hypothesis is

obtained by our findings that the BSP abnormalities observed in

abstinent alcoholics seem to "recover" with prolonged

abstinence, while the P3 deficits do not.

It remains for future research to separate those brain

aberrations that antecede alcohol abuse from those that are the

consequence of years of heavy drinking. It is not now known

which innate differences determine responsiveness to alcohol,

incluaing predisposition to alcohol abuse. Genetic differences

in strains of animals have been tound to determine whether they

were predisposed to drink alcohol or to find it aversive (Rogers

1972). Furthermore, differences in neurophysiological responses

to alcohol have been reported in different genetic rat strains

(Sorenson et al. 1980). humans have also been found to differ

in their responsiveness to alcohol, e.g., augmenting/reducing

(Spilker and Callaway 1969; Buchsbaum and Ludwig 1980), tamily

history for alcoholism (Elmasian et al. 1982; Pollock et al.

1983). and flushers/non-flushers (Fukui et al. 1981). For

example, recent findings in Japan indicate that flushers (who

manifest an adverse flushing reaction to alcohol) are more

susceptible to delayed BSPs than nonflushers when ingesting a

challenge dose of alcohol (Fukui et al.). It is possible that

the low P3 amplitudes we observe in young sons of alcoholics

represent a vulnerability marker which may only become apparent

in response to alcohol. For example, it is possible that,

although we did not observe BSP differences between boys at high

and low risk for alcoholism without the ingestion of alcohol, BSP

differences may become apparent once alcohol is introduced. It

may in fact be those boys who manifest P3 decrements without

alcohol that will respond differently to alcohol on other evoked

potential measures (e.g., BSP). Studies are under way in our

laboratory to test adolescents with family histories of

alcoholism under the influence of challenye doses of alcohol with

a full battery of evoked potential tests.

The ability to utilize sophisticated neurophysiological tools to

assess brain dysfunction in abstinent alcoholics and individuals

at risk for alcoholism may prove most valuable in separating

the deleterious effects of alcoholism on the CNS from the brain

deficits which may antecede the development of alcoholism. The

delineation of similar neurophysiological deficits in abstinent

alcoholics and children at high risk for alcoholism may be of

fundamental importance in the identification of possible genetic

marker(s). The search for a possible cluster of

neurophysiological deficits in children at high risk for

alcoholism is presently under way in our laboratory.

We have recently begun to examine recovering drug addicts with

the use of the same EP techniques. We reasoned that if these

techniques are so sensitive in delineating the effects of alcohol

on the brain (intoxication, withdrawal, long-term deficits), they

would be ideal to study whether or not other substances of abuse

92
produce long-lasting effects on the brain.

Thus far we have examined narcotic addicts with an auditory

oddball paradigm designed to examine the P3 component of the ERP.

The addicts were males (mean age 35.5) who were drug-free for a

minimum of 1 week. We found that recovering narcotic addicts

manifest lower P3 amplitudes than matched control subjects. To

the best of our knowledge, this is the first demonstration of a

neurophysiological anomaly in drug addicts. These preliminary

data from our laboratory run counter to the popular notion that

chronic intake of narcotics may not result in CNS deficits. This

popular tenet is not based on the presence of scientific evidence

but has emerged in large measure in the absence ot rigorous

clinical or scientific data.

The ability to assess the integrity of various CNS systems with

event-related brain potentials can provide valuable clinical

information about the effects of chronic drug intake on CNS

activity. ERP studies of abstinent drug addicts (heroin,

cocaine, PCP, amphetamine, etc.) should result in fundamental

information on the neurophysiological effects of various drugs of

abuse. The neurophysiological delineation of the effects of

various drugs of abuse on the human brain may help elucidate the

resulting pathophysiology as well as provide data on possible

etiological factors.

If, as appears to be the case with alcoholism, there are

antecedent brain anomalies related to drug abuse, the

neurophysiological data obtained from drug abusers would be of

fundamental utility in the search for possible vulnerability

factors.

Our current electrophysiological data in abstinent alcoholics and

subjects at risk for alcoholism, as well as our preliminary

event-related potential data in drug abusers, certainly warrant

further neurophysiological investigations of CNS function in drug

abusers. These preliminary findings encourage us to examine

recovering addicts dependent on different substances of abuse

(e.g., heroin, cocaine), to investigate possible

neurophysiological aberrations.

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AUTHORS

B. Porjesz

H. Begleiter

Downstate Medical Center

450 Clarkson Avenue

Brooklyn, NY 11203

99
Methamphetamine: Toxicity to

Dopaminergic Neurons

Lewis S. Seiden

Methamphetamine (MA) is one of the prototypic psychomotor

stimulants. In the past, it baa been widely used to auppreaa food

intake and increase mental alertness and physical endurance.

Methamphetamine and related stimulant phenethylamines can also

induce an elevation in mood. This baa led to the use of the drug

for nonmedicinal purposes. Amphetamine (AMPH) and related

compounds have also been used to treat obesity, depression,

narcolepsy, attentional deficit disorder in children, and

residual symptoms of attentional deficit disorder in adults.

Tolerance develops to the anorectic effects of AMPH and the dose

is of ten increased; therefore, the permanent (i.e., toxic)

effects of AMPH and related compounds are of concern. Among tbe

undesirable effects of AMPH that occur with large doses la

amphetamine-induced paycboaia. The amphetamine psychosis

includes symptoms of paranoid delusions, disordered thought,

inappropriate aggressive behavior, and hallucinations. When a

drug history is not available, it is difficult to discriminate an

amphetamine-induced psychosis from an acute psychotic episode

prognostic of schizophrenia (see Costa and Garattini 1970; Seiden

and Dykatra 1977; Goodman and Gilman 1980; Ellinwood and Kirby

1977).

Since the psychomotor stimulants as a class have a range of

effects, their neuropharmacoloa and behavioral pharmacology, and

possible neurotoxicity, have been focal points for studying

interrelationahips among transmitter chemistry, drugs, and

behaviors (Carlason 1970; Chiueh and Moore 1975; Creeae and

Iveraen 1973, 1975). During the early 1970s, substantial

evidence accumulated which suggested that the effects of

amphetamines and related drugs were mediated by the dopamine

(DA), norepinephrine (NE), and 5-bydroxytryptamine (5HT)

transmitter systems in the brain (Moore et al. 1970; Green and

Harvey 1974; Mabry and Campbell 1973; Crease and Iveraen 1975).

This evidence suggested a subsequent question of whether changes

in the DA, NE, or 5HT systems occur as a result of prolonged

administration of AMPH

Changes in these transmitter systems were assessed in rhesus

monkeys 3 to 6 months after the end of chronic MA administration.

The monkeys were given high doses of MA (3.5 to 6.0 mg/kg every

100
3 hours, with the total daily dose between 28 and 48 mg/kg day

administered intravenously. In spite of the high dose, the

monkeys in these studies became tolerant to the disruptive

effects of MA as measured by an operant task and feeding

(Fischman and Schuster 1977). There was a loss of DA in the

caudate nucleus but not in the hypothalamus, and also a small

change in the level of noreplnepherine (NE) in the brain stem.

The results with 5HT were not clear. The DA depletion 3 to 6

months after the last MA injection suggests that the changes in

DA were permanent (tables 1 and 2).

Since the monkeys tested in the early studies were administered

MA for a period of 3 to 6 months, we wondered how critical the

long period of high dosing was to create the apparently

irreversible damage to the DA system. Therefore, three rhesus

monkeys were treated on a 2-week regimen of MA injections. These

monkeys were injected with a total dose of 16 or 24 mg/kg per day

divided into eight intravenous (iv) injections. The control

monkeys for these studies were eight rhesus monkeys from the

Wisconsin Primate Center; these monkeys had not been given any

psychotropic drugs. After 2 weeks of MA injections, the monkeys

were MA-free for 2 weeks and then they were sacrificed and the

brains analyzed for catecholamines. As with the monkeys that were

injected for longer periods of time, the monkeys injected with

MA for 2 weeks shoved depletion of DA. Ye speculated that this

depletion may be indicative of permanent damage to the DA system.

There is strong evidence that AMPH and related drugs bloak

reuptake of DA, NE, and 5-HT; inhibit monoamine oxidase; and

cause release of DA from the cytoplasmically bound pool (see

Seiden and Dykstra 1977; Cooper et al. 1974). Release and

blockade of reuptake would’ tend to deplete levels of DA in the

nerve endings in the presence of MA. However, MA-induced

depletion would not be expected to last for more than several

hours in the absence of MA (Ricaurte et al. 1980). Therefore, on

empirical and theoretical grounds, the DA depletion seen in the

monkeys weeks or months following the cessation of MA

administration is not caused by the acute effects Of MA.

The early researah using monkeys raised several questions: 1)

Was the DA depletion Been in the rheaus monkey specific to

monkeys, or would rats, mice, guinea pigs, and other animals show

the same response to MA? 2) Was the depletion specific to MA, or

would other psychomotor stimulants (e.g., AMPH, l ethylphenldate)

that were structurally or functionally related to MA cause a

similar depletion of DA? 3) Were other transmitter systems

beside DA and possibly NE depleted by exposure to MA? 4) What

were the minimal times and doses that would produce significant

DA depletion? and 5) Were nerves destroyed, or use the synthesis

and/or storage capacity of the dopaminergic cell compromised

The data necessary to answer these five questions also helped to

elucidate the chemical mechanism by which MA and related drugs

caused the long-lasting depletion of DA and other transmitters.

101
TABLE 1

NE - 8 Injections of Methamphtamine (Meth.)

Daily for Several Months

Treatment Pons-medulla Mid.brain Hypothalamus Frontal cortex

Control 0.45 ± 0.04 0.69 ± 0.03 1.0 ± 0.26 0.21 ± 0.03

N=12

Chronic 0.23 ± 0.07(51%) 0.40 ± 0.06(67%) 0.76 ± 0.16(42%) 0.08 ± 0.23(38%)

meth. 24 h p < 0.05 p < 0.05 p < 0.05 p < 0.05

poet meth.

