Neurobiology

of Drug Abuse:

Learning

and Memory

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Neurobiology of Drug Abuse:

Learning and Memory

Editor:

Lynda Erinoff, Ph.D.

Neuroscience Research Branch

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 97

1990

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
For sale by the Superintendent of Documents. U.S. Government Printing Office, Washington, D.C. 20402
Neurobiology of Drug Abuse:

Learning and Memory
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from a technical

review on the neurobiology of drug abuse: learning and memory that took

place on September 28 and 29, 1988, in Bethesda, MD. The review

meeting was sponsored by the Neuroscience Research Branch, 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 copyrighted material is permitted

only as part of a reprinting of the entire publication or chapter. For any

other use, the copyright holder’s permission is required. All other material

in this volume except quoted passages from copyrighted sources is in the

public domain and may be used or reproduced without permission from the

Institute or the authors. Citation of the source is appreciated.

The U.S. Government does not endorse or favor-any specific commercial

product or company. Trade, proprietary, or company names appearing in

this publication are used only because they are considered essential in the

context of the studies reported herein.

DHHS publication number (ADM)90-1677

Printed 1990

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 study of the neurobiology of learning and memory has not been fully

exploited in terms of drug abuse research. Studies of learning and memory

can add to our understanding of drug abuse by demonstrating: the consequences of drug abuse on performance and cognitive function; the role(s) of

endogenous opioids on memory processes; how drug effects on perception,

arousal, and motivation modify learning and memory; and the mechanisms

involved in the development of tolerance and sensitization. There are a

number of animal models available to researchers; the choice of a model

depends on the aim of the experiment. Thus, simple, one-trial learning is

appropriate for studying effects of drugs and endogenous opioids on the

stages of memory formation, while complex tasks can be used to test cognitive function analogous to that seen in humans. By using appropriate

animal models, studies can be directed at pinpointing the anatomical, physiological, and pharmacological substrates mediating effects on learning and

memory.

This monograph is the result of a technical review that was held September

28-29, 1988 to assess the available model systems and the current research

on the neurobiology of learning and memory as it relates to the study of

drug abuse. This technical review and monograph complement Monograph

84, "Learning Factors in Substance Abuse,” which presents the clinical

perspective on this area. The reports in this monograph present studies by

researchers in the field of drug abuse and neuroscientists in the area of

learning and memory whose model systems are relevant to drug abuse

research.

A convincing. case was presented by Dr. Rosenzweig for the use of a

simple peck-aversion task in the chick in studying the different stages of

memory formation. The use of the chick model is well suited for determining dose-effects functions of pharmacological agents and mapping the regions in the chick brain that participate in memory formation. Studies are

under way using the chick model to characterize the effects of endogenous

opioids and agonists and antagonists selective for opiate receptor subtypes

on learning and memory processes.

While one-trial learning tasks can be used to advantage to elucidate the

dynamic aspects of the memory process, the use of complex tasks is

v
necessary to model human memory performance. Dr. Kesner described the

use of spatial location tasks in the rat to model human list learning. Rats

display serial position curves, serial anticipation learning, coding of temporal

information, repetition lag functions, and the use of retrospective and prospective codes that are very similar to those seen in human subjects. In

addition, there are comparable memory-deficit patterns observed in braindamaged humans and in rats with lesions in homologous brain regions.

Dr. Olton presented a strategy that might be useful for probing for deficits

produced by chronic exposure to drugs of abuse. He described the use of a

drug challenge with scopolamine to disclose a latent impairment produced

by previous chronic treatment with a cholinesterase inhibitor. While chronic

cholinesterase treatment alone produced no decrements in successive reversals of a two-choice simultaneous discrimination or in a delayed match-tosample task, previous cholinesterase treatment did potentiate the effects of

scopolamine on these tasks. Dr. Olton also delineated some of the factors

that must be considered in developing animal models for evaluating the

effects of drugs of abuse on learning and memory.

Enkephalins are endogenous opioid peptides that have a modulatory influence on the strength of the memory trace and may affect the specific sites

of memory storage by affecting hippocampal plasticity. Dr. Martinez’s laboratory is investigating both of these processes. Leu-enkephalin (LE) may

be a stress as well as a learning hormone. It has a modulatory effect on

the acquisition and retention of conditioned responses that is mediated, at

least in part, by a site outside the blood-brain barrier. In addition to this

learning modulatory role, opioid peptides are involved in the induction of

mossy-fiber long-term potentiation in the CA3 region of the hippocampus.

Long-term potentiation is a change in synaptic strength that is thought to

underlie memory storage.

Dr. Deadwyler stressed the importance of studying the underlying neurobiology of the effects of both short- and long-term drug exposure on learning

and performance. Drugs of abuse may affect cognitive and behavioral processes while the subject is intoxicated, during withdrawal, and long after

drug exposure has ceased. Since much of the study of the neurobiology of

learning and memory focuses on the hippocampus, Dr. Deadwyler’s laboratory has focused on understanding how disruptions in hippocampal function

mediate the effects of delta-9-tetrahydrocannabinol (delta-9-PTHC), the

psychoactive constituent of marijuana, on learning and memory. Dosedependent delta-9-THC-induced behavioral deficits were demonstrated in the

detection and processing of conditioned sensory stimuli as well as in the retrieval of trial-specific information. Hippocampal electrophysiological correlates were altered over the same dose range as was behavior,

The hippocampus is also involved in associative learning, and Dr. Disterhoft

describes experiments to elucidate the hippocampal cellular bases of rabbit

v i
eyeblink conditioning. To this end, behavioral testing is combined with in

vivo hippocampal electrophysiological recording and in vitro recording from

a hippocampal slice preparation. Recording from slices enables the study of

the ionic mechanisms underlying associative learning. Dr. Disterhoft reported conditioning-specific reductions in the CA1 pyramidal cell after hyperpolarization, a response that is due primarily to calcium-mediated potassium

currents. Data were also presented that nimodipine, a calcium channel

blocker, facilitates acquisition of the conditioned eyeblink response in aged

and young rabbits, suggesting that it may be possible to affect learning and

memory through pharmacological manipulation of calcium flux.

Dr. Levy presented an information computation perspective for relating

hippocampal cellular physiology and anatomy to system physiology. The

brain’s function is to ensure survival of the organism by controlling the

environment through the ability to make predictions. Prediction is possible

because of the spatiotemporal regularities and physical constraints, i.e., the

redundancies in the environment. This approach analyzes the hippocampus

in terms of the storage of redundancy at synapses to yield the predictive

representations and the reduction of signal redundancy.

The enhancement of the short-latency acoustic startle reflex by prior fear

conditioning is an excellent paradigm for investigating the effects of anxiolytic drugs as well as for studying where in the nervous system plastic

changes occur that enable a conditioned stimulus to affect behavior. In this

paradigm, a cue previously paired with a shock produces an augmented

startle response elicited by a loud tone. Dr. Davis presented data on the

temporal specificity of fear-potentiated startle that demonstrated that the conditioned stimulus in fear conditioning is not the light alone but rather the

temporal pattern of events that are paired with the unconditioned stimulus

(shock) in training. Lesion and electrical stimulation studies have mapped

the primary startle circuit as well as the point at which the visual conditioned stimulus modifies transmission in the startle pathway. Other studies

using lesions and stimulation of the central nucleus of the amygdala suggest

that a neutral stimulus will elicit a state of fear when that stimulus activates

the amygdala after being paired with an aversive stimulus.

Dr. Koob suggested that, in addition to its role in ACTH release from the

anterior pituitary, CRF is a neurotransmitter in a brain arousal system that,

under conditions of extreme activity or chronic activation, produces the behavioral manifestations of stress. In a number of behavioral tests (open

field, operant conflict, conditioned suppression, and acoustic startle) CRF

appears to be anxiogenic. CRF seems to play a role in learning where

arousal and/or an aversive stale are required for animals to learn an association such as in two-way active avoidance or the conditioned emotional response paradigm. Thus, the brain CRF system may be required for forming

associations between aversive events and previously neutral stimuli.

vii
Drawing on several studies in human subjects in which the levels of arousal

of the subjects modified drug effects on cognitive performance, Dr. Kornetsky stressed that, when studying the effects of drugs on cognitive performance, investigators should always consider whether the observed effect is

due to a direct action on the neurobiological substrate of learning and

memory or is due to alterations of motor or perceptual-attentional systems.

Dr. Kornetsky then described a series of studies in animals in which it was

possible to dissociate drug effects on the threshold for rewarding brain

stimulation from effects on the threshold for detection of intracranial stimulation. Thus cocaine lowers (and pimozide raises) the threshold for rewarding stimulation at doses that have no effect on stimulus detection. This

suggests that attentional-perceptual functioning is not impaired at doses of

cocaine that are euphorigenic.

Dr. Lê presented evidence that learning factors play a role in the development of functional tolerance, a neuroadaptive process in which there is a reduction in CNS sensitivity to the drug. Evidence that learning plays a role

in tolerance comes from the demonstration that behavioral manipulations

such as practice while intoxicated and classical conditioning and neurobio

logical manipulations (brain lesions or changing neurotransmitter levels) that

affect learning can influence tolerance development. Dr. Lê suggested that

tolerance that develops through learning involves different mechanisms than

tolerance that results pharmacologically. To clarify whether there are two

separate mechanisms, studies using neurobiological manipulation should be

conducted under conditions in which either pharmacological or learning

factors predominate.

In his treatment of the role of conditioning in sensitization produced by

psychomotor stimulants, Dr. Pert presented a historical perspective on the

development of current thinking in this area. Stimulant-induced behavioral

sensitization is due to neuropharmacological changes resulting from repeated

drug administration, such as changes in neurotransmitter release or reuptake

and conditioned drug effects elicited by situational cues including both environmental and procedure-induced interoceptive stimuli. Pharmacological and

lesion studies from Dr. Pert’s laboratory suggest that dopamine in the nucleus accumbens and amygdala are essential for the acquisition of conditioned

locomotor excitation. Dopamine in the amygdala appears to modulate the

attachment of emotional significance to a given stimulus and may determine

which stimuli gain access to structures afferent to the amygdala, while

dopamine in the accumbens determines which limbic inputs gain access to

motoric output.

I would like to thank all of the authors of chapters in this monograph for

their thoughtful presentations. This monograph highlights many areas of research that the National Institute on Drug Abuse wishes to encourage, and I

hope it will stimulate interest in these areas.

viii
Contents

Page

Preface v

The Chick as a Model System for Studying Neural

Processes in Learning and Memory 1

Mark R. Rosenzweig

New Approaches to the Study of Comparative Cognition 22

Raymond P. Kesner

Long-Term Effects of Cholinergic Agonists on Memory 37

David S. Olton and Kathleen C. Raffaele

Enkephalin Influences on Behavioral and Neural

Plasticity: Mechanisms of Action 48

Joe L. Martinez, Jr., Patricia H. Janak,

Susan B. Weinberger, Gery Schulteis, and

Brian E. Derrick

The Effects of Delta-9-THC on Mechanisms

of Learning and Memory 79

Sam A. Deadwyler, Charles J. Heyser,

Ron C. Michaelis, and Robert E. Hampson

Calcium-Mediated Events in Associative Learning 94

John F. Disterhoft

ix
Page

Hippocampal Theories and the Information/Computation

Perspective 116

William B Levy

Pharmacological and Anatomical Analysis

of Fear Conditioning 126

Michael Davis

Stress, Performance, and Arousal: Focus on CRF 163

George F. Koob, Belinda J. Cole, Neal R.

Swerdlow, Michel Le Moal, and Karen T. Britton

Attentional and Motivational Effects of

Psychoactive Drugs 177

Conan Kornetsky, Joseph E.G. Williams, and

Michael Bird

Learning as a Factor in Ethanol Tolerance 193

A.D. Lê and H. Kalant

Conditioning as a Critical Determinant of

Sensitization Induced by Psychomotor Stimulants 208

Agu Pert, Robert Post, and Susan R.B. Weiss

List of NIDA Research Monographs 242

x
The Chick as a Model System for

Studying Neural Processes in

Learning and Memory

Mark R. Rosenzweig

INTRODUCTION

Reports of learning by young chicks and other precocial birds were made in

the last century, but it was only in the 1970s and 1980s that extensive

experimental work was done on brain mechanisms of learning and memory

in the chick, and this research is gaining in volume and importance. In

1873, Spalding published the first report of what is now called imprinting in

domestic fowl. In 1896, Lloyd Morgan commented on rapid learning and

lasting memory in young chicks. He reported that if young chicks pecked

once at conspicuously colored but bad tasting cinnabar caterpillars, they

refrained from pecking if presented with the caterpillars on subsequent days.

In the late 1890s, Edward L. Thorndike (1898) studied learning in chicks as

well as in dogs and cats. For the chicks, he used simple mazes constructed

of books standing on end.

All of these kinds of learning, as well as several other paradigms, are being

studied by contemporary investigators. During September 19 through 22,

1988, the author participated in a conference at the University of Sussex

that brought together most of the investigators from around the world

studying learning, memory, and neural plasticity in chicks. There were 35

papers and about 20 posters. Several of the contributions will appear in a

book to be edited by Professor Richard J. Andrew of the University of

Sussex. Some of the participants’ conclusions about the advantages and

disadvantages of the chick subject will be summarized in the last section of

this chapter.

The two learning situations most frequently used with chicks are imprinting

and peck-aversion learning. Research on imprinting and its brain mechanisms has been reviewed recently in a book by Horn (1985). Peck-aversion

learning is related to Lloyd Morgan’s observation; it was made into an experimental method by the pharmacologist Arthur Cherkin (1969). Cherkin’s

method was to allow a chick to peck at an attractive lure-in the first experiments, a tiny light, and, in later experiments, a small metallic bead. If

1
the lure was coated with a bitter substance, the chick showed a disgust

response, and it refused to peck at a similar lure a second time, whether the

next presentation occurred seconds, minutes, hours, or days after the original

trial. The chick pecked at other targets, even a bead of a different color,

so it did acquire a discrimination in a single trial. Cherkin and his coworkers used this method to study the effects of anesthetics and other pharmacological agents on the formation of memory (Cherkin 1969; Cherkin and

Lee-Teng 1965; Davis et al. 1982). Since this learning can be timed accurately, it offers a useful technique for the study of the temporal course of

memory formation, including the stages of formation of memory. Use of

this task is affording a cohesive body of data on the processes of memory

formation. (It may be noted that this tactic is the opposite of that discussed

by Kesner (this volume) in that it eschews complexity for brevity and simplicity; both tactics are appropriate for studying different aspects of learning

and memory.)

This chapter will consist of four main parts: (1) evidence for multiple

stages in memory formation, including some new findings; (2) participation

of different regions of the chick brain in stages of memory formation;

(3) effects of opioid agonists and antagonists on learning and memory in

the chick; and (4) advantages and disadvantages of the chick for studies of

brain mechanisms of behavior and for studies of the effects of pharmacological agents.