N=5

Chronic 0.31 ± 0.08 0.39 ± 0.07(66%) 1.4 ± 0.43 0.10 ± 0.01 (46%)

meth. 3-6 p < 0.05 p < 0.05

months

post meth.

N=6

NE - 8 Injections of Methamphetamine (Meth.)

for a Period of 2 Weeks

Treatment Pons-medulla Mid-brain Hypothalamus Frontal cortex

Chronic 0.21 ± 0.11(48%) 0.41 ± 0.12(69%) 1.77 ± 0.23 (98%) 0.12 ± 0.04(57%)

meth. 24 h

post meth.

N=3

Chronic 0.40 ± 0.01(88%) 0.48 ± 0.02(81%) 1.4 ± 0.34(77%) 0.11 v 0.01(62%)

meth. 2

weeks post

meth.

N=3

Fischman and Schuster 1975/1976. Copyright 197511976, Elsevier

Biomedical Press.

102
TABLE 2

DA - 8 Injections of Methantphetamine (Meth.)

Daily for Several Months

Treatment Pons-medulla Mid-brain Hypothalamus Cudate Frontal cortex

Control 0.16 ± 0.01 0.51 ± 0.04 0.83 ± 0.12 10.1 ± 0.57 0.09 ± 0.01

N=12

Chronic 0.51 ± 0.20 0.61 ± 0.11 1.33 ± 0.30 2.0 ± 1.0 (19%) 0.19 ± 0.04

meth. 24 h ns ns ns p < 0.001 ns

post meth.

N=5

Chronic 0.13 ± 0.03 0.33 ± 0.07 0.82 ± 0.17 3.15 ± 0.64 (31%) 0.13 ± 0.05

meth. 3-6 ns ns ns p < 0.001 ns

months

post meth.

N=6

DA - 8 Injections of Methaphetamine (Meth.)

for a Period of 2 Weeks.

Treatment Pons-medulla Mid-brain Hypothalamus Caudale Frontal cortex

Chronic 0.19 ± 0.01 0.43 ± 0.07 1.14 ± 0.53 3.67 ± 0.50 (36%) 0.15 ± 0.02

meth. 24 h

post meth.

N=3

Chronic 0.63 ± 0.90 0.76 ± 0.14 1.20 ± 0.45 2.3 ± 0.37 (23%) 0.30 ± 0.01

meth 2

weeks post

meth.

N=3

Fischman and Schuster 1975/1976. Copyright 1975/1976, Elsevier

Biomedical Press.

103
Prolonged doses of MA caused depletlon of DA in the rat and the

guinea pig as long as 8 weeks after the last adminiatration of

MA. Furthermore, the depletions seen 2 weeks after discountinuing

MA were not different from the depletions seen 8 weeks after

diacontinuing MA (Wagner et al. 1980b; Ricaurte et al. 1980).

These results, summarized in tables 3 and 4, indicate that MA

toxicity la a generel phenomenon occurring in the rhesus monkey,

mice, rata, guinea pigs, and cats (Steranke and Sanders-Bush

1980; Levine et al. 1980). Since the MA-induced toxic response

(DA depletion) occurs in severel species of animals, it may also

be an important side effect of chronic administration or high

doses of psychomotor stimulants in humans.

The duration of the depletion suggested that nerve cells or nerve

terminals might. be degenerating, but decreased storage and/or

synthesis could else account for the depletions. Schmidt and

Glbb (1982) and Hotchkies and Gibb (1980) found lower total

activity of both tryptophan hydroxlylase and tyrosine hydroxylase

in rata end mice that had received high doses of AMPH or MA then

in control animals Theae two enzymes are rate-limiting for 5HT

or NE and DA synthesis, and the decreased synthetic rate could

account for the decrease in steady state levels However, these

enzyme kinetics are also consistent with oell or terminal loss.

Eillson et al. (1978) provided morphological evidence for

degeneration in rat brain. After prolonged administration of

AHPH through slow release pellets, evidence of malformed cells

appeared in the striatum.

We round that methylphenidate, a drug functionally similar to

AHPH and WA, did not cause changes in DA, NE, or 5HT systems in

rat brain Therefore, not every anorectic psychomotor stimulent

drug produces toxic effects to DA and 5HT nerve endings. In the

past 5 years, we have shown that a number of drugs, including

AMPH, cathinone, 3,4-methylenedioxyamphetamine (MDA), mazindol,

phemylpropanolamine, fenfluramine, and diethylpropion, affect the

DA, NE and/or 5HT transmitter systems. Some of the drugs are

toxic at doses which are outside the human dose range. Mazindol

oauaed smell depletions of NE at doses which were several hundred

times the ED50 for the suppression of milk intake, but

fenfluramine caused depletion of 5HT at a dose that vaa very

close to the ED50 for the suppression of milk intake. Thus, baaed

on the animal studies, one might conclude that the therapeutic

ratio for mazindol was large, but that the ratio for fenfluramine

was small. Similarly, MDA has toxic effects on 5HT neurons in the

striatum and the hippocampus of rata at doses of 3 to 6 mg/kg per

day (Ricaurte et al. 1985). Even a single administration of MDA

at a doae similar to that used by humans self-administering the

drug has long-term effects on hippocampal 5HT. In general,

drugs having structural end functional features in common with

AMPH and MA have toxic errecta upon related fiber ayatema within

the CNS.

Using kinetic analysis of DA reuptake, we have found that the

number of reuptake sites was reduoed by 50% by MA treatment. The

affinity or the carrier system remained unchanged. In these

104
TABLE 3

Regional Brain Catecholamine Levels in Rats

n Caudate Tlencephalon Midbrain Pons-medulla

DA(μg/g) NE(μg/g) NE(μg/g) NE(μg/g)

Vehicle 10 8.67 ± 0.67 0.34 ± 0.02 0.60 ± 0.04 0.50 ± 0.03

25 mg/kg per day 8 6.44 ± 0.88 0.29 ± 0.04 0.71 ± 0.05 0.45 ± 0.05

d-Meth-HCl

50 mg/kg per day 11 3.47 ±O.41 0.35± 0.03 0.66 ± 0.07 0.50 ± 0.03

d-meth-HCL

Rats received subcutaneous methamphetamine hydrochloride

(d-Meth-HCl) for 30 days, for a total of 25 or 50 mg/kg/

per day. Values reported are group means in ug/g tissue

± standard error of the mean. Pats were killed 2 weeks

after the last injection. DA = dopamine; NE = norepinrphrine.

Wagner et al. 1979. Copyright 1979, Elsevier Biomedical Press.

TABLE 4

Regional Brain Catecholamine Levels in Guinea Pigs

n Caudate Telencephalon Midbrain Pons-medulla

DA (μg/kg) NE (μg/kg) NE (μg/kg) NE (μg/kg)

Vehicle 4 9.83 ± 0.27 0.33 ± 0.03 0.50 ± 0.10 0.30 ± 0.02

6-30 mg/kg per day 6 4.62 ± 0.66 0.33 ± 0.08 0.42 ± 0.08 0.35 ± 0.05

d-Meth-HCL

Guinea pigs received subcutaneous methamphetamine

hydrochloride (d-Meth-HCl) for 30 days. Values reported

are group means in ug/g tissue ± standard error of the

mean. Guinea pigs were killed 2 weeks after the last

injection. DA = dopamine; NE = norepinephrine.

Wagner et al. 1979. Copyright 1979, Elsevier Biomedical Press.

105
experiments, rata received MA for a period of 30 days. They were

subsequently drug-free for a period of 2 to 3 weeks prior to

sacrifice. The synaptosomes were in ubated with a buffered

medium and varying concentrations of 3H-DA (Snyder and Coyle

1969). The kinetic constants, Vmax and Km, were determined by

fitting the data to a rectangular hyperbola and doing double

reciprocal analysis on the beat fitting rectangular hyperbola.

An approximate Km value of 12 x 10-um was Sound in both the

control and the MA-treated animals. In contrast, Vmax expressed

in disintegrationa per minute was 7.9 x 103m for control animals

and 5.4 x 103 for MA-treated animals,indicating a loss of DA

uptake sites (figure 1). We examined the number of high-affinity

dopaminergic binding sites using 3H-spiroperidol and found no

difference in affinity or binding sites (table 5, Ricaurte et al.

1980).

Repeated high doses of MA also produce long-lasting depletions Of

5HT in the rat brain Within the 5HT system, levels are reduced

in the amygdala, frontal cortex, and striatum. It should be

noted that the regional pattern of reduced 5HT levels after

administering MA is similar to that been after pchloroamphetamine, a vell-known 5HT neurotoxin. After both MA

and p-chloroamphetamine, the loss of 5HT synaptosomal reuptake

sites occurs in much the same way as does the loss of

dopaminergic uptake sites. Again, this evidence is consistent

with the hypothesis that the nerve terminals are degenerating

(Alcaurte et al. 1980).

Following chronic, high doses of MA levels of DA and 5HT are

reduced for a prolonged period of time, the rate-limiting enzyme

for synthesis is proportionately reduced, and the number of

uptake sites is also proportionately reduced. Them observations

are consistent with the hypothesis that the nerve. terminals

degenerate but confirmation requires morphological evidence of

neuronal degeneration.

We now have evidence that striatal move terminals degenerate in

rata after a single high doae of MA Since a single dose

produced toxicity, it was possible to check different time points

using the silver impregnation technique to measure nerve termimal

degeneration. The Fink end Heimer (1967) method depends upon the

cell being stained with silver during the period of degeneration.

Since the period Of degeneration can be relatively short, it is

necessary to do multiple time samples to find the correct period.