EVIDENCE FOR MULTIPLE STAGES IN MEMORY FORMATION

A Review of Research and Hypotheses on Multiple Stages in

Memory Formation

Evidence of many sorts has been offered since the work of Ebbinghaus for

the existence of at least two stages of memory, now called short-term

memory (STM) and long-term memory (LTM), and research with chicks is

now contributing to this topic. Parametric differences between STM and

LTM in human verbal learning convinced many that these are two quite different stages. A basis for this work had been provided by Mueller and

Pilzecker (1900) in their perseveration-consolidation hypothesis of the formation of memory. References to the term “short-term memory” began to

appear in the Psychological Abstracts by 1964, and they continued at a high

rate through the 1970s reaching a peak of over 10 per 1,000 references in

1980, then falling in the present decade to a level of about 2 per 1,000 references. Some continue to view the distinction between STM and LTM as

fundamental or at least highly useful (e.g., Horton and Mills 1984; Simon

1986), but others believe that the concept of a separate STM has outlived

its usefulness (e.g., Crowder 1982).

Neuropsychological studies also support a dissociation between STM and

LTM. Deficits of memory often affect formation of LTM but leave STM

2
intact, as in the well-known case of H.M. (Corkin 1984). On the other

hand, there are reports of impairment of STM with LTM intact (Baddeley

and Warrington 1970; Warrington 1982). Thus, the effects of certain brain

lesions may dissociate the two stages.

Studies with animal subjects on consolidation of memory began with the

work of Duncan in 1949. In the same year, Hebb (1949) and Gerard

(1949) independently set forth dual-trace hypotheses of memory. Although

neither Hebb nor Gerard mentioned Mueller and Pilzecker, their 1949 hypotheses appear to be based on that of the earlier workers; perhaps Mueller

and Pilzecker’s formulation had become so much a part of the background

knowledge of investigators in learning and memory that it had become

generic. In 1968, McGaugh published a schematic graph (figure 1) suggesting that performance after a learning trial is held very briefly by a sensory

buffer and then by three overlapping stages: STM, intermediate-term

memory (ITM), and LTM; this figure has been adapted and reprinted by

many authors, often without acknowledgment of the original.

FIGURE 1. Diagram of a multiple-trace hypothesis of memory storage

SOURCE: McGaugh 1968, copyright 1968, Academia Nazionale dei Lincei.

3
In 1972, McGaugh and Herz edited an important volume that reviewed animal research on consolidation of memory. By that time, the concept of

consolidation had almost disappeared from research on human memory.

Since then, there has been some revival of research on consolidation in normal human subjects, and there has also been interesting research on consolidation in patients with impaired memory, as is reviewed in a book edited

by Weingartner and Parker (1984). In animal research, Bennett et al.

(1972) introduced the use of the protein synthesis inhibitor (PSI) anisomycin

for work on formation of LTM. (It is true that, in the previous year,

Schwartz et al. (1971) had shown that anisomycin does not prevent shortterm sensitization in Aplysia, but they did not show that anisomycin can

prevent formation of LTM, nor did they deal with associative memory.)

The findings of Bennett el al. (1972) appeared to demonstrate that anisomycin can prevent formation of LTM for associative learning in mammals.

Anisomycin, a relatively nontoxic agent, overcame several of the difficulties

that had complicated interpretation of research using other inhibitors such as

puromycin and cycloheximide. Flood, Rosenzweig, and collaborators added

evidence that protein synthesis must occur in a narrow time window following training if LTM is to be formed (Flood et al. 1972; Flood et al. 1973;

Flood et al. 1975; Rosenzweig and Bennett 1984a; Rosenzweig and Bennett

1984b). Rosenzweig and Bennett (1984a) suggested that the protein might

be required, in part at least, for the changes seen in synaptic number and

structure as a result of differential experience (Diamond et al. 1975) or of

formal training (Chang and Greenough 1982). Control experiments were

performed to test whether the PSIs affected formation of LTM as such, or

whether the effects could be attributed to influences on factors such as perception, motivation, learning, or motor performance. For example, administration of PSIs an hour or more before training was found not to affect

learning, even though it affected other aspects of behavior. A large dose of

anisomycin 5 hours pretraining altered the motivational state of mice but did

not interfere with retention, whereas a small dose that blocked protein synthesis in the immediate posttraining period prevented formation of LTM

(Davis et al. 1980).

Meanwhile in the 1970s, the neurochemical bases of formation of the earlier

stages as well as of LTM were investigated by Mark and Watts (1971) and

Watts and Mark (1971) and then by Gibbs (formerly Watts) and collaborators (Gibbs and Ng 1978; Gibbs and Ng 1979; Gibbs and Ng 1984). This

research employed the one-trial taste avoidance training (or peck-aversion

training) of chicks introduced by Cherkin (1969). In 1977, Gibbs and Ng

set forth a comprehensive model of memory formation, including three sequentially dependent stages, each depending upon different neurochemical

mechanisms. Formation of LTM in chicks required synthesis of proteins in

the brain shortly after training, as Flood, Bennett, Rosenzweig, and others

had shown with rodents (Flood et al. 1973; Flood et al. 1975; Rosenzweig

and Bennett 1984a; Rosenzweig and Bennett 1984b). An early stage

(STM), lasting about 10 minutes, appeared to depend upon hyperpolarization

4
due to potassium conductance; a later stage (called the labile stage but in

later articles ITM), lasting about 30 minutes, was hypothesized to depend

upon hyperpolarization associated with sodium pump activity. Research in

the author’s laboratory with chicks has confirmed and extended many of the

observations of Gibbs and Ng (Patterson et al. 1986) but has also indicated

the importance of calcium influx and calmodulin for the early stages of

memory formation (Patterson 1987).

Preliminary work of Patterson et al. (personal communication) has also

yielded indications that some agents lead to a decline of memory by 30 seconds after training. If confirmed, this may reveal a stage of memory formation in the chick that resembles STM of human learners in its temporal

parameters. For the present, however, the author will continue to use the

term STM, in research with chicks, to designate a stage that lasts about

10 minutes.

The existence of three main stages in memory formation is indicated by the

fact that use of a variety of amnestic agents yields only three curves of

appearance of amnesia and not a different curve for each agent. Table 1

shows some of the agents that produce each of the three time curves.

These data suggest that there are three main stages of memory formation,

each of which can be affected by a number of different agents.

TABLE 1. Groups of amnestic agents that cause identical curves of

decay of memory

STM ITM LTM

Glutamate

LaCl3

KCl

Ouabain Anisomycin

Scopolamine Cycloheximide

Trifluoroperezine Emetine

Ethaainic Acid Aminoisobutyrate

Evidence from Animal Behavior for the Existence of Stages of Memory

Could direct behavioral evidence for the existence of stages of memory be

found? Behavioral studies by Kamin (1957a; Kamin 1957b; Kamin 1963),

and others indicated that the course of memory strength following training

may not be monotonic but may include one or more dips (Gisquet-Verrier

1983). In general, however, the complex timecourse was interpreted as

indicating motivational factors rather than stages of memory formation.

Furthermore, studies of the Kamin effect did not investigate in detail the

changes in the strength of memory within the first hour after training.

Gibbs and Ng (1977) and Gibbs and Ng (1984), however, reported that

5
memory of chicks for peck-aversion training shows dips or “fissures” at

about 12 and 55 minutes posttraining, and they interpreted the earlier dip as

marking the transition from STM to ITM and the second dip as marking the

transition from ITM to LTM. They also repotted that the timing of these

dips could be shifted by various pharmacological agents. The existence of

two sharp dips has not yet been confirmed by publications from other laboratories, and doubts have been raised about them (Roberts 1987; Ng and

Gibbs 1987).

Evidence has recently been obtained that bears upon this question. In order

to conduct research on enhancing memory formation by pharmacological

agents including opioid agonists and antagonists, Diane W. Lee, in our

laboratory, has been giving chicks weaker training by using dilute solutions

of methyl anthranilate (MeAn) as the aversive substance. The results

at 24 hours revealed, as Gherkin (1971) had reported, that animals trained

with diluted MeAn showed less retention than animals trained with 100 percent MeAn. We then wondered whether testing their memory at different

times following training might reveal successive drops in performance like

the hypothetical cures of McGaugh (1968) and, thus, indicate stages in

memory formation. This research is in progress; the results to date (based

on 20 to 25 animals per point) are shown in figure 2. They do suggest

successive stages in memory formation, but the presumed stages do not

drop off monotonically as do McGaugh’s curves. Now that we have indications of cusps around 15 minutes and 60 minutes, further work is being

done to define these time regions more clearly. It seems quite possible that

weaker training allows the transitions between stages to appear rather

clearly, whereas stronger training pushes behavior toward the ceiling at all

time points. The effects of various amnestic agents on performance after

weaker training will also be tested in an attempt to define more completely

the shapes of each of the component curves. The first component is seen

more clearly in a graph that expands the time scale (figure 3); this is a

limb that descends steeply to 60 seconds. It appears to be like the component labeled “sensory buffer” in McGaugh’s presentation, but note that it

has approximately the duration of STM in studies of human verbal learning.

Perhaps this should be labeled STM, and perhaps two intermediate-term

stages follow before protein-synthesis-dependent LTM appears.

Clearly, there is much to do along these lines. The appearance of distinct

successive stages in the behavioral curves opens up new possibilities for

research and for understanding of memory, and these possibilities have

begun to be pursued in research with chicks.

ROLES OF DIFFERENT BRAIN REGIONS IN MEMORY

FORMATION IN THE CHICK

It has been found that not all sites in the chick brain have the same significance for formation of memory. This research was made possible by the

6
FIGURE 2. Effect of training strength on retention of the peck-aversion

response

NOTE: Training strength was varied by diluting the concentration of the bitter substance on the

training bead (MeAn 100 percent, 10 percent. and 5 percent). Training-to-test intervals were

the following: 10 and 30 seconds; 1, 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes; and

24 hours. Percent avoidance is the percentage of chicks that refrained from pecking in the

10-second test period. The results are cumulative data from three successive experiment in

which 8 to 10 chicks were tested at each time point. Separate groups of chicks were tested

for each time pint.

finding that certain amnestic agents, administered in rather low doses, affect

only a small volume of tissue around the site of injection, as has already

been reported (Patterson et al. 1986). Among such agents are glutamate,

which impairs formation of STM; ouabain, which impairs formation of

ITM; and emetine, which impairs formation of LTM. (These designations

of memory stages are used here as the author and others have used them in

7
FIGURE 3. Effect of training strength on retention of the peck-aversion

response for the first eight time points

NOTE: The data of figure 2 are replotted here for the first eight time points

previous publications, although the author is not convinced that they are

correct.) The first studies on this topic in our laboratory, made by Teresa

Patterson, used injections into two regions of the chick forebrain that are

analogous to cerebral cortex in mammals: the intermediate medial hyperstriatum ventrale (IMHV) and the lateral neostriatum (LNS). With bilateral

injections, IMHV and LNS showed rather similar dose-response functions

for amnesic effects (table 2). Unless otherwise noted, all injections were

made 5 minutes before training, and testing was done 24 hours later. The

dose-response functions in the table have been abbreviated, to show some of

the main data without making the tables too full.

8
TABLE 2. Abridged dose-response curves for three amnestic agents in two

regions of chick brain (percent chicks showing avoidance at

24-hour test)

IMHV LNS

GLUTAMATE (mM)

Experimenter

TP

PS

OUABAIN (mM)

TP

PS

EMETINE (mM)

TP

PS

Sal 25 37 50 Sal 50

8.5 70 55* 38* 76 49*

86 42*

Sal 007 014 027 Sal 027

85 87 49* 25* 76 28*

82 46*

Sal 0.08 1.5 2.25 30 Sal 15 225

82 70 24* 40* 40* 76 45* 40*

4.0

85 55* 54* 50*

*Statistically significant amnesia.

NOTE: TP=T.A. Patterson; PS=P. Serrano.

The fact that IMHV and LNS yield similar results with bilateral injections

does not allow one to conclude that these two sites play identical roles in

memory formation. Unilateral injections produce mirror-image effects at

these two loci (figure 4); injections in the left IMHV cause amnesia, whereas injections into the right IMHV produce no significant effect. At the

LNS, the situation is reversed; injections into the left LNS are without

effect, but injections into the right LNS cause amnesia. At each site, each

amnestic agent causes amnesia to appear according to the temporal characteristic of that agent—STM, ITM, or LTM. Note also that effects at each

site showed sequential dependence; that is, preventing formation of one

stage of memory also prevented the appearance of the later stage or stages.

Peter Serrano, in the author’s laboratory, has been exploring further brain

sites. One of these is the cerebellum, because recent research (Thompson

1986) has shown that mechanical or chemical lesions of the deep cerebellar

nuclei prevent formation of conditioning of somatic responses. Serrano,

9
FIGURE 4. Comparison of effects of unilateral injection of amnestic

agents into the left or right IMHV or the leji or right

LNS

NOTE: Injections were made 5 minutes pretraining, and memory was tested at 24 hours. Saline control group were run with each of the 12 drug groups. Since the retention of the saline

control groups varied from 74 to 86 percent, each of these values has been normalized to 80

for clarity of presentation. but the magnitude of the difference between each drug group and

its control group has not been altered. Numbers of chicks ranged from 39 to 58 per group.

Each of the three agents caused significant amnesia when injected into the left but not the

right IMHV, each caused significant amnesia when injected into the right but not the left

LNS.

SOURCE: Patterson, unpublished doctoral dissertation.

therefore, tested the same three amnestic agents with deep cerebellar injections (table 3). The results showed a lack of amnesic effect for doses that

are amnestic in IMHV or LNS; even tripling effective doses for the other

sites did not cause amnesia when the agents were injected into the

cerebellum.

Thus far, it appeared that a brain site was either involved in memory formation for the peck-aversion response in the chick (as is the case for

IMHV and LNS) or that it was not involved (as is the case for the cerebellum). But exploration at a further site revealed another possibility (table 4):

bilateral injections of glutamate into the lobus paraolfactorium (LPO) did

not cause amnesia within or even beyond the dose range that was amnestic

in IMHV or LNS. Injections of emetine into LPO were largely without

10
TABLE 3. Abridged dose-response curves for three amnestic agents in

cerebellum (percent of chicks showing avoidance at 24-hour

test)

GLUTAMATE (mM)

OUABAIN (mM)

EMETINE (mM)

Sal 45 60 90

81 68 65 68

Sal .025 035 05

94 77 89 92

Sal 1.0 2.0 8.0

81 71 94 88

effect, although at a high dose a small amnesic effect was observed. However, ouabain produced amnesia when injected into LPO at the same dose

used for most of the experiments with IMHV and LNS. The lime curve of

amnesia produced by ouabain in LPO was similar to the curves found with

injections into IMHV and LNS. Moreover, scopolamine, another agent

found to prevent formation of ITM in IMHV, was also found to be effective in LPO. Thus, there is presumptive evidence that LPO plays a role in

formation of ITM but not in the formation of STM or LTM. It should be

noted that, although LPO does not appear to be involved directly in formation of LTM, the same sequential dependence is seen here as at IMHV and

LNS; that is, prevention of formation of ITM by intervention at LPO causes

failure of LTM, since the test was made at 24 hours.

TABLE 4. Abridged dose-response data for three amnestic agents in lobus

paraolfactorium (percent of chicks showing avoidance at

24-hour test)

GLUTAMATE (mM)

OUABAIN (mM)

EMETINE (mM)

Sal 35 50 70

89 78 77 83

Sal .02 .03 .04 .05

87 65 50* 38* 40*

Sal 2.25 3.0 6.0

82 79 72 61*

*Statistically significant amnesia.

11
Exploration is continuing at other brain sites. Possibly some will be found

that appear to have special significance for formation of STM or LTM, as

LPO appears to have for ITM. Even among the sites already identified, it

will be necessary to trace their connections and intereactions and to try to

find how each fits into an overall picture of the formation of the stages of

memory.

A note of caution must be added here. The dosages stated in tables 2

through 4 are based upon amounts injected into the chick brain, but we

found recently that half or more of the injected material may leak rapidly

out of the brain through the track of the hypodermic needle unless special

precautions are taken. This was discovered when a measurement of the rate

of metabolism of radioactively labeled [leu]enkephalin injected into one

hemisphere of the chick brain was sought. Chicks were sacrificed from 1

to 60 minutes after injection, the brains were dissected into several samples,

and the radioactivity was determined by scintillation counting. It soon

became apparent that much of the activity could not be recovered from the

brain, even at the earliest analyses. Activity was found on the surface of

the head and on the headholder. This problem had not been encountered in

work with rodents, where injections were made through indwelling cannulas.

The loss of material from the chick brain was not only relatively large but

was also was variable from animal to animal. Because of these losses, the

doses stated in this article and in published reports from the author’s

laboratory and other laboratories are probably too high by at least a factor

of two.

In spite of the low and variable recovery, it has been possible to determine

the transfer of radioactivity from the site of injection to neighboring regions,

to the other hemisphere, and into the hindbrain. Over 90 percent of the recovered activity is found in the injected hemisphere, and nearly 90 percent

of this amount is found close to the site of the injection. The distribution

does not appear to be time dependent. It should be noted that preliminary

studies have shown that [leu]enkephalin is metabolized to smaller peptides

within 1 to 2 minutes after injection into the chick brain. When radioactively labeled glutamate or ouabain were injected into one hemisphere,

data for localization of the recovered activity were similar to those found

for [leu]enkephalin. Thus, in spite of the loss of material, the data support

the localization of the regions of the brain involved in memory formation.

EFFECTS OF OPIOID AGONISTS ON LEARNING AND MEMORY

IN THE CHICK

Because many similarities were found between chicks and laboratory rodents

in their responses to amnestic agents, the author collaborated with Dr. Joe

L. Martinez, Jr., and his research group to determine whether chick learning

and memory formation might provide a useful model system for the study

of the effects of opioid agonists and antagonists. Preliminary findings

12
appear in Patterson et al. (1989), and other findings have been reported at

the Society for Neuroscience (Bennett 1988); some of the results will be

reported briefly here.

ß-endorphin (ß-END) injected bilaterally 5 minutes before training (10 μl/

hemisphere into the region of the IMHV) made the chicks amnesic at tests

conducted 24 hours later. Three doses of ß-END produced significant

amnesia, compared to the retention of control animals that received injections of physiological saline solution: 0.01 nmole/hemisphere, O-END vs.

saline, (4, n=63)=4.8, p<.05.; 0.10 nmole/hemisphere, ß-END vs. saline,

(4, n=64)=5.8, p<.05; 1.0 nmole/hemisphere, ß-END vs. saline,

n=63)=9.5, p<.01.

To test whether the effect of P-END was an opioid effect and not due to

some other type of action of the drug, we tested to see whether it could be

reversed by naloxone in experiments conducted like the previous one.

P-END given alone or in combination with a low dose of naloxone (16.0

nmole/hemisphere) produced significant amnesia compared to saline controls.

But a higher dose of naloxone (50.0 nmole/hemisphere) reversed the

amnesia caused by P-END. Flood et al. (1987) have reported that naloxone

administered immediately posttraining enhanced memory formation in both

chicks and mice. In their experiments, naloxone was approximately 1,000

fold more potent when administered intracerebroventricularly than subcutaneously, so it appears to exert its effect within the central nervous system.

[Leu5]enkephalin (LEU), employed in similar experiments, was found to be

amnestic at a dose of 1.0 nmole/hemisphere. Since LEU works mainly on

delta receptors, the effects of an agonist that is highly selective for delta

receptors, [D-Pen2, L-Pen5]enkephalin (DPLPE), were tested. Injections of

10 @/hemisphere of DPLPE produced significant amnesia.

The reversal of amnesia produced by either LEU or DPLPE by the use of

an agonist that is selective for the delta receptor, ICI 174,864, was then

attempted. Figure 5 shows that LEU and DPLPE again produced significant

amnesia compared with saline-injected controls. ICI 174,864 reversed these

amnesias in a dose-dependent manner.

These indications of clear effects of opioid agonists and antagonists on

learning/memory formation in chicks have encouraged the author to further

develop a chick model to investigate effects and mechanisms of drugs of

abuse. Several lines of research are being followed. Whether the effects

are on learning or on memory formation is being investigated, and, in the

latter case, the effects on different stages of memory formation will be

tested. These experiments are studying enhancement as well as impairment

of learning and memory. Different kinds of learning and different tests of

memory are being investigated for their effectiveness with chicks. The

13
FIGURE 5. Reversal of amnesia produced by DPLPE or LEU by the

delta selective antagonist ICI 174,864

NOTE: ICI administered alone did not produce either enhancement or amnesia. LEU or DPLPE

given alone produced significant amnesia compared to saline controls (LEU vs. saline

[4,N=41]=6.7; DPLPE vs. saline [4,N=43]=8.9, p<.01). ICI 174.864 reversed amnesia

produced by either LEU or DPLPE in a dose-dependent manner [4, N=45]=6.2, mixture of

LEU and ICI compared to LEU alone; [4,N=41)=6.4. mixture of DPLPE and ICI compared

to DPLPE alone; , p<.05). All injections (10 μl/hemisphere) were made bilaterally into the

region of the IMHV 5 minutes before training, and chicks were tested 24 hours after training.

SOURCE: Patterson et al. (1989), copyright 1989, American Psychological Association.

concentrations of endogenous opioids and of different opioid receptors in

the chick brain are being mapped. Among other things, this mapping will

suggest further sites to investigate for their roles in learning and memory

formation. The effects of peripheral administration of opioid agonists and

antagonists in the chick are also being investigated, following findings of

effects of peripheral injections in rodents. Clearly there is much to explore

and investigate with the chick preparation, and the author will be happy to

communicate with others who wish to work in this field.

ADVANTAGES AND DISADVANTAGES OF THE CHICK

LEARNING MODEL

Following are some of the advantages and disadvantages of the chick learning and memory system for investigation of effects of pharmacological

agents and for the study of brain processes in learning and memory formation, drawn from the author’s observations and from the comments of his

colleagues at the Sussex conference.

14
Advantages

Chicks are readily available and inexpensive. Many hatcheries keep

only female chicks and dispose of cockerels, so they are willing to sell

cockerels cheaply. Some investigators incubate their chicks in laboratory brooders in order to know the exact times of hatching.

Commercially available chicks are F1 hybrids between inbred lines and

they are phenotypically and genetically standard. For this reason,

experimental results tend to be robust. Information about the exact

crosses is proprietary and, therefore, not available.

The young chick is highly competent; it sees, hears, and tastes well, and

it makes well-coordinated pecking and locomotor responses. It also

learns and remembers well. Its precocial state at hatching means that it

can be tested readily shortly after hatching. This in turn means low

costs of housing and maintenance.

Previous experience of the chick is limited and can readily be controlled, especially if the chick is hatched in the laboratory. The small

amount of prior experience may make early training particularly significant and its cerebral effects relatively easy to measure.

The brain is relatively large for a small animal. About the size of the

brain of an adult mouse, the chick brain is suitable for localized lesions

or recording, for localized intracerebral injections, and for biochemical

studies. There is little blood-brain barrier in the young chick.

The unossified skull roof permits rapid intracerebral injections without

the necessity of anesthesia. This allows training and/or testing shortly

after injections, affording precise and close timing of events following

administration of drugs.

Because young chicks can be injected rapidly and can learn discriminations in a single trial, as many as 200 can be injected and trained in a

single session, so 8 to 10 experimental conditions can be compared

directly within a single batch of chicks,

Chicks recover rapidly from surgery, and they are resistant to operative

infection.

Although detailed mapping of concentrations of endogenous opioids and

of opioid receptors in the brain of the chick remains to be done, preliminary reports indicate that opioid receptors are present in relatively high

concentrations in the brains of chicks and other birds (Bardo et al.

1982; Felix et al. 1979; Pert et al. 1974).

1 5
Chicks have complete optic decussation, so that visual input can be

restricted to one cerebral hemisphere by covering one eye. There is

evidence that the two hemispheres process visual information differently.

Although the brain of the young chick is immature and is developing

rapidly, the young chick can be substituted for juvenile and adult mammals in many behavioral and physiological studies. Thus, Thompson et

al. (1983), in a review of “Cellular Processes of Learning and Memory

in the Mammalian CNS,” cited work with chicks in several places, and,

in a footnote to their first page (p. 447), they conferred honorary membership in the class Mammalia upon the class Aves.

A final advantage of the young chick, pointed out by Kastin (Kastin et

al. 1981), an investigator who had used rats in his previous research,

was that chicks do not bite.

Disadvantages

Because the young chick is clearly immature in its behavior, neuroanatomy, and neurochemistry, it is possible that, although it is capable

of learning and of other behaviors that resemble those of juvenile and

adult mammals, the underlying processes may differ from those that are

obtained in juvenile and adult mammals.

The neuroanatomy and neurochemistry of the chick have been less completely investigated than have those of mammals.

Some differences are found from batch to batch of chicks from the

same hatchery, and there are seasonal differences in activity and excitability of chicks.

Chicks grow rapidly into large animals that are expensive to feed and

house, so studies of posthatch development are costly and are rarely

undertaken.

When injections are made into the brain of the unanesthetized chick

with a hypodermic needle, much of the injected material promptly leaks

out unless special precautions are taken. For this reason, published

dose-response data must be interpreted with caution.

The chick is not yet well known as a research subject, so investigators

and granting agencies may be wary of undertaking or sponsoring projects with them. The publication of the Sussex conference and the

increasing numbers of research reports with chicks should help to overcome this reluctance to use chicks.

16
CONCLUSION

On balance, the young chick has much to recommend it for studies of

neural bases of learning and memory; in particular, it may serve as an

excellent model system to study drugs of abuse. The author invites other

investigators to join him in developing and exploiting the advantages of the

chick system.

REFERENCES

Bardo, M.T.; Bhatnagar, R.K.; Gebhart, G.F.; and Hughes, R.A. Opiate

receptor development in midbrain and forebrain of posthatch chicks.

Brain Res 255:568-673, 1982.

Baddeley, A.D., and Warrington, E.K. Amnesia and the distinction between

long and short-term memory. J Verbal Learn Verbal Behav 9:176-189,

1970.

Bennett, E.L.; Orme, A.; and Hebert, M. Cerebral protein synthesis inhibition and amnesia produced by scopolamine, cycloheximide, streptovitacin

A, anisomysin and emetine in rat. Fed Proc 31:838, 1972.

Bennett, E.L.; Patterson, T.A.; Schulters, G.; Martinez, J.L.; and

Rosenzweig, M.R. Beta-endorphin administration impairs acquisition in

the chick. Soc Neurosci Abstr 14:1029, 1988.

Chang, F.-L.F., and Greenough, W.T. Literalized effects of monocular

training on dendritic branching in adult split-brain rats. Brain Res

232:282-292, 1982.

Cherkin, A. Kinetics of memory consolidation: Role of amnesic treatment

parameters. Proc Natl Acad Sci USA. 63:1094-1101, 1969.

Cherkin, A. Biphasic time course of performance after one-trial avoidance

training in the chick. Commun Behav Biol 5:379-381, 1971.

Chcrkin, A., and Lee-Teng, E. Interruption by halothane of memory consolidation in chicks. Fed Proc 24:328, 1965.

Corkin, S. Lasting consequences of bilateral medial temporal lobectomy:

Clinical course and experimental findings in H.M. Semin Neural 4:249-

259, 1984.

Crowder, R.G. The demise of short-term memory. Acta Psychol (Amst)

50:291-323, 1982.

Davis, J.L.; Pico, R.M.; and Cherkin, A. Memory enhancement induced in

chicks by L-prolyl-L-leucyl-glycineamide. Pharmacol Biochem Behav

17:893-896, 1982.

Davis, H.P.; Rosenzweig, M.R.; Bennett, E.L.; and Squire, L.R. Inhibition

of cerebral protein synthesis: Dissociation of nonspecific effects and

amnesic effects. Behav Neural Biol 28:99-104, 1980.

Diamond, M.C.; Lindner, B.; Johnson, R.; Bennett, E.L. and Rosenzweig,

M.R. Differences in occipital cortical synapses from environmentally

enriched, impoverished, and standard colony rats. J Neurosci Res 1:109-

119. 1975.

1 7
Duncan, C.P. The retroactive effect of electroshock on learning. J Comp

Physiol Psychol 42:32-44, 1949.

Felix, D.; Henke, H.; and Frangi, U. Opiate receptors in the pigeon optic

tectum. Brain Res 175:145-149, 1979.

Flood, J.F.; Bennett, E.L.; Orme, A.E; and Rosenzweig, M.R. Relation of

memory formation to controlled amounts of brain protein synthesis.

Physiol Behav 15:97-102, 1975.

Flood, J.F., Bennett, E.L.; Rosenzweig, M.R.; and Orme, A.E. Influence of

training strength on amnesia induced by pretraining injections of cycloheximide. Physiol Behav 9:589-600, 1972.

Flood, J.F.; Bennett, E.L.; Rosenzweig, M.R.; and Orme, A.E. The influence of duration of protein synthesis inhibition on memory. Physiol

Behav 10:555-562, 1973.

Flood, J.F.; Cherkin, A.; and Morley, J.E. Antagonism of endogenous

opioids modulates memory processing. Brain Res 422:218-234, 1987.

France, C.P., and Woods, J.H. Antagonistic and rate-suppressing effects of

opioid antagonists in the pigeon. J Pharmacol Exp Ther 235:442-447,

1985.

Gerard, R.W. Physiology and psychiatry. Am J Psychiatry 106:161-173,

1949.