The rata were killed 2 days after drug treatment since we had

found from preliminary studies that the silver impregnation of

degenerating rat nigroatriatal DA terminals is beat achieved

after a 2-day survival period (figures 2 and 3). The reaults

clearly show degeneration in the striatum suggesting that the DA

fibers are degenerating. Since it was possible to block

degeneration of 5HT fibers by pretreating with amfonelic acid, me

were relatively certein that the obaened degenerating cells

contained DA. We were also able to aee degeneration in the

106
FIGURE 1

Douple-reciprocal plot of 3H-dopamine uptake by

rat striatal homogenates 2-3 weeks after the high

dose methamphetamine treatment (50 mg/kg/day for

4 days). Dopamine uptake was determined at dopamine

concentrations ranging from 0.25 to 5.0 uM. The km

values of saline-treated (1.11 uM) and methamphetaminetreated (0.18 uM) were not significantly different.

The difference in dopamine uptake site density (Vmax)

is significant (P<0.05). Data are from one representative

experiment replicated twice.

Wagner et al. 1980. Copyright 1980, Elsevier Biomedical Press.

107
TABLE 5

Specific Binding of 3H-Spiroperidol to Striatal

Membranes Obtained from Rats.

Post-drug woising Bmax Kd

period(weeks) (fmol/mg prot.) (nM)

2 saline 92 ± 8 0.24 ± 0.02

methamephetamine 107±11 0.19 ± 0.03

4 saline 127±9 0.19 ± 0.04

methamphetamine 116 ± 6 0.22 ± 0.03

8 saline 107±12 0.21 ± 0.02

methamephetamine 102±14 0.23 ± 0.04

Rats were treated with 100 mg/kg/day of methamphetamine

for 4 days. They were killed 2, 4, or 8 weeks after the

last injection. Kd and Bmax values were derived from

Scatchard analyses of specific 3H-spiroperidol binding

at concentrations ranging from 0.15 to 2.4 nM. None of the

differences in dissociation constants (Kd) or receptor

densities (Bmax) between saline-treated and methamphetaminetreated rats were significant. Each of the values shown is

the mean ± S.E.M. of 3 separate determinations.

Ricaurte et al. 1980. Copyright 1980, Elsevier Biomedical

Press.

108
FIGURE 2

Fine granular degeneration in the rat neostriatum

following the high dose (50 mg/kg) regimen of

methylamphetamine. Four-day survival period.

Fink-Heimer method I with cresyl violet counterstain x 1190.

Ricaurte et al. 1982. Copyright 1982, Elsevier Biomedical

Press.

109
FIGURE 3

Absence of fine granular degeneration in

the neostriatum of a rat treated with saline

and killed after a 4-day survival period.

Fink-Heimer method I with cresyl violet

counter-stain x 1190.

Ricaurte et al. 1982. Copyright 1982, Elsevier Biomedical

Press.

110
hippocampus where there are very fev DA fibers. In addition, we

found massive degenerating pyramidal cells in the sensory motor

cortex. The chemical transmitter lo these cells is unknown at

the present time, but it is of considerable interest that we find

degeneration in this sensory motor area of tbe cortex. Chronic

MA users often have sensory motor abnormalities which result lo

lesions to the skin from excessive scratching or picking.

Whether this is related to the degeneration of pyramidal cells is

as yet unknown.

Alpha-methyltyrosine (AMT) attenuates the neurochemical changes

induced by MA, but reserplne enhances these changes Thus, the

AMT and reserpine results imply that the action of MA on the

cytoplasmic transmitter pool may be respooslble for the MAinduced neuronal damage. AMT reduces the cytoplasmlc transmitter

pool through synthesis inhibition whereas reserplne increases the

cytoplasmlc pool through destruction of the transmitter storage

vesicles. It has been suggested that MA preferentially releases

these cytoplasmlc transmitters by reversing the direction of the

high-affinity transport pump (Raiteri et al. 1979). Reserploe

and AMT evidence suggests that the MA-induced toxicity is

dependent upon cytoplasmic DA stores. Therefore, we reasoned

that massive DA release induced by MA may in some way be

neurotoxic.

Senoh and Witkop (1959) and Seoob et al. (1959) showed that DA

could be non-ensymatically converted to tribydroxyphenethylamines

such as 2,4,5-tribydroxyphenethylamine (6-hydroxydopamine, 6-

OHDA). If DA is released by MA and MA also blocks both DA

reuptake and monoamine oxldase (MAO), the amount of DA in the

synaptic cleft would be large, and an oxidative reaction could

occur. To test this hypothesis, male rate vere injected with 100

mg/kg of MA and than sacrificed after a time interval of either

20 minutes, 30 minutes, 1, 2, 4, 8, 16, or 24 hours. We found

that between 30 minutes and 2 hours, 6-OHDA vas formed in the

caudate nucleus. The 6-OHDA amounted to approximately 5% of tbe

DA that was present. The declining levels of

dihydroxyphenylacetic acid (DOPAC) in the caudate nucleus

indicated that the MA vas a strong MAO inhibitor at this dose

(table 6). In addition, we injected 6-OHDA intraventricularly

which causee about the same level of depletion of DA as tbe dose

of MA that we used, and found that the levels of 6-OHDA vere at

least roughly comparable to tbe levels of 6-OHDA formed after MA

Further experimentation showed that the AMT attenuates tbe

effects of MA and we, therefore, reasoned that AMT could prevent

tbe formation of 6-OHDA. This hypotbesis was confirmed (as shown

in figure 4).

In summary, we bare shown that MA is toxic to DA and 5HT fibers

in the central nervous system and that this toxic response is

manifested by degenaration of nerve terminals. Furthermore, the

toxicity to the DA system is mediated by DA itself which is

oxidized to 6-OHDA due to increased release from the vesicles and

blockade of inactivation. Whether this conversion of DA to 6-

OHDA in vivo is a normal metabolic pathway that can occur in the

111
TABLE 6

Levels of 6-OHDA and DOPAC in Rats Treated with

100 mg/kg Methylamphetamine

mg/kg tissue

Treatmnet Time of Kil 6-OHDA DA DOPAC DOPAC/DA

Saline 20 minutes/ 0 8.1 0.87

N=8 0 post injection ±0.25 ±0.09 10.6

100 mg/kg 30 minutes/ 0.20 8.7 0.72

±0.17 0.68 0.07 10.2 N=8 post injection

100 mg/kg 1 hours/ 0.39 6.1 0.62

N=5 post injection 0.31 0.60 0.07

100 mg/kg 2 hours/ 0.24 6.4 0.44

N=6 post injection 0.21 0.64 0.56

100 mg/kg 4 hours/ 0 5.7 0.37

N=5 post injection 0 0.65 0.08

100 mg/kg 8 hours 0 6.9 0.33

N=6 post injection 0 0.54 0.04

100 mg/kg 16 hours 0 6.3 0.78

N=4 post injection 0 2.6 0.25

100 mg/kg 24 hour/ 0 4.1 0.77

N=10 post injection 0 1.2 0.29

1.3

6.8

6.4

4.8

12.4

18.8

Seiden and Vosmer 1984. Copyright 1984, Ankho International

Press.

112
FIGURE 4

Caudate dopamine levels for 6 groups of rats

treated with methamphetamine (M) (100 mg/kg/day

for 4 days) or with saline (S) (2 ml/kg for 4

days). Reserpine (R), aiphamethyltyrosine (A),

or saline (S) were administered as pretreatment

agents (see text). * = Significantly different

from S-S group.

Wagner et al. 1983. Copyright 1983, Elsevier Biomedical Press.

113
absence of a drug, is certainly an interesting question and one

that needs to be pursued. Several diseases, including

Parkinson’s disease and Huntington’s chorea, are marked by the

degeneration of the dopaminergic system. Whether a toxic

metabolite of DA is responsible for this is a question that

romaine open, but certainly in light of the data that we have

promoted, it is a reasonable possibility.

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AUTHOR

Lewis S. Seiden, Ph.D.

The University of Chicago

Department of Pharmacological and

Physiologioal Sciences

947 East 58th Street

Chicago, Illinois 60637

ACKNOWLEDGMENTS

This research was supported by PHS grant MR-011191 and DA-00025.

L.S. Seiden is a recipient of Research Scientist Award MH-10562.

116
Molecular Mechanisms of Opiate

Tolerance and Dependence in a

Clonal Cell Line

Alan K. Louie, Ping-Yee Law, and Horace H. Loh

The neuroblastoma x glioma NGl08-15 hybrid cell line

provides a model system of neuronal origin for

studying molecular mechanisms of opiate tolerance

and dependence. This hybrid cell line has retained

many neuronal properties, including excitable

membranes and light and dense core vesicles

(Hamprecht 1977), thus serving as an in vitro

system suitable for biochemical investigations.

particular relevance to opiate research is the fact

that the neuroblastoma x glioma NGl08-15 hybrid

cells contain specific opiate binding sites of the

delta subtype (Klee and Nirenberg 1974; Chang et al.

1978) These receptors bind enkephalin and its

D-Ala2 analogues high aff affinity (Law et al.

1983a). The effects of opiates have been well

studied in this system. The opiate receptor is

coupled to adenylate cyclase (Sharma et al. 1975;

Traber et al. 1975). When opiate agonists are

given acutely, intracellular cyclic AMP levels are

lowered (Klee et al. 1975), an effect that is

stereospecific and reversed by naloxone (Traber et

al. 1975). With longer exposure to opiates, in

contrast, the intracellular cyclic AMP level rises

over a 24-hour period until it is equal to the

baseline level prior to opiate exposure (Sharma et

al. 1975; Klee et al. 1975; Lampert et al. 1976;

Sharma et al. 1977). At this point, if the opiate

is withdrawn, or naloxone is added, an immediate

increase or rebound above baseline levels of cyclic

AMP is observed (Sharma et al. 1975). This

normalization of cyclic AMP levels after chronic

opiate exposure , and the increase above baseline

levels after opiate removal, have similarities to

the phenomena of tolerance and dependence,

respectively, that are observed when whole animals

are administered opiates chronically. Thus, to some

extent, these cellular phenomena may serve as useful

models of the whole animal effects. Biochemical

117
investigations based on this view are subsequently

discussed.