Gibbs, M.E., and Ng, K.T. Psychobiology of memory formation. Biobehav

Rev 1:113-136, 1977.

Gibbs, M.E., and Ng, K.T. Memory formation for an appetitive visual

discrimination task in young chicks. Pharmacol Biochem Behav 8:271-

276, 1978.

Gibbs, M.E., and Ng, K.T. Behavioral stages in memory formation.

Neurosci Lett 13:279-283, 1979.

Gibbs, M.E., and Ng, K.T. Hormonal influence on the duration of shortterm and intermediate-term stages of memory. Behav Bruin Res 11:109-

116, 1984.

Gibbs, M.E.; Richdale, A.L.; and Ng, K.T. Effect of excess intracranial

amino acids on memory: A behavioral survey. Neurosci Biobehav Rev

11:331-339, 1987.

Gisquet-Verrier, P. L’effet Kamin: Description, analyse et interpretations.

Revue critique. Cahiers de Psychologie Cognitive 3:3-34, 1983.

Gold, P.E., and McGaugh, J.L. A single-trace, two-process view of

memory storage process. In: Deutsch, D., and Deutsch, J.A., eds.

Short-Term Memory. New York: Academic Press, 1975.

Hebb, D.O. The Organization of Behavior. New York: Wiley, 1949.

Horn, G. Memory, Imprinting, and the Brain. Oxford: Clarendon Press,

1985.

Horton, D.L., and Mills, C.B. Human learning and memory. Annu Rev

Psychol 35:361-394, 1984.

Kamin, L.J. The retention of an incompletely learned avoidance response.

J Comp Physiol Psychol 50:457-440, 1957a.

Kamin, L.J. The retention of an incompletely learned avoidance response:

Some further analyses. J Comp Physiol Psychol 56:713-718, 1957b.

18
Kamin, L.J. Retention of an incompletely learned avoidance response.

J Comp Physiol Psychol 56:713-718, 1963.

Kastin, A.J.; Honour, L.C.; and Coy, D.H. Kyotorphins affect aversive

pecking in chicks. Physiol Behav 27:1073-1076, 1981.

Keppel, G. Consolidation and forgetting theory. In: Weingartner, H., and

Parker, E.S., eds. Memory Consolidation: Psychobiology of Cognition.

Hillsdale, NJ: Lawrence Erlbaum Associates, 1984. pp. 149-161.

Mark, R.F., and Watts, M.E. Drug inhibition of memory formation in

chickens. I. Long-term memory. Proc R Soc Lond [Biol] 178:439-454,

1971.

McGaugh, J.L. A multi-trace view of memory storage processes. In:

Bovet, D., ed. Attuali Orientamenti della Ricerca sull’Apprendimento e la

Mentoria. Quaderno N. 109. Rome: Accademia Nazionale dei Lincei,

1968.

McGaugh, J.L., and Herz, M.J. Memory Consolidation. San Francisco:

Albion, 1972.

Mizumori, S.J.Y.; Rosenzweig, M.R.; and Bennett, E.L. Long-term working

memory in the rat. Effects of hippocampally applied anisomycin. Behav

Neurosci 99:220-232, 1985.

Morgan, L. Habit and Instinct. London: Edward Arnold, 1896.

Mueller, G.E., and Pilzecker, A. Experimentelle Beitrage zur Lehre vom

Gedächtniss. Z Psycho1 [Suppl] 1:1-288, 1900.

Ng, K.T., and Gibbs, M.E. Less-than-expected variability in evidence for

three stages in memory formation: A response. Behav Neurosci

101:126-130, 1987.

Patterson, T.A. Neurochemical processes in memory formation in the chick.

University of California at Berkeley. Unpublished doctoral dissertation.

Patterson, T.A.; Alvarado, M.O.; Warner, I.T.; Bennett, E.L.; and

Rosenzweig, M.R. Memory stages and brain asymmetry in chick learning. Behav Neurosci 100:850-859, 1986.

Patterson, T.A.; Rosenzweig, M.R.; and Bennett, E.L. Amnesia produced

by anisomycin in an appetitive task is not due to conditioned aversion.

Behav Neural Biol 47:17-26, 1987.

Patterson, T.A.; Schulteis, G.; Alvarado, M.C.; Martinez, J.L.; Bennett, E.L.;

Rosenzweig, M.R.; and Hruby, V.J. Influence of opioid peptides on

memory formation in the chick. Behav Neurosci 103:429-437, 1989.

Pert, C.B.; Aposhian, D.; and Synder, S.H. Phylogentic distribution of

opiate receptor binding. Brain Res 75:356-361, 1974.

Postman, L. Transfer, interference and forgetting. In: Kling, J.W., and

Riggs, J.A., eds. Woodworth and Schlosberg’s Experimental Psychology.

3rd ed. New York: Holt, Rinehart and Winston, 1971.

Roberts, S. Less-than-expected variability in evidence for three stages in

memory formation. Behav Neurosci 101:120-125, 1987.

Rosenzweig, M.R. Experience, memory, and the brain. Am Psychol

39:365-376, 1984.

19
Rosenzweig, M.R., and Bennett, E.L. Direct processes and modulatory

influences in the stages of memory formation. In: Lynch, G.; McGaugh,

J.L.; and Weinberger, N.M., eds. Neurobiology of Learning and Memory.

New York: Guilford Press, 1984a.

Rosenzweig, M.R., and Bennett, E.L. Studying stages of memory formation

with chicks and rodents. In: Butters, N., and Squire, L.R., eds. The

Neuropsychology of Memory. New York: Guilford Press, 1984b.

Schwartz, J.H.; Castellucci, V.F.; and Kandel, E.R. Functioning of identified neurons and synapses in abdominal ganglion of Aplysia in absence of

protein synthesis. J Neurophysiol 34:939-963, 1971.

Simon, H. The parameters of human memory. In: Klix, F., and

Hagendorf, H., eds. Human Memory and Cognitive Capabilities: Symposium in Memoriam Hermann Ebbinghaus: Vol. A. Amsterdam: Elsevier

Science Publishers, 1986. pp. 299-309.

Spalding, D.A. Instinct, with original observations on young animals.

MacMillan’s Magazine 27:282-293, 1873. [Reprinted: Br J Animal

Behav 2:2-1], 1954.]

Thompson, R.F. The neurobiology of learning and memory. Science

233:941-947, 1986.

Thompson, R.F.; Berger, T.W.; and Madden, J., IV. Cellular processes of

learning and memory in the mammalian CNS. Annu Rev Neurosci 6:447-

491, 1983.

Thorndike, E.L. Animal intelligence. An experimental study of the associative processes in animals. Psychol Rev Monogr Suppl 2(4, Whole

8):1-109, 1898.

Warrington, E.K. The double dissociation of short- and long-term memory.

In: Cermak, L.S., ed. Human Memory and Amnesia. Hillsdale, NJ:

Lawrence Erlbaum Associates, 1982.

Watts, M.E., and Mark, R.F. Drug inhibition of memory formation in

chickens. II. Short-term memory. Proc R Soc Lond [Biol 178:455-464,

1971.

Weingarter, H., and Parker, E.S. eds. Memory Consolidation: Psychobiology of Cognition. Hillsdale, NJ: Lawrence Erlbaum Associates,

1984.

ACKNOWLEDGMENTS

All of the research reported here was done in collaboration with

Edward L. Bennett; parts of the research were done in collaboration with

Teresa A. Patterson, Diane W. Lee, Peter Serrano, and Duncan Beniston;

the opioid research was done in collaboration with Joe L. Martinez, Jr.,

Teresa A. Patterson, and Gerhart Schulteis. The research was supported by

grant R01 DA 04795 from the National Institute on Drug Abuse and grant

BNS-86-06938 from the National Science Foundation.

20
AUTHOR

Mark R. Rosenzweig, Ph.D.

Department of Psychology

University of California

3210 Tolman Hall

Berkeley, CA 94720

21
New Approaches to the Study of

Comparative Cognition

Raymond P. Kesner

INTRODUCTION

There has been and there continues to be a genuine interest in the development of animal models of human memory. This interest is based on the

assumption of evolutionary continuity between animals and humans in terms

of brain-memory functional relationships. To the extent that the above

assumption has validity, animal models can provide the impetus (a) to

understand the neurobiological basis of memory in animals and humans;

(b) to develop new neurobiological theories of memory representation;

and (c) to develop potential therapeutic approaches, including pharmacological ones, aimed at alleviating memory problems in humans.

Of the many animals that have been studied, rodents have been used most

extensively. Can the memory performance of rats provide the basis of an

animal model of human memory? There have been a number of approaches. One approach emphasizes the importance of learning and memory storage or retrieval within tasks that can be learned in a single trial, e.g., active

avoidance, inhibitory avoidance, and taste aversion learning. The advantage

of this approach is that, because acquisition is rapid, one can evaluate the

efficacy of a pharmacological treatment with a single drug dose. By injecting animals before or after training or before retention testing, one can

assess whether the treatment produced state-dependent effects or affected

storage, short-term memory, long-term memory, or retrieval. One can thus

evaluate the dynamic aspects of the memory process. The disadvantages

are that one often needs large numbers of animals, variability in memory

performance is often quite high, and one often needs to use negative reinforcement. Finally, these tasks are not used to assess memory function in

humans, in part because acquisition is too rapid.

A different approach emphasizes the use of complex tasks, e.g., list-learning

tasks. The advantages of this approach are that fewer subjects are required,

performance is quite stable, and comparisons can be made more readily

with human memory performance. The disadvantages are that training takes

a long time, requiring multiple injections of specific pharmaceutical agents

or the use of long-acting agents. Even though the latter approach has not

22
been used extensively, the ease of between-species comparisons makes this

approach attractive.

In this chapter, a number of examples of animal memory performance in a

variety of list-learning tasks will be presented. These examples include the

display of serial position curves, serial anticipation learning, coding of temporal information, repetition lag function, and utilization of retrospective and

prospective codes.

SERIAL POSITION CURVES

One commonly used method to assess memory function in humans is to

present a list of items (e.g., pictures, words) of information to a subject and

ask for recall or recognition of the items. In normal subjects, one observes

a serial position curve with better performance for the first items (primacy

effect) and the last items (recency effect) compared to items located in the

middle of the list. It has been proposed by some theorists that the primacy

effect reflects information storage in long-term memory, while the recency

effect reflects information processing in short-term memory (Atkinson and

Shiffrin 1968). However, it should be noted that there are other interpretations for the serial position effect. For example, it has been suggested

that the recency effect reflects automatic availability of information requiring

little attention, whereas the primacy effect reflects the utilization of controlled or effortful attention-directing activity (Hasher and Zacks 1979;

Shiffrin and Schneider 1977). Others have suggested that the ends (primacy

and recency) of a list are more distinctive and, therefore, are remembered

better than the interior positions (Ebenholtz 1972).

How do rats perform on an item- or order-recognition task for a list of

spatial locations? Rats were placed on an eight-arm radial maze and were

given experience exploring all eight arms. Reinforcements could be obtained at the end of each arm and consisted of pieces of sweetened cereal.

After receiving extensive exposure to the radial arm maze, the itemrecognition memory task was begun. Each animal was allowed to visit a

sequence of five arms on each trial (one per day), which was selected on a

pseudorandom basis. The sequencing of the five arms was accomplished by

sequential opening of Plexiglas doors (one at a time) located at the entrance

of each arm. This constituted the study phase. Immediately after the animal had received reinforcement from the last of the five arms, the test

phase began. Only one test was given for each trial; it consisted of opening two doors simultaneously, with one door representing an arm previously

visited for that trial and the other door representing a novel arm for that

trial. For half of the animals, the rule to be learned, leading to an additional reinforcement, was to choose the arm that had been previously visited

during the study phase of the trial (win-stay). For the other half of the

animals, the rule leading to a reinforcement was to choose the arm that had

not previously been visited during the study phase of the trial (win-shift).

23
Each animal received 2 sets of 40 trials, with 8 tests for each of the serial

positions.

For the order-recognition memory task, each animal was allowed on each

trial (one per day) to visit all eight arms in an order that was randomly

selected for that trial. The sequencing of the eight arms was accomplished

by the sequential (one at a time) opening of Plexiglas doors located at the

entrance of each arm. This constituted the study phase. Immediately after

the animal had received reinforcement from the last of the eight arms (i.e.,

completed the study phase) the test phase began. Only one test was given

for each trial; it consisted of opening two doors simultaneously. On a

random basis, either the first and second, fourth and fifth, or seventh and

eighth doors that occurred in the sequence were selected for the test. The

rule to be learned, leading to an additional reinforcement, was to choose the

arm that occurred earlier in the sequence.

The animals were initially given 8 trials per serial position for a total of 24

trials. If, after these 24 trials, the animal had not performed at 75 percent

or better on tests of both the first and second (primacy) and seventh and

eighth (recency) serial positions (criterion), then additional blocks of 6 trials

(2 trials per serial position) were given until the criterion was met for the

most recent block of 24 trials or until 100 trials total had been given.

The results of the item-recognition memory task are shown in figure 1 and

indicate that by the second block of 40 trials rats display a serial position

curve for memory of spatial locations given that a win-stay rule is required.

However, when a win-shift rule is required, animals display only a recency

effect, which can already be observed within the first block of 40 trials.

With continued training, rats display excellent retention for all serial positions for either the win-stay or win-shift rules. DiMattia and Kesner (1984)

have interpreted these results as reflecting differential processing requirements for the win-stay and win-shift rules. They suggest that the win-stay

rule necessitates relatively more effortful, elaborative processing than does

the win-shift rule, which is used automatically. A more detailed description

of the experimental methods can be found in DiMattia and Kesner (1984).

The results of the order-recognition memory task are shown in figure 2 and

indicate that rats again display a serial position curve for the sequential

order of spatial locations. This serial position function is maintained even

with extensive training (Kesner et al. 1984). Recently, Dale (1987) tested

humans in a task analogous to the order-recognition memory study for rats.

In this case, eight lights were turned on (one at a time) in a specific sequence. Subjects were then tested for memory for the order of the first and

second, fourth and fifth, or seventh and eighth serial positions. The results

are shown in figure 2 and indicate a serial position function parallel to that

obtained with rats. Thus, it is clear that rats, like humans, display serial

position curves for a list of items, suggesting that list-learning experiments

24
can provide an excellent paradigm in which to develop animal models of

human memory.

FIGURE 1. Mean percent correct as a function of serial position for the

win-stay (closed circles) and win-shift (open circles) for the

first 40 trials (a) and for the second 40 trials (b)

SOURCE: DiMattia and Kesner 1984, Copyright 1984, American Psychological Association.

Since similar serial position curves for item memory for lists of information

have been reported for pigeons and monkeys (Sands and Wright 1980;

Wright et al. 1985), these animals could also serve as useful animal models.

SERIAL ANTICIPATION LEARNING

Another commonly used procedure to assess human memory is to present a

single nonvarying list of items that must be recalled in the order presented.

One method used to study serial recall requires that a subject remember the

second item in the list when the first is presented alone, the third item in

the list when the second is presented, and so on. This method is called the

25
FIGURE 2. Mean percent correct as a function of serial order position in

rats and humans

anticipation method. Thus, a subject is required to anticipate the next item

of a nonvarying list. During the learning of such a list, subjects often

make errors by reporting an incorrect serial position item within the list.

These errors are called intralist intrusion errors. The results of an experiment with 12 words using the serial anticipation method are shown in

figure 3. The results indicate that there are more errors in the middle

compared to the end items of the list (Deese and Kresse 1952).

In order to test how rats perform in a serial list task using the serial anticipation method, rats were tested on an eight-arm radial maze. After extensive experience with the maze, animals were given 40 trials in the S-arm

maze. On each trial, the animals were presented with a constant sequence

of five arms. Serial anticipation memory was measured as a pattern of correct or incorrect orienting responses in anticipation of the ensuing doors in

the constant sequence. Since the sequence of spatial locations did not vary

from trial to trial, it was assumed that the emitted orienting responses reflected the operation of memory for sequential spatial information. An

intralist intrusion error reflected an orienting response in front of an incorrect door that was part of the set of spatial locations within the constant sequence. The results based on 40 trials are shown in figure 4 and indicate

that most of the errors occurred for the middle serial position compared to

the end positions (Kesner and Beers 1988). A more detailed description of

the experimental methods can be found in Kesner and Beers (1988). Thus,

performance of the rats parallels the results achieved with humans, suggesting that measurement of intralist intrusion error in a serial anticipation

26
learning task might represent another example of a potential animal model

of human memory.

FIGURE 3. Mean number of intralist intrusion errors as a function of

serial position in humans

SOURCE: Deese and Kresse 1952, copyright 1952, American Psychological Association.

CODING OF TEMPORAL INFORMATION

In a somewhat different experiment, Estes (1985) has summarized data in

humans demonstrating that order or sequential information is remembered

better with more items (lag) between any two items to be tested for order

memory. In order to test this temporal lag function for spatial location

information, rats were trained on an eight-arm maze as described for the

order-recognition memory task. After extensive training, the rats were

allowed on each trial (one per day) to visit all eight arms in an order that

was randomly selected for that trial. This constituted the study phase. Immediately after the animal had received reinforcement from the last of the

eight arms (i.e., completed the study phase), the test phase began. Only

one test was given for each trial; it consisted of opening two doors

27
FIGURE 4. Mean number of intralist intrusion errors as a function of

serial position in rats

SOURCE: Kesner and Beers 1988, copyright 1988, Academic Press.

simultaneously. On a random basis, lags of zero, one, two, three, four,

five, or six arms were selected for the test. In the case of a lag of zero

arms, the arms followed each other in the study phase; in the case of a lag

of six arms, six items occurred between the two selected arms in the study

phase. The rule to be learned, leading to an additional reinforcement, was

to choose the arm that occurred earlier in the sequence. The animals

received a total of 56 trials, with 8 tests randomly selected for each lag

28
condition. An identical procedure was used for college students, but, in this

case, spatial locations were presented one at a time as X’s on a grid of 16

squares. The study phase consisted of the presentation of X’s in eight different locations. During the test phase, subjects had to select from two X’s

that occurred during the study phase and that varied in terms of lag. There

were 56 trials, with 8 tests randomly selected for each lag condition. The

results are shown in figure 5 and indicate that both rats and college students

perform poorly for lag of zero, but perform well for lags of one to six.

College students, however, continue to improve with longer lags, whereas

rats do not show such an improvement. Again, there appear to be comparable memory functions for rats and humans.

FIGURE 5. Mean percent correct as a function of lag between occurrences of items selected for the test phase in college

students and rats

REPETITION LAG FUNCTION

In a final experiment emphasizing the importance of temporal coding of

information, it has been shown that memory for a repetition or frequency of

occurrence of specific items is a function of lag (number of items) between

29
repetitions. The more items between a repetition or the greater the lag, the

better the memory for the repetition. Results of such an experiment conducted with humans are shown in figure 6 (Madigan 1969). It is clear that

there is a monotonic increasing-repetition-lag function.

FIGURE 6. Recall probability as a function of repetition and the lag

between occurrences of repeated items in humans

NOTE: 1P is one presentation.

SOURCE: Madigan 1969. Copyright 1969, Academic Press

In order to test whether rats could show a repetition lag function, rats were

tested for memory for the frequency of occurrence of specific spatial locations. In this task, the animals were tested on an eight-arm radial maze.

On each trial (one per day), each animal was allowed to visit four arms to

receive reinforcement in an order that was randomly selected for that trial.

One of the arms was repeated with a lag of one, two, or three arms in between a repetition. This constituted the study phase. Upon completion of

the study phase, the door in front of the repeated arm and a door in front

of a nonrepeated arm were opened simultaneously. This constituted the test

phase. The rule to be learned, leading to an additional reinforcement, was

to choose the arm that had been repeated in the study phase sequence.

The results after about 60 trials of training are shown in figure 7 and indicate that animals show excellent memory for the repetition with a lag of 3

30
arms between a repetition, but poor performance for a lag of 1 or 2 arms,

even when the data were analyzed for those trials on which the repetition

occurred in the last serial position. This appears to be yet another example

of a comparable memory function in animals and humans.

FIGURE 7. Mean percent correct as a function of repetition and the lag

between occurrences of repeated items in rats

RETROSPECTIVE AND PROSPECTIVE CODING OF

INFORMATION

As a final example of the possibility of comparability of memory functions

between animals and humans, one can point to the recent observation that

rats can use retrospective and prospective memory codes when asked to re31
member items within long lists (Cook et al. 1985). More specifically, on

any one trial, a rat is presented with 2, 4, 6, 8, or 10 items (places) on a

12-arm radial arm maze followed 15 minutes later by 2 win-shift tests comprising choices between a place previously visited and a novel place. The

animal is reinforced for entering the novel spatial location. During learning,

animals showed an increase in errors as the number of places to be remembered increased from two to six, reflecting the use of a retrospective memory code. These animals also showed a decrease in errors as the number of

places to be remembered increased from 6 to 10, reflecting the use of a

prospective memory code.

In an analogous mnemonic task, college students were presented with either

2, 4, 6, 8, 10, 12, or 14 consecutive pages of X’s marked on a grid of 16

squares. On subsequent tests, college students were asked to choose between a novel X and one that had appeared previously. Subjects were

asked to circle the novel X. Based on verbal reports, subjects were divided

into two groups reflecting different memory coding strategies. The results

arc shown in figure 8 and indicate that the subjects reporting a retrospective

coding strategy showed an increase in errors as the list length increased.

The subjects reporting a retrospective and prospective strategy showed an

initial increase in errors for a list length of 2 to 8 items, followed by a

decrease in errors for a list length of 8 to 14 items. This latter error

pattern is seen in rats, with the largest number of errors occurring in the

middle of the list. Even though there are a few differences in procedure

between the rat and human study, a short vs. long delay between study

phase and test phase and the use of a different type of reinforcement, there

are a sufficient number of similarities in the procedure, including the use of

spatial location information and two tests for each study phase, that a comparison between rats and humans can be made. Thus, the similarity in the

pattern of results suggests the possibility that rats indeed utilize both retrospective and prospective codes in remembering long lists of information.

This explanation could account for the excellent ability to remember all spatial locations in a 17-arm maze (Olton et al. 1977). In conclusion, the

present study provides for yet another demonstration that comparable mnemonic functions can be found in animals and humans.

There arc other memory and cognitive phenomena that have been studied in

animals and humans that provide for nearly equivalent functional patterns.

For example, rats, monkeys, and humans display similar functions in memory scanning tasks based on the Sternberg paradigm (Ellis et al. 1984;

Sands and Wright 1981). Pigeons display the use of mental imagery similar

to representational imagery in humans (Neiworth and Rilling 1987; Shepard

and Cooper 1982; Jagacinski et al. 1983). Pigeons and monkeys can, like

humans, learn to categorize items utilizing conceptual schemas (Hermstein

1984; Medin and Dewey 1984).

32
FIGURE 8. Total number of errors as a function of point of interpolation

(list length) for humans using retrospective and

retrospective/prospective codes

SOURCE: Kesner and DeSpain 1988, Copyright 1988, Psychonomic Society, Inc.

NEURAL MEDIATION OF MEMORY FUNCTION

Since the rat can serve as an animal model for a variety of memory functions based on lists containing temporal-spatial information, it should be

possible to compare the performance of animals with specific lesions or

drug treatment with humans that have sustained brain damage or are under

the influence of specific drugs. A couple of examples will suffice. Damage to the medial prefrontal cortex in the rat impairs memory for order

information, as evidenced by a total deficit in the order-recognition memory,

temporal coding, and repetition lag tasks (Kesner and Holbrook 1987;

33
Kesner, in press). Similar order-memory deficits have been found in

humans with frontal lobe damage (Milner 1971; Milner et al. 1985).

In the item-recognition memory task, small lesions of the medial septum or

dorsal hippocampus in rats produce a deficit in the primacy but not recency

component of the serial position curve (Kesner et al. 1988). A similar deficit pattern is seen in humans with damage to the hippocampus and patients

diagnosed as having mild dementia of the Alzheimer’s type (Milner 1978;

Kesner et al. 1989). Large lesions of the medial septum, dorsal hippocampus, or parietal cortex in rats produce a deficit in both the primacy and

recency component of the serial position curve (Kesner et al. 1986;

DiMattia and Kesner 1988). A similar deficit pattern has been observed in

patients diagnosed as having moderate dementia of the Alzheimer’s type

(Kesner et al. 1987). These data demonstrate not only specific neural mediation of memory function but also comparable memory-deficit patterns between brain-damaged humans and lesioned rats.

SUMMARY

The rat’s memory capacity and performance render it an excellent model of

human memory. Rats display serial position, serial anticipation learning,

temporal coding, and repetition lag functions as well as utilization of retrospective and prospective codes that are nearly equivalent to that observed

for humans. Furthermore, based on the above-mentioned memory performance functions, there are comparable memory-deficit patterns between braindamaged rats and humans. Thus, the rat can serve as an excellent animal

model to evaluate the efficacy of pharmacological treatments or brain damage upon memory.

REFERENCES

Atkinson, R.C., and Shiffrin, R.M. Human memory: A proposed system

and its control processes. In: Spence, K.W., and Spence, J.T., eds. The

Psychology of Learning and Motivation: Vol. 2. Advances in Research

and Theory. New York: Academic Press, 1968.

Cook, R.G; Brown, M.G.; and Riley, D.A. Flexible memory processing by

rats: Use of prospective and retrospective information in the radial maze.

J Exp Psychol [Anim Behav] 11:453-469, 1985.

Dale, R.H.I. Similarities between human and animal spatial memory: Item

and order information. Anim Learn Behav 15:293-300, 1987.

Deese, J., and Kresse, F.H. An experimental analysis of errors in rats serial

learning. J Exp Psychol 43:199-202, 1952.

DiMattia, B.V., and Kesner, R.P. Serial position curves in rats: Automatic

vs. effortful information processing. J Exp Psychol [Anim Behav]

10:557-563, 1984.

34
DiMattia, B.V., and Kesner, R.P. The role of the posterior parietal association cortex in the processing of spatial event information. Behav

Neurosci 102:397-403, 1988.

Ebenholtz, S.M. Serial learning and dimensional organization. In: Bower,

G.H., ed. The Psychology of Learning and Motivation. Vol. 5. New

York: Academic Press, 1972.

Ellis, M.E.; Clegg, D.K.; and Kesner, R.P. Exhaustive memory scanning in

Rattus Norvegicus: Recognition for food items. J Comp Psychol 98:194-

200, 1984.

Estes, W.K. Memory for temporal information. In: Michon, J.A., and

Jackson, J.L., eds. Time, Mind and Behavior. Heidelberg: SpringerVerlag, 1985. pp. 112-130.

Hasher, L., and Zacks, R.T. Automatic and effortful processes in memory.

J Exp Psychol [Gen] 108:356-388, 1979.

Herrnstein, R.J. Objects, categories, and discriminative stimuli. In:

Roitblat, H.L.; Bever, T.G.; and Terrace, H.S., eds. Animal Cognition.

Hillsdale, NJ: Erlbaum, 1984. pp. 233-262.

Jagacinski, R.J.; Johnson, W.W.; and Miller, R.A. Quantifying the cognitive

trajectories of extrapolated movements. J Exp Psychol [Hum Percept]

9:43-57, 1983.

Kesner, R.P. Neuronal organization of memory functions in animals and

humans. In: Delacour, J., and Levy, J.C.S., eds. Systems With Learning

and Memory Abilities. Amsterdam: Elsevier, in press.

Kesncr, R.P.; Adelslein, T.; and Crutcher, K.A. Rats with nucleus basalis

magnocellularis lesions mimic mnemonic symptomatology observed in

patients with dementia of the Alzheimer’s type. Behav Neurosci 101:451-

456, 1987.

Kesner, R.P.; Adelstein, T.A.; and Crutcher, K.A. Equivalent spatial location memory deficits in rats with medial septum or hippocampal formation lesions and patients with dementia of the Alzheimer’s type. Brain

and Cogn 9:289-300, 1989.

Kesner, R.P., and Beers, D. Dissociation of data-based (declarative)

memory and expectancy-based (procedural) memory following hippocampal lesions in rats. Behav Neural Biol 50:46-60, 1988.

Kesner, R.P.; Crutcher, K.A.; and Beers, D. Serial position curves for item

(spatial) information: Role of the dorsal hippocampus and medial septum.

Brain Res 454:219-226, 1988.

Kesner, R.P.; Crutcher, K.A.; and Measom, M.O. Medial septal and

nucleus basalis magnocellularis lesions produce order memory deficits in

rats which mimic symptomatology of Alzheimer’s disease. Neurobiol

Aging 7:287-295, 1986.

Kesner, R.P., and DeSpain, M.J. Correspondence between rats and humans

in the utilization of retrospective and prospective codes. Anim Learn

Behav 16(3):299-302, 1988.

Kesner, R.P., and Holbrook, T. Dissociation of item and order spatial

memory in rats following medial prefrontal cortex lesions. Neuropsychologia 25:653-664, 1987.

35
Kesner, R.P.; Measom, M.O.; Forsman, S.L.; and Holbrook, T.H. Serial

position curves in rats: Order memory for episodic spatial events. Anim

Learn Behav 12:378-382, 1984.

Madigan, S.A. Intraserial repetition and coding processes in free recall.

J Verb Learn Verb Behav 8:828-835, 1969.

Medin, D.L., and Dewey, G.I. Learning of ill-defined categories by

monkeys. Can J Psychol 38:285-303, 1984.