When it was first observed, the development of

tolerance to opiate inhibition of adenylate cyclase

in NG108-15 hybrid cells was considered a simple

homeostatic process, involving compensatory

increase in adenylate cyclase activity which brought

intracellular cyclic AMP back up to baseline levels

(Sharma et al. 1977). According to this line of

thinking, the opiate receptors are still coupled to

and inhibit adenylate cyclase, but a compensatory

increase in intrinsic activity of adenylate cyclase

masks this inhibition. Thus, when the opiate

inhibition is removed by addition of naloxone, the

compensatory adenylate cyclase activity would

immediately manifest itself as an increase above

baseline of intracellular Cyclic AMP levels. In

this model, tolerance and dependence are intimately

related and mediated by the same mechanism.

Further research, however, brought this model into

question. The underlying hypothesis predicts that

the degree of tolerance should directly correlate

with the observed increase in adenylate cyclase

activity after naloxone addition. This was not the

case, however, when NGl08-15 cells, chronically

treated with 10nM etorphine, were compared with

cells treated with 1nM etorphine. Since a greater

degree of opiate inhibition had to be reversed with

the former treatment, the naloxone-induced increase

adenylate cyclase activity should have been

greater, but in fact it was the same as with the

latter treatment (Law et al. 1982a).

Additional data also indicate that these two

processes--loss of opiate inhibition and

naloxone-induced increase in adenylate cyclase

activity--are not closely correlated. For instance,

the conditions of chronic opiate treatment necessary

to elicit these processes vary. During a 24-hour

treatment with etorphine, 50% of the maximal

increase in adenylate cyclase activity occurred at a

dose of 1 nM, which was significantly lower than the

14nM dose required for 50% of maximal tolerance (Law

et al. 1983b). In a related cell

line--neuroblastoma N18TG2--the two processes are

completely dissociated. In these cells, chronic

opiate treatment results in a loss of opiate

inhibition of adenylate cyclase, but the addition of

naloxone at that point does not lead to an increase

in adenylate cyclase activity above baseline levels

(Law et al. 1982b). All of these data, therefore,

suggest that loss of opiate inhibition and the

118
naloxone-induced increase in adenylate cyclase

activity are independent processes and their

molecular mechanisms should be investigated

separately.

POSSIBLE MECHANISMS OF OPIATE TOLERANCE

If loss of opiate activity is not due to a

compensatory stimulation of the effector, in this

case adenylate cyclase, then the most attractive

explanation is that some change in the opiate

receptor occurs. For example, there could be a

decrease in the number of receptors, known as

down-regulation. Investigations of this possibility

have indeed revealed a significant decrease in

opiate binding sites after chronic treatment with

opiate agonists (Law et al. 1982a; Chang et al.

1982) . Further studies demonstrated that, during

the down-regulation, the opiate receptors are

internalized; they were associated with material

co-migrating with a lysosomal fraction on a Percoll

Gradient (Law et al. 1984). Studies of other

receptors which internalize, such as the low-density

lipoprotein (LDL) and insulin receptors, have shown

that this process is part of the normally occurring

recycling of the receptor, from the cell surface,

into the cell, and then back out to the cell

surface. The available data, interpreted in this

light, suggest that chronic opiate treatment

interferes with this recycling in such a manner that

the number of receptors the cell surface

decreases. Some data suggest that there is a

decrease in the return of receptors to the cell

surface (Law et al. 1985).

Down-regulation of opiate receptors does not,

however, appear to fully explain how opiate

tolerance is achieved. Down-regulation of the

opiate receptor is not necessary for loss of opiate

inhibition of adenylate cyclase. Some opiate

ligands act in this system as partial agonists, such

as 1 evorphanol , cyclazocine, and morphine: they

induce opiate tolerance but do not, unlike full

opiate agonists, down-regulate the receptors (Law et

al. 1983b). Furthermore, when full opiate agonists

such as etorphine are used, the loss of opiate

inhibition develops before down-regulation is

observed (Law et al. 1982a; Law et al. 1983b). In

other words, a two-step process seems to exist.

First, the receptor’s affinity for opiate agonists

decreases about tenfold: this occurs without a

change in receptor density. Subsequently, the

density of opiate receptors decreases to

approximately 20 to 30% of control levels. If the

119
chronic opiate is then removed, the cells

resensitize in a similar stepwise fashion: first,

opiate inhibition of adenylate cyclase reappears;

and second the density of receptors increases.

Thus, while down-regulation may contribute to opiate

tolerance, it does not always accompany and usually

follows the loss of opiate activity.

As discussed above, the initial change induced by

chronic opiate treatment, and the change best

correlated with loss of opiate inhibition of

adenylate cyclase, is a decrease in receptor

affinity for opiate agonists. This loss of affinity

suggests that the receptor is being uncoupled from

adenylate cyclase, as described by Lefkowitz et al.

(1980) for beta-adrenergic receptors. In this view,

when the opiate receptor is coupled to adenylate

cyclase, it is in a conformational state with high

affinity for agonists. As the receptor is

uncoupled, it changes to a conformational state with

low affinity for agonists. Agonists are able to

induce the high-affinity-coupled state, while

antagonists bind the coupled and uncoupled receptors

with equal affinities and cannot induce one or the

other state.

In this model, tolerance blocks receptor coupling

with adenylate cyclase, keeping the receptors in a

low-affinity-uncoupled state. Binding studies

provide support for this model. when NG108-15 cells

were treated for 3 hours with 100nM D-Ala

D-Leu5 enkephal in (DADLE) opiate tolerance

developed prior to the occurrence of significant

down-regulation of the receptors. Competition

binding to membranes of these tolerant cells was

compared to that of control cells. High- and

low-affinity sites, representing 75% and 25% of the

total binding sites, respectively, were revealed by

computer analysis of the binding data from control

membranes. In contrast, membranes from cells

chronically treated with opiates demonstrated a

shift of binding sites from high-affinity sites, now

only 45% of the total, to low-affinity sites, now

55% of the total (table 1). Presumably, in the

tolerant state receptors are shifted into an

low-affinity-uncoupled state in which the opiate

receptors cannot mediate opiate inhibition of

adenylate cyclase. These low-affinity sites, unlike

those in control membranes cannot be shifted back

into high-affinity sites by the presence of Mg++

(table 1).

120
TABLE 1

opiate Receptor Affinities for D-Ala2, D-leu5

Enkephalin (DADLE) in Control and Opiate-Treated

NG108-15 Cells

High-Affinity Sites

Kd (nM) 2.5+0.3

% of total sites 75

Low-Affinity Sites

% of total sites 25

K 206.5+49.9 d(nM)

High-Affinity Sites

Kd (nM)

% of total sites

1.88+0.16

100

Low-Affinity Sites

Kd (nM)

% of total sites

2.0+0.6

45

663+104.4

55

0.6+0.15

49

280+240

51

Opiate-treated cells were exposed to 1OnM or 100nM

DADLE for 3 or 4 hours at 24°C, which was removed

by repeated washings before binding studies.

Affinities were determined by computer analysis of

competition binding studies carried out at 24°c

for 90 minutes in the presence Of 1OmM Na+ and

100 uM GTP or 10mM Mg++. Opiate receptors were

labeled with 3H-diprenorphine, 2nM, in the

presence of various concentrations of DADLE.

An additional feature of this model is that opiate

inhibition of adenylate cyclase is now thought to be

mediated through a transducer protein, which binds

guanine nucleotides. various receptor systems,

including the beta-adrenergic, alpha2-adrenergic,

prostaglandin, muscarinic and opiate, all regulate

adenylate cyclase through such nucleotide-binding

proteins, or N proteins. The regulation is negative

or positive, depending on whether the receptor

system couples with, respectively, an inhibitory

(Ni) or stimulatory (Ns) protein (Gilman 1984).

Thus, the opiate receptor, in its high-affinity

121

484-443 O - 85 - 5

Binding in Presence of

10mM Na+ and 100um GTP Control

100nM DADLE

(24°C, 4hr)

Binding in Presence of

10mM Mg++ Control

10nM DADLE

(24°C, 4hr)
state, couples with the Ni. protein, which mediates

the inhibition of adenylate cyclase.

It follows, then, that during tolerance the opiate

receptor is unable to couple the Ni protein,

either because of an alteration of the receptor or

of the n protein. Alteration of the receptor

seems more likely since evidence indicates that the

Ni. protein can still mediate adenylate cyclase

inhibition during opiate tolerance. For example, if

NG108-15 cells are treated for 24 hours with 10nM

etorphine, they become completely tolerant to opiate

inhibition of adenylate cyclase, but the ability of

alpha2-adrenergic and muscarinic a agonists to

inhibit adenylate cyclase remains uneffected (Law et

al. 1983b). Since the inhibitory actions of these

latter agonists are also mediated through the N.

protein, opiate tolerance appears to leave the Ni

protein intact.

This suggests, then, that receptor uncoupling

results from an alteration of the receptor itself.

In order to explore this possibility, chemical

requirements for tolerance were defined in a

cell-free system. Purified plasma membranes were

prepared from NG108-15 cells and chronically treated

with opiates in a chemically defined incubation

mixture (Louie et al. 1984).

Tolerance to Opiate inhibition of adenylate cyclase

was demonstrated to be dose, time, and temperature

dependent. The presence of both Mg++ and Na+

were absolutely reququired for tolerance development.

Since these ions are necessary for coupling, the

development of tolerance may require coupling of the

receptor. This is consistent with the fact that

antagonists, which do not induce coupling, do not

cause tolerance.

Other data obtained with the cell-free system

suggest that a decrease in phosphorylation, or a

dephosphorylation, may be important to the

development of tolerance (Louie et al. 1984).

Conditions which favored dephosphorylation

potentiated tolerance development. An association

between opiates and a decrease in phosphorylation

has been previously described using synaptic

membranes from rat striatum (O’Callaghan et al.

1977; Clouet and O’Callaghan 1979). In this system,

chronic morphine treatment, in vivo, resulted in as

much as a 55% decrease in phosphorylation in vivo by

endogenous protein kinases of particular protein

bands. These changes only occurred if the morphine

treatment had been chronic. Thus, the observed

122
decrease in phosphorylation appeared to correlate

with tolerance to an opiate. Further studies will

be required, of course, to investigate the

possibility that dephosphorylation of the receptor

has direct relevance to the uncoupling of the opiate

receptor during tolerance.

POSSIBLE MECHANISMS OF OPIATE DEPENDENCE

As discussed above, the naloxone-induced increase in

adenylate cyclase activity does not correlate well

with the development of tolerance and may be viewed

as a different adaptive process occurring during

chronic opiate treatment. A mechanism is required

to account for how the displacement of an opiate,

from its binding site on an opiate receptor, results

in the stimulation of adenylate cyclase. Such a

mechanism presumes that the receptor remains coupled

to some effector which can directly or indirectly

stimulate adenylate cyclase. If during tolerance

development the opiate receptor becomes uncoupled

from the Ni protein, then Ni must not be

directly involved in mediating the naloxone-induced

increase in adenylate cyclase. Some other

mechanism involving other cellular components

should be sought.

One hypothesis is that some cytosolic component

which accumulates during chronic opiate treatment

stimulates adenylate cyclase. This was suggested by

experiments in which membranes and cytosol from

control and opiate - treated cells were isolated and

then mixed in every possible combination. The

cytosol from opiate-treated cells provided greater

stimulation of adenylate cyclase in membranes than

did cytosol from control cells (Griffin 1983).

Analysis of cytosol from chronic opiate-treated

cells indicated two components capable of

stimulating adenylate cyclase, both individually,

and synergistically when combined (table 2).

One of the cytosolic components was determined to

be adenosine. Its stimulation of adenylate cyclase

was inhibited by treatment with adenosine deaminase

or with the adenosine antagonist, theophylline.

Furthermore, neither this cytosolic component nor

exogenous adenosine stimulated adenylate cyclase in

NlBTG2, a neuroblastoma cell line which does not

demonstrate a naloxone-induced increase in

adenylate cyclase activity.

123
TABLE 2

Characterization of Cytosolic Components from

NGl08-1 Cells Chronically Treated with Etorphine

Component 1 Component 2

Molecular weight 300 >10,000

Heat stable + +

Calcium dependent +

Stimulation of

adenylate cyclase as

% of control 220 280

Putative identity Adenosine Calmodulin

Cells were exposed to 1uM etorphine for 4 hours.

Then cytosol and membranes were separated and the

former was resolved into two components, which

stimulated adenylate cyclase, by gel chromatography

with a Bio-Gel P-6DG desalting column. Stimulation

of adenylate cyclase in the presence or absence

(control) of the components was determined using

chronically etorphine-treated membranes in the

presence of 400uM naloxone. When components 1 and 2

were combined, the stimulation of adenylate cyclase

was 670% of control.

The exact relationship between increased

intracellular adenosine levels and naloxone-induced

increases in adenylate cyclase activity remains

unclear. If it is assumed that intracellular

adenosine increases because opiates prevent its

release from NG108-15 cells, however, one could

postulate that during chronic opiate treatment,

intracellular adenosine gradually accumulates. Upon

addition of naloxone and, thus, antagonism of

opiate inhibition of adenosine release, the

adenosine would be released into the extracellular

space. Then, these high levels of adenosine would

stimulate the cell's adenylate cyclase through the

adenosine receptors (A2) on the cell surface.

This naloxone-induced induced increase in adenylate cyclase

activity would not be observed after acute opiate

treatment because the accumulation of intracellular

adenosine would be insignificant at that point.

In support of the above hypothesis, some studies

indicate that the basal adenylate cyclase activity

is in part maintained by continuous release of

124
adenosine from NG108-15 cells (Griffin 1983). If

chronic opiate treatment disrupted this release of

adenosine, the development of adenosine receptor

super-sensitivity might be expected. This, in

fact, was demonstrated by the finding that chronic

opiate treatment is accompanied by an increase in

the coupling of adenosine receptors (Griffin 1983).

This increase in adenosine-receptor coupling and

the increased levels of adenosine might

synergistically stimulate adenylate cyclase after

naloxone addition, As predicted by the hypothesis,

the prior desensitization of the cells to the

effects of adenosine, by chronic adenosine

treatment, abolished the naloxone-induced increase

in adenylate cyclase activity (Griffin 19831.

The second cytosolic component from opiate-treated

cells was found to be heat stable and calcium

dependent, and to have a molecular weight of

greater than 10,000 daltons. While this component

has not been rigorously identified, it resembles

calmodulin in many of its properties. In

particular, this cytosolic component’s stimulation

of adenylate cyclase is enhanced greatly by the

presence of calcium. Similarly, naloxone-induced

increases in adenylate cyclase activity can be

Effected by manipulation of the intracellular

level (Griffin 1983). If this component is

calmodulin, these findings would be consistent with

the reports that calmodulin may act directly on

adenylate cyclase’s catalytic subunit (Salter et

al. 1981). Opiate treatment has been shown to

decrease the amount of calmodulin bound to the

plasma membrane of NG108-15 cells and this might

influence calmodulin stimulation of adenylate

cyclase (Nehmed et al. 1982).

Any proposed mechanism of dependence must involve

an opiate receptor that is coupled to an effector

system which will directly or indirectly stimulate

adenylate cyclase when naloxone is added. This

system might involve adenosine, calmodulin, or

other unidentified components. This implies that

the opiate receptor remains coupled to effectors

responsible for dependence while, simultaneously,

being uncoupled to effectors involved in tolerance

development. This hypothesis is not unreasonable

since receptors may interact with multiple

effectors and these interactions may be effected

differentially by chronic drug treatment. Studies

have indicated that while the opiate receptor may

be completely uncoupled from the inhibitorynucleotide binding protein (Ni), it is still

coupled to other N proteins, whose functions are as

125
yet unidentified. when NG108-15 cells are

pretreated with pertussis toxin, the Ni protein

is uncoupled from opiate receptors, yet opiate

binding is still sensitive to the addition of

guanine nucleotides, like guanosine triphosphate

(GTP) (Law & Loh, unpublished). This suggests that

the receptor is still coupled to other

guanine-nucleotie-binding proteins, because the

decrease in opiate binding, caused by guanine

nucleotides, is generally mediated through an N

protein. Similar binding data are obtained from

cells chronically treated with DADLE. In this case

the opiate receptor is uncoupled from the N.

protein during tolerance development, but appears

still to be coupled to other N proteins. These

other N proteins may be involved in mediating the

dependence aspects of chronic opiate effects.

CONCLUSION

The molecular mechanisms of tolerance and

dependence appear to be extremely complex. A

unitary mechanism to explain both processes has not

been found and some investigations have indicated

that separate mechanisms are in fact involved. The

neuroblastoma x glioma NG108-15 hybrid cells

provide a relatively simple model system with which

these complex mechanisms may be elucidated. Only

until some understanding is mastered on this single

cell level, will more intricate brain systems be

comprehensible.

REFERENCES

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Opioid peptides induce reduction of enkephalin

receptors in cultured neuroblastoma cells.

Nature 296:446-448, 1982.

Chang, K.J.; Miller, R.J.; and Cuatrecasas, P.

Interaction of enkephalin with opiate receptors

in intact cultured cells. Mol Pharmacol 14:961-

971, 1978.

Clouet, D.H., and O'Callaghan, J.P. Role of protein

kinases in opiate actions in brain. In: Ross, D.,

and Loh, H.H., eds. Neurochenmical Mechanisms

of Opiates and Endorphins. New York Raven

Press, 1979. pp. 281-300.

Gilman, A.G. G proteins and dual control of

adenylate cyclase. Cell 36:577-579, 1984.

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Griffin, M.T. Chronic opiate regulation of

adenylate cyclase activity in neuroblaqtoma x

glioma NG108-15 hybrid cells. Unpublished Ph.D.

thesis, Department of Pharmacology, University of

California San Francisco, 1983. 271 pp.

Hamprecht, B. Structural, electrophysiological,

biochemical and pharmacological properties of

neuroblastoma x glioma cell hybrids in cell

culture. Int Rev Cystol 49:99-170, 1977.

Rlee, W.A., and Nirenberg, M. A neuroblastoma x

glioma hybrid cell line with morphine receptors.

Proc Natl Acad Sci USA 71:3474-3477, 1974.

Klee, W.A.; Sharma, S.A.; and Nirenberg, M.

Opiate receptors as regulators of adenylate

cyclase. Life Sci 16:1869-1874, 1975.

Lampert, A.; Nirenberg, M.; and Klee, W.A.

Tolerance and dependence evoked by an endogenous

opiate peptide. Proc Natl Acad Sci USA

73:3165-3167, 1976.

Law, P.Y.; Horn; D.S.; and Loh, H.H. Loss of opiate

receptor activity in neuroblastoma x glioma NG

108-15 hybrid cells after chronic opiate

treatment: multiple step process.

Mol Pharmacol 22:l-4, 1982a.

Law, P.Y.; Horn, D.A.; and Loh, H.H. Opiate

regulation of adenosine 3' ,5' -monophosphate level

in neuroblastoma x glioma NG108-15 hybrid cells -

relationship between receptor occupancy and

effect. Mol Pharmacol 23:25-35, 1983a: -

Law, P.Y.; Hom, D.S.; and Loh, H.H. Opiate

receptor- down-regulation and desensitization in

neuroblastoma x glioma NG108-15 hybrid cells are

two separate cellular adaptation processes.

Mol Pharmacol 24:413-424, 1983b.