Milner, B. Interhemispheric differences in the localization of psychological

processes in man. Brit Med Bull 27:272-277, 1971.

Milner, B. Clues to the cerebral organization of memory. In: Buser, P.A.,

and Rougeul-Buser, A., eds. Cerebral Correlates of Conscious Experience. Amsterdam: Elsevier, 1978.

Milner, B.; Petrides, M.; and Smith, M.L. Frontal lobes and the temporal

organization of memory. Hum Neurobiol 4:137-142, 1985.

Neiworth, J.J., and Rilling, M.E. A method for studying imagery in animals. J Exp Psychol [Anim Behav] 13:203-214, 1987.

Olton, D.S.; Collison, C.; and Werz, M.A. Spatial memory and radial arm

maze performance of rats. Learn Motiv 8:289-314, 1977.

Sands, S.F., and Wright, A.A. Serial probe recognition performance by a

rhesus monkey and a human with 10- and 20-item lists. J Exp, Psychol

[Anim Behav] 6:386-396, 1980.

Sands, S.F., and Wright, A.A. Monkey and human pictorial memory scanning. Science 216:1333-1334, 1981.

Shepard, R.N., and Cooper, L.A. Mental Images and Their Transformations. Cambridge, MA: MIT Press, 1982.

Shiffrin, R.M., and Schneider, W. Controlled and automatic human

information processing: II. Perceptual learning, automatic attending and

a general theory. Psychol Rev 84:127-190, 1977.

Wright, A.A.; Santiago, H.C.; Sands, S.F.; Kendrick, D.F.; and Cook, R.G.

Memory processing of serial lists by pigeons, monkeys, and people.

Science 229:287-289, 1985.

AUTHOR

Raymond P. Kesner, Ph.D.

Department of Psychology

University of Utah

Social and Behavioral Science, Room 502

Salt Lake City, UT 84112

36
Long-Term Effects of Cholinergic

Agonists on Memory

David S. Olton and Kathleen C. Raffaele

INTRODUCTION

Long-term exposure to drugs and other chemical compounds can produce

significant and enduring changes in neurotransmitter systems. Of particular

interest to the current project is the decrease in the number of receptors following prolonged exposure to a neurotransmitter agonist. The agonist produces excessive stimulation of postsynaptic receptors, which decrease in

number, a phenomenon called “downregulation of receptors.” This downregulation decreases the sensitivity of the postsynaptic neuron to endogenously released neurotransmitters, which should in turn produce a decrease

in the output of the postsynaptic neuron for any given input that stimulates

those receptors. This decrease in the “gain” of the synaptic receptor

mechanism might be expected to produce substantial functional impairment.

However, the brain is a very plastic organ and maybe able to compensate

for at least some alterations in a neurotransmitter system. Long-term exposure to a drug provides ample opportunity for both neural and behavioral

compensation. Indeed, some recovery of function is the expected outcome

of brain damage (Finger and Stein 1982). If excessive stimulation by a

neurotransmitter agonist is viewed as abnormal brain activity, then some

kind of compensation is expected.

In a relatively benign environment, one that does not challenge the organism excessively, the impairments expected from decreased synaptic gain

may be fully compensated by recovery of function so that no behavioral

impairment follows. However, the neural system may still have a significant latent impairment, one that can be made manifest by challenging the

system in some way. One obvious challenge is to give an antagonist for

the neurotransmitter system in question. The downregulation of receptors

should produce increased sensitivity to the antagonist, resulting in a leftward

shift of the dose-response curve, as compared to the normal condition (one

in which no exposure to the drug has taken place prior to testing with the

antagonist).

37
In the context of learning and memory, the cholinergic neurotransmitter

system is an obvious one in which to carry out this manipulation (Bartus

et al. 1985; Davies 1985; Pontecorvo et al. 1985). Long-term exposure to a

cholinergic agonist should result in excessive stimulation of the postsynaptic

neurons, downregulation of the postsynaptic receptors, increased sensitivity

to an anticholinergic drug such as scopolamine, and a leftward shift of the

dose-response curve, so that a given dose of scopolamine causes a greater

impairment in tasks requiring memory.

The experiments summarized here tested this hypothesis (Raffaele et al., in

press). The chronic treatment used diisopropylfluorophosphate (DFP), a

compound that irreversibly inhibits acetylcholinesterase (AChE), the enzyme

that breaks down acetylcholine. Chronic treatment with DFP produces

downregulation of muscarinic receptors (Lim et al. 1986; Sivam et al. 1983;

Yamada et al. 1983). Scopolamine, a cholinergic antagonist, produces significant impairments in many different tests of memory (Bartus et al. 1985;

Pontecorvo et al. 1985). Consequently, following chronic exposure to DFP,

rats given scopolamine should show a leftward shift of the dose-response

curve.

SUCCESSIVE REVERSALS

Successive reversals of a two-choice simultaneous discrimination (TCSD)

were conducted in an apparatus that had a runway connected to a goal platform with two interchangeable goal boxes. One goal box had black and

white stripes, the other had black spots on a white background.

Each rat was first shaped in the usual testing procedures to obtain reinforcement, which was chocolate milk placed in a cup at the far end of each goal

box. Next, each rat was given a TCSD. Each day’s testing had two parts.

The first part was retention. During retention, the goal box that was correct

at the end of the previous day remained correct. If the rat met any one of

several different criteria, indicating accurate memory of the previous day’s

discrimination, the second part, reversal, was begun. For the discrimination

reversal. the previously correct goal box was incorrect, and the previously

incorrect goal box was correct. Testing continued until the rat reached

criterion in the reversed discrimination or a total of 30 trials had been

given. This procedure was continued for each rat until stable performance

was reached, using an intertrial interval of 1 minute.

The cholinergic antagonist, scopolamine, was used to produce an impairment

of choice accuracy in this task, Scopolamine, at doses of .15, .35, or

.75 mg/kg, was given at least twice prior to treatment with DFP to establish

a pretreatment baseline; at least four times during treatment with DFP to

determine the effects of this treatment on the sensitivity to scopolamine;

and at least twice following treatment with DFP to determine the recovery

from the DFP treatment. Scopolamine was given once every 6 days.

38
Methylscopolamine or saline was given on the third day following the scopolamine treatment. No acute treatment injections were given during the

intervening days.

DFP was given in two doses. For the first day of DFP treatment, the dose

was 1.0 mg/kg. For all subsequent days of DFP treatment, the dose was

0.5 mg/kg. DFP was given after behavioral testing every third day. Consequently, in a block of 6 days, the drug treatment procedure looked like that

outlined in table 1.

TABLE 1. Summary of experimental procedures

Days Chronic Treatment Acute Treatment

1 DFP or Saline None

2 None None

3 None Methylscopolamine or Saline

4 DFP or Saline None

5 None None

6 None Scopolamine or Saline

NOTE: The chronic treatment was given after the day’s behavioral testing. The acute challenge was

given 20 minutes prior to the day’s behavioral testing.

The use of a between-subjects design produced eight groups, as outlined in

table 2. These groups are identified by the abbreviations SAL-O, SAL-15,

SAL-35, SAL-75, DFP-0, DFP-15, DFP-35, and DFP-75. The first half of

each abbreviation indicates the chronic treatment (SAL or DFP), the second

half indicates dose of scopolamine (0, .15, .35, and .75 mg/kg (table 2)).

TABLE 2. Summary of the experimental groups

Chronic Treatment

Saline

DFP

Acute Challenge: mg/kg Scopolamine

0 .15 .35 .75

SAL-0 SAL-15 SAL-35 SAL-75

DFP-0 DFP-15 DFP-35 DFP-75

At the end of behavioral testing, each rat was sacrificed. Total muscarinic

receptor binding was measured by modifications of [

3H]-QNB binding

(Yamamura and Snyder 1974). M1 muscarinic receptor binding was

39
measured by modifications of [

3H]-pirenzepine binding (Watson et al. 1984).

AChE activity was determined spectrophotometrically (Ellman et al. 1961).

Each rat learned the discrimination rapidly, and the mean number of errors

decreased with continued behavioral testing. Prior to the first injection of

scopolamine, the mean number of errors to criterion was 2.4 for retention

and 5.2 for reversal.

Scopolamine produced a slight increase in the number of errors, with

.75 mg/kg producing the greatest increase. However, this difference was

not statistically significant. Methylscopolamine and saline had no effect on

choice accuracy. A similar pattern of performance occurred during reversal.

DFP itself produced no significant change in behavior. The DFP-0 group

performed similarly to the SAL-0 group. Thus, the chronic treatment itself

did not produce a significant change in behavior.

However, the chronic treatment did produce a marked increase in sensitivity

to scopolamine, especially at .75 mg/kg (figure 1). During retention, the

control group (SAL-75) had a mean of five errors or less. In contrast, the

DFP-75 group had a mean of seven errors or more. As can be seen in

figure 1, this increase in the number of errors was consistent throughout

testing during DFP treatment. Furthermore, it continued to the posttreatment days, after DFP treatment was stopped. The magnitude of the effect

showed some signs of decreasing during this posttreatment phase and might

have eventually disappeared if testing was continued. However, it clearly

persisted for at least 2 weeks after DFP was stopped.

A similar pattern of results occurred during the reversal of the discrimination; the DFP-75 group was more sensitive to scopolamine than was the

saline group. The effects at other doses (.15 and .35 mg/kg scopolamine)

were not as large. The lowest dose (.15 mg/kg) produced no differential

effect. The intermediate dose (.35 mg/kg) produced a slight increase in

errors in rats receiving chronic DFP, but the magnitude was significant for

only one block of tests.

The neurochemical assessments indicated a decrease in cholinergic receptors

in the cortex of rats given DFP. On the second day following the last DFP

treatment, muscarinic receptor concentration was reduced by 22 percent, M1

receptor concentration was reduced by 18 percent (not a significant difference), and AChE activity was reduced by 60 percent.

DELAYED MATCH-TO-SAMPLE

An additional experiment tested performance in delayed match-to-sample.

The apparatus was a rectangular tank filled with water. The start area was

located at one end. Two choice areas were located at the other end. In

each choice area, there was a goal box. Each goal box had a distinctive

4 0
FIGURE 1. Response of rats to a scopolamine challenge

NOTE: Rats given a chronic treatment of DFP show an enhanced response to an acute challenge with

.75 mg/kg scopolamine. The data are the number of errors made during retention testing

when the acute challenge was given before the treatment with DFP (“pre”), during the treatment with DFP (1 and 2, each two injections of scopolamine), and after the treatment with

DFP had ended (“post”). As indicated in the bottom right graph, .75 mg/kg scopolamine produced a significant increase in the number of errors for all injections during treatment with

DFP and for the two injections after DFP was stopped.

pattern, similar to the ones used in the preceding experiment. One goal had

vertical black and white stripes, the other had large black spots against a

white background. In the correct goal box, a platform was hidden under

the water, allowing the rat to escape. In the incorrect goal box, no platform was available, so the rat could not escape.

Each trial began with a forced swim, during which the rat was forced to go

to the goal box containing the platform. After reaching the goal box, the

rat climbed onto the platform and escaped from the water. For the subsequent choice swim, the rat was allowed to go to either goal box. However,

only the goal box entered during the immediately preceding forced swim

had the platform. Consequently, the correct response for the rat was to

return to the goal entered during the immediately preceding forced swim.

Because the preceding experiment had established a dose-response curve and

the major goal of the present experiment was to test the generality of the

interaction between chronic exposure to DFP and acute exposure to scopolamine, only a single dose of scopolamine (3.0 mg/kg) was used in the

41
present experiment. One group of rats received chronic treatment with

DFP, the other did not. All other aspects of the procedure were similar to

those described for the previous experiment.

DFP produced a similar increase in sensitivity to scopolamine. Rats receiving saline had a mean of 68 percent correct during scopolamine test days,

rats receiving DFP had a mean of only 51 percent correct.

These results consistently demonstrate a significant increase in sensitivity to

a cholinergic antagonist as a result of chronic treatment with DFP, a compound that inhibits AChE, which normally breaks down acetylcholine. DFP

produced a downregulation of cholinergic receptors, which in turn produced

an increased sensitivity to scopolamine in two different tasks. Consequently

the observed phenomenon is probably a general one and may occur in many

different situations.

ANIMAL MODELS

Benefits and Risk

Animal models, like all models, have both benefits and risks. The benefits

include the opportunity to manipulate and measure parameters that cannot

be examined with people. Indeed, the very term “abuse” implies an inappropriate use of drugs, one that should not be undertaken voluntarily by a

person, or imposed by one person upon another person. In addition, animals provide the opportunity for important and necessary controls, because

all aspects of the experiment can be controlled, including the genetic characteristics, developmental experience, drug exposure, etc.

Animal models also provide important opportunities for carrying out measurements that cannot be conducted with people. Ultimately, drugs interact

with elements in the brain, and these neural events are the critical links

between the drug and behavior. Although considerable information can be

obtained from purely psychological and behavioral analyses, these analyses

arc ultimately limited by their inability to examine directly each event that

intervenes between the stimulus and the response (the behavioral language)

or each of the cognitive modules lying between the input and the output of

the system (the cognitive/computational language). Although a variety of

strategies have been developed to try to assess the intervening steps in the

chain, such steps are fundamentally limited in their ability to access the

details of the mechanisms interrelating stimuli and responses.

In contrast, neurobehavioral analyses in animal models can examine directly

each element in the chain via manipulation or measurements. An electrode

or a cannula can be placed at every point along the pathway between the

receptors and effectors, allowing each step to be described completely.

42
Animal models, like all models, have the risk that the phenomena observed

in the model do not generalize to the situation of primary interest, namely

human drug abuse. Awareness of these risks has led to considerable effort

to create procedures to minimize them. Especially in the analysis of animal

models in memory, substantial progress has been made in two directions.

First, criteria to evaluate the validity of any given model have been identified. Second, individual models have used these criteria to design procedures that are highly valid (Ingram 1985; Kesner 1985; Olton 1985; Squire

and Zola-Morgan 1985). These points are illustrated by some animal

models that examine recent memory. Further examples are provided by

Kesner (this volume).

Recent memory, as described earlier, is the memory for information presented several minutes to hours previously. In everyday life, it is involved in

answering questions such as: What did you have for breakfast? When did

you last see your brother? Where did you park your car? In the laboratory, it is usually assessed by giving a person some information to be remembered, such as a list of words, set of pictures, or information in a short

story. After a delay interval, during which the person is engaged in other

kinds of tasks, retrieval of the information presented earlier is assessed.

Some of the dimensions used for assessing validity and a specific example

of a manipulation using that dimension are given in table 3. The discussion

below comments briefly on each of these.

TABLE 3. Types of validity and examples from tests of recent memory

Type of Validity

Operational

Example

Number of items to be remembered

Length of time each item is to be remembered

Psychological Interference: proactive, retroactive

Serial order effects

Ethological Optimal foraging in natural environment

Neuroanatomical Hippocampus

Neurotransmitter Cholinergic system

Validity Assessment

Operational validity describes the specific parameters manipulated in an

experiment. These parameters include the amount of information to be

43
remembered and the delay interval during which the information is to be

remembered. As might be expected, choice accuracy decreases as the

amount of information and the delay interval increase.