Law, P.Y.; Horn, D.S.; and Loh, H.H. Downregulation

of opiate receptors in NG108-15 hybrid cells:

chloroquine promotes accumulation of tritiated

enkephal in in the lysosomes. J Biol Chem

259:4096-4104, 1984.

Law, P.Y.; Koehler, J.E.; and Loh, H.H. Comparison

of opiate inhibition of adenylate cyclase

activity in neuroblastoma N18TG2 and

neuroblastoma x glioma NG108-15 hybrid cell

lines. Mol Pharmacol 21:483-491, 1982b.

Law, P.Y.; Ungar, H.G.; Horn, D.S.; and Loh, H.H.

Effects of cycloheximide and tunicamycin on

opiate receptor activities in neuroblastoma x

glioma NG108-15 hybrid cells. Biochem Pharmacol

34:9-17, 1985.

Lefkowitz, R.J.; Wessels, M.R.; and Stadel, J.M.

Hormones, receptors and cyclic AMP: their role in

target cell refractoriness. Top Cell Regulation

17:205-230, 1988.

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Louie, A.X. ; Law, P.Y.; and Loh, H.H. "Opiate

receptor desensitization in a cell-free system."

Paper presented at the International Narcotics

Research Conference Cambridge, England, 1984.

Nehmed, R.; Nadler, H.; and Simantov, R. Effects

of acute and chronic morphine treatment on

calmodulin activity of rat brain. Mol Pharmacol

22:389-394, 1982.

O'Callaghan, J.P.; Williams, N.; and Clouet, D.H.

Endogenous protein kinase activity associated

with synaptic membrane proteins: inhibition by

morphine. Fed Proc 36:968, 1977.

salter, R.S.; Krinks, M.H.; Xlee, C.B.; and Neer,

E.J. Calmodulin activates the isolated catalytic

Unit of brain adenylate cyclase. J Biol Chem

256:9830-9833, 1981.

Sharma, S.X.; Klee, W.A.; and Nirenberg, M.

Opiate-dependent modulation of adenylate cyclase.

Proc Natl Acad Sci USA 74:3365-3369, 1977.

Sharma, S.; Nirenberg, M; and xlee, W. Morphine

receptor as regulators of adenylate cyclase

activity. Proc Natl Acad Sci USA 72:590-594,

1975.

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cells. Nature 253:120-122, 1975.

ACKNOWLEDGEMENTS

Supported in part by NIDA Grants DA-00564 and

DA-01696. H.H. Loh is a recipient of NIDA

Research Scientist Career Award 5-K05-DA 70554.

A.K. Louie is a recipient of NIMH National

Research Service Award, MH 08960-02.

AUTHORS

Alan K. Louis, M.D.

Ping-Yee Law, Ph.D.

Horace H. Loh, Ph.D.

Departments of Psychiatry and Pharmacology

School of Medicine

University of California

San Francisco, CA 94143

128
Consequences of Perinatal Drug

Administration on Brain

Development: Striatal Opiate

Receptors and Dopamine

Sandra Lynn Moon

INTRODUCTION

Perinatal exposure to certain drugs and other agents can alter

the development of the organism, and the consequences of these

alterations may be long lasting. We have become more sophisticated in the analysis of these effects because we are beginning

to look at the subtle molecular changes in multiple systems in

addition to gross mutagenic capacity (from Nair 1974). The

following study outlines the normal development of mu opiate

receptors and dopamine in the striatum and demonstrates that

after prenatal administration haloperidol induces changes in

the normal course of development.

The striatum is an ideal model for examining drug-induced

changes. The corpus striatum is now recognized as a heterogeneous structure that can be compartmentalized on the basis of

its diverse neurochemistry, cytoarchitecture in some animals,

and anatomical connections (see Moon Edley and Herkenham 1984a).

In experimental animals, these compartments are: (1) variably shaped zones called "patches", including a band along the

dorsolateral perimeter of the structure, and (2) the zone

outside the patches, called the "matrix." In the rat, the

distribution of mu opiate receptors is an excellent marker for

the striatal compartments because these receptors appear very

early in striatal development, and are quickly transformed

into dense patches on a sparsely labeled matrix. This pattern

is maintained from a late prenatal age throughout adulthood.

Although dopamine is evenly distributed in the adult rat

caudate-putamen, during early development dopamine is clustered

in so-called "islands" (Loizou 1972; Olson et al. 1972). All

striatal topographic discontinuities are not in alignment,

however (e.g., see Graybiel et al. 1981), and the topographic

relation of the transient dopamine "islands" with opiate

receptor patches had not been established, even though chemical

systems that share a compartment might be expected to have

functional links.

129
THE NORMAL DEVELOPMENT OF STRIATAL OPIATE RECEPTORS AND DOPAMINE

IN THE RAT

To investigate further the development of the compartmentalization of opiate receptors and dopamine in the striatum, the

temporal and spatial sequences of these two markers were examined in the same brain. In the rat, the first striatal cells

are born on the 12th day in a gestational period that lasts

22 days. By means of glyoxylic acid-induced catecholamine

fluorescence (De la Torre 1980) and in vitro receptor autoradiography using emulsion-coated tissue sections and tritium

sensitive film (Herkenham and Pert 1982), dopamine and opiate

receptors labeled with either 3

H-naloxone or 125I-enkephalin

were compared on alternate striatal sections beginning at

embryonic day (E) 13 through adulthood.

The Caudate-Putamen

Opiate receptors and dopamine, which are two of the earliest

appearing striatal neurochemical markers, emerge in the caudateputamen 1 full week before birth. At that time, neither is

arranged in patches. The nigral fiber system, the source of

striatal dopamine, appears to be the earliest striatal afferent

connection (see Moon Edley and Herkenham 1984a). Other components related to these striatal chemical systems arrive later.

For example, 3H-DALA enkephalin-labeled opiate receptors (Kent

et al. 1982) arrive on E18, and enkephalinlike immunoreactivity

is not apparent until E20 (Pickel et al. 1981). Striatal dopamine receptors labeled by 3

H-spiperone do not arrive until E18.

On E14, dopamine fluorescence is dim and scattered throughout

the caudate-putamen. Small irregularly shaped clumps of fluorescence have no resemblance in size or position to the islands

that have yet to emerge. Opiate receptors appear on E14, also.

They are distributed throughout the caudate-putamen with a

slightly greater density in the caudal portion. Some opiate

labeling is seen in the subependymal layer among cells that have

yet to migrate into the striatal anlage.

The first signs of a discontinuous pattern appear a few days

before birth. On E19, dopamine begins to aggregate along the

lateral striatal perimeter (figure 1A) and patches are apparent

on E20 (figure 1B). High density opiate receptor patches and a

band along the perimeter emerge on E20 (figure 2). At the

earliest time of striatal heterogeneity, opiate receptor and

dopamine patches appear to be aligned, although the vague

boundaries of the aggregates make an accurate determinate of the

degree of alignment difficult. From this point on, opiate

receptor and dopamine developments are nearly synchronous.

Dopamine patches are at the height of their visibility on

postnatal day (P) 0. Figure 3 shows that they stand out as

aggregates composed of bright fluorescent dots on a dark and

dimly fluorescent matrix.

130
Dopamine patterning is generally more distinct in the lateral

aspect of the striatum. By contrast, opiate receptor patches,

although quite distinct on PO, are still embedded in a welllabeled matrix. There is now a clear one-to-one correspondence

between dopamine patches and opiate receptor patches in adjacent

tissue sections, as seen in figure 4.

During the second postnatal week, dopamine fluorescence in the

caudate-putamen loses much of its dotted quality, moderates in

intensity in the patches, and increases in intensity outside the

patches. These events, which begin to obscure dopamine patches,

will lead to their eventual disappearance. On the other hand,

the opiate receptor pattern continues to mature into discrete and

dense patches with definite borders on a lightly labeled matrix.

By P16, opiate receptors in the patches and matrix closely approximate the adult distribution, while nearly all dopamine fluorescent patches have become obscured. At this late stage in

the life of dopamine patches, an occasional patch can be discerned, and it is still aligned with an opiate receptor patch

(figures 5A and 5B). But, by P18, the dopamine fluorescence in

the caudate-putamen is virtually homogeneous, except for a bright

stripe along its medial border (figure 5C).

It has been suggested that dopamine plays a role in striatal

development (Pickel et al. 1981). Because the existence of

dopamine patches spans most of the period of striatal development, a role as a developmental cue is suggested, particularly

for those striatal systems that acquire the same patch/matrix

scheme. However, since dopamine and opiate receptor developments are nearly coincident, one can only speculate about a

developmental role for either opiate receptors or dopamine in

the topographic arrangement of striatal compartments.

The Nucleus Accumbens

Whereas the caudate-putamen has strong sensorimotor ties, the

nucleus accumbens is thought to be limbic-related. Their

connections are generally in parallel, although distinguishable.

Their neurochemistry is extremely similar.

The development of the nucleus accumbens lags behind that of the

dorsal striatum. It has some features in common with the

caudate-putamen, but there are some major differences as well.

Both dopamine and opiate receptors are evenly distributed when

they first arrive: opiate receptors on El8 and dopamine on E20.

Just as in the caudate-putamen, opiate receptors coalesce into

dense patches that are apparent by P6 (figure 6B). The dopamine

fluorescence pattern, however, is comprised of low intensity

"holes" surrounded by bright and dotted fluorescence in the

matrix. Thus, the major difference between the dorsal striatum

and the nucleus accumbens is that in the caudate-putamen, bright

fluorescence and opiate receptor-rich patches are aligned until

dopamine patches are obscured; but, from the earliest stages of

131
patterning in the nucleus accumbens, opiate receptor-rich patches

fill holes in the dopamine fluorescent distribution (figure 7).

These dopamine-poor zones and opiate receptor dense patches remain aligned throughout maturity (also see Herkenham et al. 1984).

Summary

Thus, dopamine systems in the caudate-putamen and in the nucleus

accumbens can be distinguished from each other by their: (1) time

of appearance, (2) early pattern, (3) subsequent adult distribution, and (4) fluorescent quality at maturity. In contrast,

opiate receptors obey the same rules of pattern formation in both

the dorsal striatum and in the later developing nucleus accumbens.

Striatal dopamine can be subdivided on the basis of many other

criteria, including source of origin, turnover rate, and susceptibility to pharmacological manipulation (see Moon Edley and

Herkenham 1984a). The positive register of opiate receptors and

dopamine in the caudate-putamen and the negative correlation in

the nucleus accumbens is another criterion by which to subdivide

striatal dopamine, and it is a contrast between the caudateputamen and nucleus accumbens. It seems clear that the opiate

and dopamine systems can be suspected of interacting differently

in the compartments of the caudate-putamen, where there has been

established a positive correlation between dense, opiate labeling and dopamine, and in the compartments of the nucleus accumbens where the correlation is negative. Such differences in

chemoarchitecture must certainly underlie regional dissimilarities in function.

PRENATAL DRUG ADMINISTRATION

The previous study outlined the developmental period of striatal

opiate and dopamine topographic organization. The role that

enkephalins play in the striatum is not understood; however,

numerous studies document striatal opiate/dopamine interactions.

The register of opiate receptors and dopamine early in development could result from the localization of opiate receptors on

dopamine terminals, as some biochemical studies have implied

(Biggio et al. 1978; Pollard et al. 1977). Destruction of

nigral terminals results in a large decrease in opiate receptors

found in the patch zones (Bowen et al. 1982). Two alternatives

could explain the loss: some opiate receptors could have a

terminal localization and are thereby lost when the terminal is

destroyed, or the decreased opiate binding may be mediated

transsynaptically through the loss of dopamine interactions

with its receptor. It is not possible to deplete rat fetuses of

dopamine through the use of 6-hydroxydopamine (Imamoto et al.

1980; Lidou et al. 1982) to study the effect of the absence of

dopamine on striatal development. However, interactions in the

developing striatum can be investigated further by means of

other pharmacologic manipulations during the time of early

striatal pattern formation. The dopamine antagonist haloperidol

was administered prenatally to investigate whether dopamine

132
receptor-mediated events are important for the normal development and maintenance of dopamine and opiate receptor distributions (see Moon 1984).

Haloperidol-filled osmotic minipumps were implanted subcutaneously in pregnant Sprague-Dawley rats that had been lightly

anesthetized with ether. The pumps released haloperidol (0.71

mg/day) from El5 through PO. As controls, some pregnant rats

were implanted with saline pumps and others were left untreated.

The litters from the haloperidol-treated group were small, containing usually from 3 to 7 pups, as compared to the normal litters of 6 to 12 pups. On PO and P16, rat pups were taken from

each litter and their brains processed for in vitro receptor

autoradiography and dopamine fluorescence. Sections prepared

for autoradiography were incubated in 3H-naloxone, 3H-spiperone,

or 3H-muscimol to label opiate! dopamine, and gamma aminobutyric

acid (GABA) receptors, respectively. After film exposure, the

binding densities in sections from the anterior half of the

striatum were measured by computer-assisted optical densitometry

(Goochee et al. 1980). Optical density (O.D.) measurements were

used as estimates of binding values.l For the statistical analysis of drug effects, nonspecific binding--estimated as the

binding to white matter dorsolateral to the striatum--was subtracted. The measurements were pooled to yield average specific

binding for each drug condition per film. Student's t-test was

used to indicate significance. In all binding conditions, tissue from the group of animals pretreated with saline was identical to untreated tissue.

Dopamine Receptor Binding

That chronic antagonist treatment results in increased receptor

binding is a corollary of receptor theory that has been well

demonstrated many times in many systems, but most often in the

adult. There have been demonstrations of exceptions to this

rule. In the case of chronic dopamine antagonism in the adult

rat, striatal spiperone binding increases (e.g., Burt et al.

1976). The relationship between increased dopamine receptor

binding, the pathogenesis of schizophrenia, and abnormal involuntary movements is not well understood. It would be interesting to know whether in the present experimental paradigm the

developing animal responded with increased binding, since

changes in dopamine receptor binding may be related to either

psychiatric or neurologic disease states.

Spiperone binding is normally arranged in a high-lateral to lowmedial density gradient in the striatum and is apparent at PO

and P16 (figure 7). Chronic haloperidol treatment did not alter

the pattern of 3H-spiperone binding. However, binding was uniformly elevated over saline control measures at PO by 27%

(p<0.02) and at P16 by 34% (p<0.05). These results suggest that

the neonate and the adult respond to dopamine receptor blockade

in the same way.

133
However, Rosengarten and Friedhoff (1979) reported that spiperone

binding decreases after chronic prenatal haloperidol treatment.

There are a number of differences in the experimental protocols

between that study and the present one that may explain the differences in results. In particular, the region of striatum analyzed and the binding conditions are different. In the present

study, only the anterior caudate-putamen was sampled and ketanserin was used to block spiperone binding to serotonin receptors,

whereas it seems that the entire striatum, including nucleus

accumbens, was analyzed in the previous study and no serotonin

blocker was used. It is unlikely that the lack of drug wash-out

contributed significantly to the variant results since, as figure

7 illustrates, 16 days after drug withdrawal (on P16). striatal

spiperone is elevated by virtually the same amount (34%) as immediately following drug withdrawal on PO (27%).

Nigral terminals were presumed intact in all cases since all

brains showed normal dopamine fluorescence in the striatum.

Since it has been postulated that some striatal opiate receptors

are located on dopamine-containing terminals, the normal fluorescence suggests that the decrease in opiate binding was not a

mere secondary effect caused by the degeneration of dopaminecontaining terminals.

Opiate Receptor Binding

Haloperidol has no affinity for mu opiate receptors. Thus,

changes in opiate receptor binding after haloperidol treatment

presumably are due to transsynaptic interactions between systems

containing dopamine and opiate receptors.

In normal animals, opiate receptor binding is arranged in

patches at PO; however, the matrix binding is only slightly

sparser (Kent et al. 1982). At this age, average O.D. values

were taken from patch and matrix, together, to perform the

analysis. In the haloperidol-treated animals, the pattern of

opiate receptor binding was unaffected, but the binding density

was substantially reduced. Naloxone binding in the caudateputamen was decreased by 45% (p<0.02) on PO.

Opiate receptors have nearly reached their adult distribution by

P16. At this age, separate measurements of patch and matrix

binding were analyzed. At P16, in the haloperidol-treated

animals, the drug-induced depression of opiate binding still

lingered, but its magnitude was diminished. Moreover, patch

binding was more stunted (-17%, p<0.05) than matrix binding

(-5%, p<0.05), suggesting different recovery mechanisms in each

of these two compartments. Likewise, opiate receptor binding is

preferentially reduced in striatal patches after destruction of

the nigrostriatal pathway in the adult (Bowen et al. 1982).

In the present experiment, dopamine terminals have been left

intact, suggesting that the decrease in patch opiate binding is

not merely due to mechanical destruction of the neuronal element

134
on which opiate receptors might be found, e.g., the dopamine

terminal (see discussion, Moon Edley and Herkenham 1984a). As

evidenced by the different rates of recovery of opiate receptor

binding in patch and matrix, a different recovery mechanism may

operate in each of these two striatal compartments. One suggestion is that these different rates of recovery in the compartments may be related to different connectivies. The projection to the striatum from the substantia nigra, proper, has a

patchy distribution (Wright and Arbuthnott 1981). Preliminary

evidence suggests that these patches are aligned with opiate

receptor-dense patches, and that the outlying dopaminergic cell

groups, the ventral tegmental area of Tsai, and the retrorubral

nucleus project preferentially to the matrix of the caudateputamen and nucleus accumbens (Moon Edley and Herkenham 1984b).

GABA Receptor Binding

It could be questioned whether these haloperidol-induced changes

represent a disruption of ontogeny due to a general vulnerability

of the developing striatum, rather than specific disruptions owing to opiate/dopamine interactions. As a test of this hypothesis, some sections were used for in vitro 3H-muscimol binding.

Muscimol binding is also distributed in patches on PO. There

was no significant difference between haloperidol and control

tissue, although a tendency for reduced muscimol binding in

haloperidol-treated tissue was noted. Like dopamine, 3H-muscimol-labeled GABA receptors take on a uniform distribution in

the striatum by P16. The specificity of the haloperidol-induced

changes is therefore supported by the lack of an effect on

striatal GABA receptors.

Functional Implications

Treatment with haloperidol induces a large increase in striatal

enkephalin content in the adult (Hong et al. 1978). If the same

mechanism operates in the neonate, perhaps the reduced opiate

receptor binding shown here is correlated with elevated enkephalin. Both enkephalin content (Hong et al.) and receptor binding

follow a similar course of normalization after drug withdrawal.

In this regard, it is clear that investigations of relative

transmitter levels and receptor density will be necessary to

access drug-induced changes in "set point" of individual chemical

systems and the functional implications of their regulation.

Haloperidol-induced behavioral responses are known to follow

different time courses. In man, haloperidol (1) can produce a

parkinsonian-like syndrome after treatment; (2) has antipsychotic

effects after repeated doses; and (3) can lead to the development of tardive dyskinesia after long-term administration. With

multiple effects that follow different time courses, it is likely

that nondopaminergic systems are involved in producing some of the

haloperidol-induced actions. Some of the nondopaminergic responses may be found within the striatum. The changes in the opiate

system in the dorsal striatum suggest that striatal opiates may

135
have a role in these effects. In experimental animals, haloperidol produces catalepsy, and opiates administered into the lateral ventricle also have been shown to produce a cataleptic-like

state (Bloom et al. 1976). Haloperidol is now the primary antipsychotic in use, and pregnant women have taken haloperidol

either as an antipsychotic or antiemetic. The present results

demonstrate that prenatal haloperidol treatment alters striatal

development. There may be functional consequences to these alterations, particularly since all affected systems do not recover

at the same rate and these consequences have yet to be studied.