Psychological validity describes the psychological/cognitive/computational

processes that mediate performance in the memory tasks. Proactive interference refers to processes that result in negative transfer, so that previously

leamed information interferes with currently learned information. Retroactive interference refers to processes that underlie another form of negative

transfer, the fact that currently learned information can interfere with previously learned information.

Ethological validity reflects considerations of optimal foraging and ethological analyses of animal behavior. The issue is whether the kinds of problems given to animals in the laboratory are so odd that they distort the

normal behavior of the animal. Examination of the kinds of problems faced

by animals in their natural habitat has consistently shown that many animals

are faced with situations in which recent memory is required. The ability

to solve these problems influences the ability of the animal to survive, and

different species of animals have developed highly sophisticated foraging

strategies to solve these problems (Sherry and Schacter 1987).

Neurological validity refers to the brain mechanisms involved in recent

memory. Considerable evidence implicates the hippocampus and associated

temporal lobe structures in the circuit for recent memory.

Neurotransmitter validity refers to the transmitter substances involved in

recent memory. As mentioned earlier, the cholinergic system is implicated

as one of the important neurotransmitters required for normal recent

memory (Bartus et al. 1985). Kesner (this volume) provides numerous

examples of the similarity in operational manipulation and measurement and

psychological/cognitive/computational description. The other references in

this chapter provide documentation for ethological, neuroanatomical, and

neurotransmitter validity.

A large number of experimental procedures and conceptual frameworks are

available to bring to the study of drug abuse. Obviously, some alterations

will have to be made to address the issues important in drug abuse. However, the fact that these analyses of animal models have been undertaken so

extensively in other situations means that much of the preliminary work has

already been completed, and the additions to address issues in drug abuse

can proceed quickly and effectively. Finally, of course, the behaviors in

these models have the advantage of direct similarity to many of the

behaviors of interest in humans. Thus, the results obtained from these

models should be more readily applicable to the human situation than the

results from models that use behaviors for which the human analog is less

44
clear, and for which the issues of validity have not yet been as carefully

considered.

SUMMARY

Animal models provide important information about strategies that can be

used to assess the effects of chronic exposure to drugs or other compounds.

Futhermore, when used in conjunction with analyses designed to utilize the

many advantages of animal models to examine learning and memory, these

kinds of experiments can have significant ramifications for assessing the

mnemonic effects of chronic drug use in people. Finally, given that aging

is accompanied by significant deterioration of neurochemical systems, which

may compromise the functions of those systems, the results from the experiments with DFP predict that changes in learning and memory abilities that

are not apparent in young individuals may begin to surface as those individuals age or may appear following any other kind of insult to their nervous

system.

REFERENCES

Bartus, R.T.; Dean, R.L.; Pontecorvo, M.J.; and Flicker, C. The cholinergic

hypothesis: A historical overview, current perspective and future directions. In: Olton, D.S.; Gamzu, E.; and Corkin, S., eds. Memory

Dysfunctions: An Integration of Animal and Human Research from

Preclinical and Clinical Perspectives. Vol. 444. New York: New York

Academy of Sciences, 1985. pp. 332-358.

Davies, P. A critical review of the role of the cholinergic system in human

memory and cognition. In: Olton, D.S.; Gamzu, E.; and Corkin, S., eds.

Memory Dysfunctions: An Integration of Animal and Human Research

from Preclinical and Clinical Perspectives. Vol. 444. New York: New

York Academy of Sciences, 1985. pp. 212-217.

Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; and Featherstone, R.M. A

new and rapid calorimetric determination of acetylcholinesterase activity.

Biochem Pharmacol 7:88-95, 1961.

Finger, S., and Stein, D.G. Brain Damage and Recovery. New York:

Academic Press, 1982.

Ingram, D.K. Analysis of age-related impairments in learning and memory

in rodent models. In: Olton, D.S.; Gamzu, E.; and Corkin, S., eds.

Memory Dysjiictions: An Integration of Animal and Human Research

from Preclinical and Clinical Perspectives. Vol. 444. New York: New

York Academy of Sciences, 1985. pp. 312-331.

Kesner, R.P. Correspondence between humans and animals in coding of

temporate attributes: Role of hippocampus and prefrontal cortex. In:

Olton, D.S.; Gamzu, E.; and Corkin, S., eds. Memory Dysfunctions: An

Integration of Animal and Human Research from Preclinical and Clinical

Perspectives. Vol. 444. New York: New York Academy of Sciences,

1985. pp. 122-136.

45
Lim, D.K.; Hoskins. B.; and Ho, I.K. Correlation of muscarinic receptor

density and acetylcholinesterase activity in repeated DFP-treated rats after

the termination of DFP administration. Eur Pharmacol 123:223-228,

1986.

Olton, D.S. Strategies for the development of animal models of human

memory impairments. In: Olton, D.S.; Gamzu, E.; and Corkin, S., eds.

Memory Dysfunctions: An Integration of Animal and Human Research

from Preclinical and Clinical Perspectives. Vol. 444. New York: New

York Academy of Sciences, 1985. pp. 113-121.

Pontecorvo, M.; Spencer, Jr., D.G.; Heise, G.A. Central cholinergic

involvement in working memory: Effects of scopolamine on continuous

nonmatching and discrimination performance in the rat. Behav Neurosci

99:1049-1065, 1985.

Raffaele, K.; Olton, D.; and Annau, Z. Repeated exposure to diisopropylflurophosphate (DFP) produces increased sensitivity to cholinergic antagonists in discrimination retention and reversal. Psychopharmacology, in

press.

Sherry. D.F., and Schacter, D.L. The evolution of multiple memory systems. Psycho1 Rev 94:439-454, 1987.

Sivam, S.P.; Norris, J.C.; Lim, D.K.; Hoskins, B.; and Ho, I.K. Effect of

acute and chronic cholinesterase inhibition with diisopropylfluorophosphate

on muscarinic, dopamine, and GABA receptors of the rat striatum.

Neurochem 40:1414-1422, 1983.

Squire, L.R., and Zola-Morgan, S. The neuropsychology of memory: New

links between humans and experimental animals. In: Olton, D.S.;

Gamzu, E.; and Corkin, S., eds. Memory Dysfunctions: An Integration

of Animal and Human Research from Preclinical and Clinical

Perspectives. Vol. 444. New York: New York Academy of Sciences,

198.5. pp. 137-149.

Watson, M.; Roeske, W.R.; Johnson, P.C.; and Yamamura, H.I. [

3H]Pirenzepine identifies putative M1

 muscarinic receptors in human stellate

ganglia. Brain Res 290:179-182, 1984.

Yamada, S.; Isogai, M.; Okudaira, H.; and Hayashi, E. Regional adaptation

of muscarinic receptors and choline uptake in brain following repeated

administration of diisopropylfluorophosphate and atropine. Brain Res

268:315-320. 1983.

Yamamura, H.I., and Snyder, S.H. Muscarinic cholinergic binding in rat

brain. Proc Natl Acad Sci USA 71:1725-1729, 1974.

AUTHORS

David S. Olton, Ph.D.

Professor

Department of Psychology

The Johns Hopkins University

Baltimore, MD 21218

46
Kathleen C. Raffaele, Ph.D.

Staff Fellow

Laboratory of Neurosciences

National Institute on Aging

National Institutes of Health

Bethesda, MD 20892

47
Enkephalin Influences on

Behavioral and Neural Plasticity:

Mechanisms of Action

Joe L. Martinez, Jr., Patricia H. Janak,

Susan B. Weinberger, Gery Schulteis, and

Brian E. Derrick

INTRODUCTION

The anatomical basis of memory storage must involve the functional connections of neurons. The concept of circuits of neurons forming the neural

substrate of memory was clearly elaborated by Hebb (1949) in his postulation of a dual trace mechanism. In this conception, memory was first encoded as a reverberating cellular assembly of neurons. The activity of this

assembly would decrease over time unless exercised by repetition; this assembly formed the basis of short-term memory. Through exercise, this

assembly would become a functionally intact system through the growth of

synaptic knobs and would become long-term memory (Hebb 1949). Although it is easy to imagine a cellular assembly forming the neural substrate

of a memory, this cellular assembly is not of much use on its own. It

must be functionally connected to other nervous system components; for instance, an effector system is necessary. In addition, there also must be an

input from a sensory or other modality that regulates in some way the activity of the circuit and, hence, determines its strength. Conceptualized in

this way, this modulatory input can be recognized as an integral part of the

memory storage process.

In this view of learning, nervous system changes most certainly involve specific neural sites of information storage as well as those modulatory inputs

that serve to mark the importance of the event to the organism. This chapter will examine the role of enkephalins in both of these processes, beginning with a consideration of [leu]enkephalin (LE) as a component of a

modulatory input system and concluding with a discussion of enkephalin

influences on neural plasticity in the hippocampus.

ENKEPHALINS AND BEHAVIORAL PLASTICITY

Kety suggested in 1970 that there might be modulatory inputs to the memory trace; that is, an input might act to make memory traces stronger. Of

48
course, if this modulatory input can make memory traces stronger, then it is

possible that this input can make memory traces weaker as well (Martinez

1986). Although the memory trace itself is probably located within the central nervous system (CNS), the inputs that modulate the activity of this trace

may originate from locations more widely distributed throughout the body.

Extensive evidence supports the role of some hormonal systems as modulators of learning and memory, such that administration of low doses of a

hormone may increase or decrease the strength of training. These hormones

may therefore play a role in normal learning by providing the modulatory

input that influences the strength of a memory trace.

An examination of the opioid peptide, LE, in the periphery suggests that it

may be a stress hormone as well as a learning modulatory hormone

(McGaugh and Martinez 1981; Martinez et al. 1988b). For example, the

enkephalins are coreleased with the adrenal medullary hormones epinephrine

and norepinephrine (Chaminade et al. 1984; Corder et al. 1982; Hanbauer

et al. 1982; Viveros et al. 1980); in addition, enkephalins are localized with

norepinephrine in sympathetic nerve terminals (Klein et al. 1982). Conditioning studies in animals suggest that LE has a modulatory influence on

the memory circuit, and that this modulatory influence originates, at least in

part, outside the blood-brain barrier (BBB).

Peripherally Administered LE Alters Conditioning

LE influences the acquisition or retention of a variety of conditioned

responses in a number of species (deWied et al. 1978; Yamamoto et al.

1982; Rigter 1978; Izquierdo and Dias 1981; Martinez and Rigter 1982;

Martinez et al. 1981; Rigter et al. 1980a; Rigter et al. 1980b; Rigter et al.

1981; Martinez et al. 1984; Martinez et al. 1988a). For example, figure 1,

panel A, shows the impairing effect of peripheral administration of LE administered prior to the acquisition of a one-way active avoidance response

in mice (Schulteis et al. 1988). In these studies, mice are given 10 seconds

to avoid delivery of a footshock by crossing to a safe chamber. The number of avoidances an animal makes during 14 training trials is taken as a

measure of acquisition performance.

An examination of these same animals 1 day after training suggests that LE

affects learning rather than performance variables. As seen in figure 1,

panel B, the animals showing impaired acquisition on the first day of

training still exhibited impaired performance in the same paradigm 24 hours

later. Thus, the interaction of the peptide treatment and the training

experience produced an as-yet-unidentified enduring effect that outlasts the

residence time in the body of the exogenously administered LE.

LE produced a U-shaped dose-response function (figure 1) in this study, as

it does in most studies; that is, doses lower and higher than the behaviorally

active dose had no effect. This type of function is commonly seen in

49
FIGURE 1. LE dose-response functions

*p<.01

**p<.0001

NOTE: A LE impairs acquisition of a one-way active avoidance task in mice at doses of 30 and

100 μg/kg. Saline control group. n=26; all LE groups, n=10 to 12. B. These same doses

(30 and 100 μg/kg.) result in poor retention when animals are given a one-trial retention test

24 hours after injection and training.

SOURCE: Adapted from Schulteis et al. 1988, copyright 1988, American Psychological Association.

studies examining the effects of hormone administration on conditioning and

is consistent with the proposed modulatory influence of LE on the actual

memory trace (see Martinez 1986 and Koob 1987 for further discussion of

U-shaped curves and learning and memory).

In addition to mice, enkephalin effects on conditioning are seen in three

strains of rats (Rigter et al. 1980a; Rigter et al. 1981; Martinez et al.

1985a), in chicks (Patterson et al. 1989), and in primates (Olson et al. 1979;

Olson et al. 1981). Therefore, LE conditioning effects exhibit considerable

generality across species.

LE’s conditioning effects also exhibit generality across tasks. For example,

LE impairs Y-maze conditioning in mice. In a discriminated Y-maze

shock-escape task, mice receiving a pretraining injection of 100 μg/kg. LE

made significantly more errors than saline-treated control animals (Martinez

50
et al. 1984). Likewise, in an appetitively motivated Y-maze discrimination

task, posttraining injections of LE (300 μg/kg) impaired retention of the

maze response. The impairing effect of LE has also been observed by

others in a nonshock-motivated, nonassociative-learning situation in which

LE (10 μg/kg IP) injected into rats following habituation training impairs

later performance (Izquierdo et al. 1980). However, it is important to note

that LE has been reported to produce enhancement of learning and memory

as well (Rigter et al. 1980b).

These data suggest that LE’s conditioning effects may be important to

learning in general, since, in addition to affecting conditioning in many

species, LE alters conditioning in both aversive and nonaversive tasks.

Control Studies That Strengthen the Interpretation That LE Affects

Learning

All of the conditioning studies described thus far involve multiple-trial tasks

during which the experimenter must handle the animal multiple times. Plasma levels of stress hormones such as corticosterone (Ader et al. 1967;

Dobrakovova and Jurcovicova 1984) and catecholamines (McCarty and Gold

1981) are elevated in rodents when they are handled. We therefore studied

the effects of LE on acquisition of an automated shelf-jump active avoidance response in rats. Handling of the animals in this paradigm occurs

only during drug administration and initial placement of the animal into the

testing chamber. As in the standard active avoidance paradigm, LE (1 and

3 μg/kg) impairs acquisition of the automated shelf-jump avoidance response

(Weinberger et al., in press; Weinberger et al. 1988). Experimenter handling, therefore, probably does not contribute significantly to the effect of

LE on behavior in our experiments.

A number of additional control studies strengthen the suggestion that LE

affects learning. Since enkephalins are opioid agonists, impaired performance could be due to analgesia seen when they are administered before

training in a shock-motivated situation. However, since LE also impairs

conditioning in the appetitively motivated Y-maze task, this is unlikely.