Moreover, the present study appears to be a good model to study

the effects of changes on the development of the nervous system.

It is noteworthy that only by means of visualization techniques

have the functional units of the striatum become appreciated.

The utility of in vitro autoradiography for this type of study

is that multiple systems can be examined quantitatively with a

sensitivity comparable to standard biochemical procedures using

tissue homogenates, but at the level of the functional unit.

FOOTNOTE

1

The extent of drug-induced changes, estimated from O.D. values,

has probably been underestimated in the study, owing to the relatively low overall density values of these data (see bar in figures 7 and 8) and the properties of LKB film (see Herkenham, this

volume).

REFERENCES

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and Stille, G. Tolerance phenomena with neuroleptics catalepsy, apomorphine stereotypies and striatal dopamine metabolism

in the rat after single and repeated administration of loxapine and haloperidol. Eur J Pharmacol 22:287-294, 1973.

Biggio, G; Casu, M.; Lorda, M.G.; DiBello, C.; and Gessa, G. L.

Stimulation of dooamine svnthesis in caudate nucleus bv intrastriatal enkephalins and antagonism by naloxone. Science

200:552-554, 1978.

Bloom, F.; Segal, D.; Ling, N.; and Guillermin, R. Endorphins:

profound behavioral effects in rats suggests new etiological

factors in mental illness. Science 194:630-632, 1976.

Bowen, W.D.; Pert, C.B.; and Pert, A. Nigral 6-hydroxydopamine

lesions equally decrease mu and delta opiate binding to striatal patches: further evidence for a conformationally malleable type 1 opiate receptor. Life Sci 31:1679-1682, 1982.

Burt, D.R.; Creese, I.; and Snyder, S.H. Antischizophrenic drugs:

chronic treatment elevates dopamine receptor binding in brain.

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Davison, A.N., and Dobbina. J. The developing brain. In:

Davison, A.N., and Dobbing, J., eds. Applied Neurochemistry.

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Goochee, C.; Rasband, W.; and Sokoloff, L. Computerized densitometry and color coding of C-deoxyglucose autoradiographs. Ann Neurol 7:359-370, 1980.

Graybiel, A.M.; Ragsdale, C.W., Jr.; Yoneoka, E.S.; and Elde,

R.P. An immunohistochemical study of enkephalins and other

neuropeptides in the striatum of the cat with evidence that

the opiate peptides are arranged to from mosaic patterns in

reqister with the striosomal compartments visible by acetylcholinesterase staining. Neuroscience 6:377-397, 1981.

Herkenham, M., and Pert, C.B. Light microscopic localization of

brain opiate receptors: a general autoradiographic method

which preserves tissue quality. J Neurosci 2:1129-1149, 1982.

Herkenham, M.; Moon Edley, S.; and Stuart. J. Cell clusters in

the nucleus accumbens of the rat, and the mosaic relationship

of opiate receptors, acetylcholinesterase and subcortical

afferent terminations. Neuroscience 11:561-593, 1984.

Hong, J.S.; Yang H.Y.T.; Fratta, W.; and Costa, E. Rat striatal

methionine-enkephalin content after chronic treatment with

cateleptogenic and noncateleptogenic antischizophrenic drugs.

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Imamoto, K.; Nagai,; Kimura, H.; and Maeda, T. Effect of

neonatal administration of 6-OHDA on the brain development. I.

Alteration in the striatum. Arch Histol Jpn 43:347-364, 1980.

Kent, J.L.; Pert, C.B.; and Herkenham, M. Ontogeny of opiate

receptors in rat forebrain: and visualization by in vitro

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Lidou, G.W., and Molliven, M. The structure of cerebral cortex

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Loizou, L.A. The postnatal ontogeny of monoamine-containing

neurons in the central nervous system of the albino rat.

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Levine, R. Long-term haloperidol treatment and hydroxylase. In:

Cattebeni, F.; Spano, P.F.; Racagine, G.; and Costa, E., eds.

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pp 9-15.

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with cateleotogenic and noncateleotoaenic antischizophrenic

drugs. Neuropharmacology 17:589-596, 1979.

Moon, S.L. Prenatal haloperidol alters striatal dopamine and

opiate receptors. Brain Res 323:109-113, 1984.

Moon Edley, S., and Herkenham M. Comparative development of

striatal opiate receptors and dopamine revealed by autoradiography and histbfluorescence. Brain Res 305:27-42, 1984a.

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208:12OA, 1984b.

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Nair, V. Prenatal exposure to drugs: effect on the development

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Plenum Press, 1974, pp 171-198.

Olson, L.; Seiger, A.; and Fuxe, K. Heterogeneity of striatal

and limbic dopamine innervation: highly fluorescent islands

in developing and adult rats. Brain Res 44:283-288, 1972.

Penney, J.B.; Pan, H.S.; Young, A.B.; Frey, K.A.; and Dawth, G.W.

Quantitative autoradiography on 3H-muscimol binding in rat

brain. Science 214:1036-1038, 1981.

Pickel, V.M.; Beckley, S.C.; Sumal, K.K.; Joh, T.H.; Reis, D.J.;

and Miller, R. J. Light and electron microscopic localization

of enkephalin and tyrosine hydroxylase in neostriatum of fetal

and adult rat brain. Acta Histochem [Suppl] (Jena) XXIV:S97-

105, 1981.

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receptors on dopaminergic neurons in rat striatum. Nature

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Quirion, R.; Bayorh, M.A.; Zerbe R.L.; and Pert, C.B. Chronic

phencyclidine treatment decreases phencyclidine and opiate

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1982.

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dopamine receptor cells of pups from drug administration to

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the corpus striatum by the-substantia nigra. Neuroscience 6:

2063-2067, 1981.

ACKNOWLEDGMENTS

Dr. Moon was a senior staff fellow at the National Institute of

Mental Health when these experiments were performed. The study

of normal striatal development was performed in collaboration

with Dr. Miles Herkenham.

AUTHOR

Sandra Lynn Moon, Ph.D.

Bristol-Myers Company

Pharmaceutical Research and Development Division

Evansville, IN 47721

Indiana University School of Medicine

Evansville Center for Medical Education

Evansville, Indiana, 47732.

138
FIGURE 1

Photomontage of the earliest signs of dopamine aggregations in

coronal sections. A: arrow points to the formation of a lateral rim on E19. B: On E20, dense patches of fluorescence are

apparent within the nucleus.

139
FIGURE 2

Emerging opiate receptor patches labeled with 3H-naloxone coronal

section through the striatum on E20. Bar=500μm.

140
FIGURE 3

Photomontage of dopamine fluorescent patches and a band along

the lateral border in a coronal section on PO (open arrow points

to a dense aggregate of fluorescence in the caudal nucleus

accumbens).

141
FIGURE 4

Patch alignment in adjacent sections on P2 (A, B) and P6 (C, D).

Photomontages show dopamine fluorescent patches, at white arrows

in A, C. They perfectly overlap opiate receptors highlighted by

black arrows in B, D. Film autoradiographs of 3H-naloxone

binding in B and 125I-enkephalin binding in D.

142
FIGURE 5

The disapperance of domamine

patches in postnatal week 3

A: arrows point to last

remaining dopamine patches in

dorsal striatum. B: a

dark-field photomicogrpah

of an adjacent section

labeled with 125I-enkephalin

and emulsion-coated. White

arrows point to opiate

receptor patches that are in

register with dopamine in A.

Asterisks point to band along

the dorsolateral striatal

border. C: At P18 no

dopamine patches are apparent. Open arrow points

to bright fluorescence along

medial striatal wall.

Bar=200μm.

FIGURE 6

In the nucleus accumbens,

dopamine at P6 (A), P1O (C),

and P22 (E) shows zones of

dim fluorescence (white

arrows) that are in register

with 125I-enkephalin-labeled

patches shown in film autoradiographs from adjacent

sections, B, D, F respectively. Bar=500μm.

143
FIGURE 7

Photograph of TV monitor showing computer reconstructed film

images of 3H-spiperone binding to the striatum at P16. 0.D.s

are represented as 5 shades of gray on the bar. Striatal

spiperone binding in the haloperidol-treated animals is greater

than in saline-treated animals. Images represent total binding

and thus visually underestimate the difference between the two

conditions. Specific binding was 60%. Incubation procedure

followed the protocol of Quirion et al. 1982.

144
FIGURE 8

Photograph of TV monitor showing computer reconstructed images

of 3H-naloxone binding to the left striatum at PO. The relative

0.D.s are represented in the 5-shade gray scale on the bar.

When compared to saline treatment, haloperidol reduced opiate

binding. Specific binding was 93%. Incubation procedures are

detailed by Moon (1984). Images are of total binding. Arrows

point to emerging patches.

145
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49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $22

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed.

GPO Stock #017-020-01214-9 $4 NTIS PB 85-150381/AS $14.50

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed. NCDAI out of stock

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $17.50

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas. Ph.D., eds.

GPO Stock #017-024-01204-1 $4.25 NTIS PB 85-150373/AS $16

150
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed.

GPO Stock #017-024-01213-1 $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #017-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S., and Louise G. Richards, Ph.D., eds.

GPO Stock #017-024-01246-7 $4.25

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W., ed.

GPO Stock #017-024-01247-5 $4.25

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert, M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

62 THE NEUROSCIENCE OF DRUG ABUSE. Roger M Brown, Ph.D., and

David P. Friedman, Ph.D., eds.

IN PRESS

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John

Grabowski, Ph.D., ed.

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang,

Ph.D., and Charles C. Lee, Ph.D., eds.

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECTIVES. Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S.,

eds.

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

ADOLESCENTS. Catherine S. Bell, M.S.; and Robert J. Battjes,

D.S.W., eds.

151

U.S. GOVERNMENT PRINTING OFFICE : 1985 O - 484-443
DHHS Publication No. (ADM) 85-1415

Printed 1985