More direct evidence is supplied by experiments in which the effect of LE

on shock sensitivity was examined. LE, at doses up to 10 times the

avoidance-impairing doses, injected IP immediately before testing of flinchjump thresholds, did not produce evidence of analgesia in rats (Rigter et al.

1980b) or mice (Schulteis et al. 1988). Thus, changes in shock sensitivity

cannot explain the avoidance-impairing effects of LE.

Since we commonly administer the peptide before training, it is also important to examine possible effects of LE on locomotor activity. In mice,

100 μg/kg LE usually does not alter shock-induced locomotor activity

(Martinez et al. 1984; Schulteis et al. 1988; Weinberger and Martinez 1988)

although, in one study, 100 μg/kg LE was found to enhance locomotion

5 1
(Weinberger and Martinez 1988). In addition, 10 μg/kg LE can enhance

shock-induced locomotor activity in rats (Martinez et al. 1985). These findings raise the possibility that motor activity changes could contribute to the

avoidance conditioning impairment seen after LE administration. However,

an increase in locomotor activity is difficult to reconcile with an overall

decrease in the number of avoidances seen in the LE-treated animals.

In summary, the above data suggest that LE can alter the strength of a

memory trace. This effect is seen in many species and tasks, suggesting

that LE may play an important role in learning in general. How might LE

act to modulate learning? The following discussion examines the possible

mechanisms involved in the effects of LE on conditioning.

Delta Receptors Mediate the Effects of LE on Conditioning

LE exhibits greater affinity for delta than for mu opioid receptors; LE’s

affinity for kappa receptors is negligible (Kosterlitz and Hughes 1978;

Kosterlitz and Paterson 1985; Paterson et al. 1983). Thus, it is reasonable

to hypothesize that the effects of LE on avoidance conditioning are mediated by delta receptors. Indirect support for this hypothesis comes from the

finding that a number of opioid peptides and opioid analogs with affinity

for mu receptors do not impair acquisition of the one-way active avoidance

response when injected peripherally over a wide dose range. These peptides

include [d-ala2-met]enkephalin and [d-met2,pro

5

]enkephalinamide; betaLPH62-65 and beta-LPH62-60; alpha, gamma, and beta endorphin (Rigter et al.

1981); and Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (Schulteis and Martinez

1988).

More direct evidence for the involvement of delta receptors comes from

recent studies employing delta-selective agonists and antagonists. As can be

seen in figure 2, peripheral injections of [D-Pen2,D-Pen5]enkephalin

(DPDPE), an enkephalin analog with very high selectivity for delta receptors

(Hruby 1986; Mosberg et al. 1983; Corbett et al. 1984; Cotton et al. 1985),

impair acquisition of one-way active avoidance conditioning in mice at onetenth the maximally impairing dose of LE (Schulteis et al. 1988). The

greater potency of DPDPE in this study is most likely due to its resistance

to the peptidases that rapidly degrade LE and [met]enkephalin (ME)

(Corbett et al. 1984; Weinberger and Martinez 1988).

DPDPE also impairs acquisition of an automated shelf-jump avoidance task

in male Sprague-Dawley rats (Weinberger et al., in press; Weinberger et al.

1988). Furthermore, chicks receiving DPDPE prior to training on a tasteavoidance task show amnesia when tested 24 hours later (Patterson et al.

1989), although these effects are seen after intracerebral, rather than IP,

injection. Like LE, DPDPE produces a U-shaped dose-response curve in all

three tasks and species. Evidence to date, therefore, indicates that delta

receptor activation can impair conditioning in several tasks and species.

52
FIGURE 2. DPDPE dose-response function

*p<.001

NOTE: DPDPF, a selective delta receptor agonist. impairs acquisition of one-way active avoidance

tasks in mice at 11.6 μg/kg. a dose equimolar to 10 μg/kg. of LE; thus DPDPE is more potent

than LE in impairing learning (figure 1). Saline control group, n=24; all DPDPE groups,

n=10 to 12.

SOURCE: Adapted fmm Schulteis et al. 1988, copyright 1988, American Psychological Association.

Consistent with the above data, ICI 154,129 (Schulteis et al. 1988) and ICI

174,864 (Schulteis and Martinez 1988) two delta-selective antagonists

(Cotton et al. 1985; Gormley et al. 1982; Shaw et al. 1982), enhance oneway active avoidance conditioning in mice when injected systemically shortly before training (see figure 3). ICI 174,864 enhances learning at a lower

dose than ICI 154,129. a finding that is consistent with the in vitro bioassay

potencies of these two compounds (Cotton et al. 1985). Thus, activation of

delta opioid receptors by selective agonists impairs learning in a manner

similar to that of LE, whereas selective blockade of delta receptors has the

opposite effect.

53
FIGURE 3. ICI 174,864 dose-response functions

*p<.02

NOTE: ICI 174,864. a selective delta receptor antagonist, enhances acquisition of one-way active

avoidance tasks in mice at 1.0 mg/kg. Thus, blocking delta receptors produces an effect on

learning opposite to that seen with stimulation of delta receptors using agonists (figures 1

and 2). Saline control group, n=17; all ICI 174,864 groups. n=15 to 19.

SOURCE: Developed from data presented in Schulteis and Martinez 1988

Perhaps the most compelling evidence that delta receptors mediate LE’s

conditioning effects comes from recent studies in which the effects of LE

54
on avoidance responding in mice were reversed in a dose-dependent manner

by the concurrent administration of ICI 174,864 (Schulteis and Martinez

1988). A comparison of figures 3 and 4 reveals that a 1 mg/kg dose of

ICI 174,864 that completely reverses the impairment produced by LE is

without effect when administered by itself. This suggests that the reversal

of LE’s effect is due to a competitive receptor antagonism. The findings in

mice are supported by studies in chicks, in which the amnesia for tasteavoidance training produced by LE is blocked by concurrent administration

of ICI 174,864 (Patterson et al. 1989). Taken together, these studies provide strong evidence that the effects of LE on avoidance conditioning

involve delta receptor activation.

FIGURE 4. Reversal of LE effects by ICI 174,864

NOTE: The effects of LE (100 μg/kg) on one-way active avoidance acquisition in mice are attenuated

by concurrent administration of a 0.10 mg/kg dose of ICI 174,864 and are completely reversed by a 1.0 mg/kg dose of ICI 174,864. All possible pairwise comparisons were made

using a Tukey test, and the results are summarized in the legend above the histogram. Saline

control group, n=16; all other groups, n=14 to 16.

SOURCE: Developed from data presented in Schulteis and Martinez 1988

55
LE Affects Conditioning Through a Site Outside the BBB

As the above evidence indicates, either peripheral or central administration

of LE affects conditioning. This suggests that both central and peripheral

opioid systems influence learning. The discussion below will focus on

peripheral mechanisms of action following IP administration of LE.

Enkephalins affect avoidance conditioning when administered peripherally in

microgram doses. When given in these amounts, it is unlikely that LE

crosses the BBB in sufficient quantities to be of physiological significance.

Studies measuring penetration of LE into the brain using the brain uptake

index (BUI) failed lo reveal significant entry of LE into the CNS (Pardridge

and Mietus 1981; Cornford et al. 1978; Zlokovic et al. 1985). Furthermore,

LE is rapidly hydrolyzed by aminopeptidases present on the walls of brain

microvessels (Pardridge and Mietus 1981; Zlokovic et al. 1985), and any

radioactivity found in the brain following peripheral administration of

labeled LE is almost entirely in the form of free tyrosine. The preponderance of evidence to date suggests that LE crosses the BBB poorly, if at all,

and, therefore, effects on conditioning seen following peripheral administration of LE likely occur at a site outside the BBB (for other interpretations

see Kastin et al. 1976; Zlokovic et al. 1987).

A number of behavioral pharmacology studies support the notion that LE

acts to impair conditioning at a site or sites outside the BBB. Intracerebroventricular ((ICV) administration of LE fails to alter passive-avoidance retention (Belluzzi and Stein 1981), active avoidance learning (Martinez et al.

1985a), and extinction of a pole-jump response (Yamamoto et al. 1982).

Similarly, D-ala2-met-enkephalinamide, an analog of ME, impairs acquisition

of a conditioned heart-rate response in rabbits when injected directly into

the amygdala, but equivalent doses of the LE analog D-ala2-[D-leu] enkephalin (DADLE) are without effect (Gallagher et al. 1982).

The conditioning effects of LE are attenuated by opioid antagonists that do

not cross the BBB. Mcthylnaloxonium, a quatemary form of naloxone with

limited ability to cross the BBB (Brown et al. 1983), attenuates the effects

of LE on active avoidance conditioning (Martinez et al. 1985b) and place

preference conditioning (Heinrichs and Martinez 1986) in mice. Similar

results were reported by Introini et al. (1985), who found that a quatemary

form of naltrexone, naltrexone methyl bromide, reversed the impairment of

passive avoidance retention produced by posttraining injections of ME or

LE in mice. Thus, the lack of effect of centrally administered LE, combined with the reversal of the effects of peripherally injected LE by quaternary antagonists, strongly suggests that the receptors that mediate the effects

of LE on conditioning lie outside the BBB. The precise locations and nature of these opioid receptors are currently under investigation.

56
The Role of Endogenous Peripheral Enkephalin Systems in the

Conditioning Effects of LE

Administration of quaternary antagonists prevents endogenous opioids access

to their receptors in the periphery and, therefore, allows for an assessment

of the role of these endogenous peptides in learning and memory. In this

regard, it is interesting to note that methylnaloxonium enhances acquisition

in Y-maze and one-way active avoidance conditioning tasks in mice

(Martinez and De Graaf 1985; Mendieta and Martinez 1988), as well as

enhancing retention of this same active avoidance task in mice (Mendieta

and Martinez 1988). Mendieta and Martinez (1988) also report that naltrexone methylbromide enhances both acquisition and retention of one-way

active avoidance conditioning, whereas passive avoidance in mice (Introini

et al. 1985) and conditional opioid analgesia in rats (Calcagnetti et al.

1987) are unaffected by peripheral injedions of naltrexone methyl bromide.

These different results may reflect differences in the conditioning tasks

employed. Although these results require further investigation, the data suggest that, in some cases, preventing access of opioids to their receptors in

the periphery produces an action opposite to that seen with exogenously

administered LE. This provides evidence that endogenous opioid systems

normally participate in the acquisition and retention of conditioned responses. Since neither mcthylnaloxonium nor naltrexone methyl bromide is

notably selective for specific opioid receptor types, these results do not

indicate which endogenous opioid peptide ligand or ligands are important.

However, studies using an antiserum to LE indicate that LE is an endogenous peptide important in avoidance conditioning. Passive immunization of

endogenous peripheral LE systems enhances acquisition of active avoidance

conditioning in mice (Martinez et al. 1985), an effect qualitatively the same

as that observed with methylnaloxonium and opposite that produced by LE.

This suggests that an important source of endogenous LE exists in the periphery and that this LE normally acts to impair conditioning.

The source of this LE might well be the adrenal medulla (Yang et al. 1980;

Pelto-Huikko et al. 1985; Schultzberg et al. 1978; Viveros et al. 1980).

Martinez and Rigter (1982) found that the effects of both ME and LE were

abolished by adrenal medullectomy (ADMX). Tenfold to a hundredfold

increases in the dose of LE restored its impairing action in demedullated

rats, suggesting that ADMX had removed an endogenous source of LE that

normally acts in concert with the injected peptide. In contrast, the effect of

ME could not be restored. In summary, the available evidence suggests

that peripheral enkephalin systems normally act to modulate conditioning

and that an important source of these enkephalins might be the adrenal

medulla. Since hormones are distributed to their targets by the circulatory

system, the findings discussed thus far suggested that we examine LE in the

blood circulatory system.

57
Changes in Plasma LE Concentrations With Conditioning May Be

Associated With a Regulatory Enzyme System

The changes in conditioning seen with systemically injected enkephalins

suggest that peripherally located endogenous enkephalin systems may participate in regulating an animal’s response to a conditioning situation. In

accordance with this suggestion, evidence has been found in rats for a

plasma enzyme system whose activity, as measured by LE hydrolysis, is

highly correlated with performance in an active avoidance conditioning task.

As seen in figure 5, a correlation of 0.82 was measured between the latency

of rats to escape on the first active avoidance trial and the rate at which LE

is hydrolyzed in plasma (Martinez and Weinberger 1988). Of particular

interest are the changes we observed in the activity of this enzyme system

following the conditioning experience. Following the first avoidance trial

and shock exposure, hydrolysis activity in plasma is significantly altered

(figure 6) (Martinez and Weinberger 1988). Importantly, both increases and

decreases in hydrolytic activity were seen. These data point to the existence in plasma of a regulatory enzyme system that modulates behavior, and,

accordingly, we have begun to characterize the enzymes that hydrolyze LE

in rat plasma and that may be involved in this modulatory relationship with

conditioning behavior.

Rat plasma was found to have its own unique pattern of enkephalin hydrolysis. These findings are based on differences in total metabolite accumulation measured in the presence or absence of selective peptidase inhibitors,

as assayed by thin layer chromatography. Blood was collected through an

indwelling femoral artery cannula. Eighty-five to 90 percent of the hydrolysis of LE is attributed to the combined action of aminopeptidase M

(E.C. 3.4.11.2) and angiotensin-converting enzyme. The remaining hydrolysis of LE in plasma is at present unaccounted for; it does not involve

“enkephalinase” (E.C. 3.4.24.1) or aminopeptidase MII activity, but it could

include dipeptidyl aminopeptidases (Weinberger and Martinez 1988). These

data suggest that either aminopeptidase M or angiotensin-converting enzyme

potentially could be involved in the relationship observed in rats between

the rate of LE hydrolysis in plasma and performance in an avoidanceconditioning situation.

LE is Rapidly Metabolized After Peripheral Injection

In order to understand the mechanisms responsible for the effects of LE on

conditioning, it is important to characterize the fate of LE after peripheral

injection. Recent studies have examined the kinetics of uptake and metabolism of radiolabeled LE and DADLE following IP administration in rats

(Martinez et al. 1988a; Schulteis et al., in press). Interestingly, 1 minute

following IP injection, 95 percent of the LE in plasma is in the form of

metabolites. By 2 minutes a plateau is reached, at which approximately

97.5 percent of the LE is metabolized.

58
FIGURE 5. Correlation between escape latency and LE metabolism

NOTE: Corellation between the latency of rats to escape on the first trial of a one-way active avoidance task and the amount of LE metabolized in 1 minute, as corrected for age. in plasma collected through an indwelling femoral artery cannula immediately before the conditioning trial;

r(19)=.82, p<.001. Correlation between age and LE metabolism rate: r(19)=.58, p<.005.

SOURCE: Martinez and Weinberger (1988), copyright 1988, American Psychological Association.

In contrast, LE is 50 percent metabolized by 2.5 minutes in plasma

in vitro (Martinez et al. 1988a; Martinez and Weinberger 1988; Schulteis

et al., in press). DADLE is metabolized more slowly than LE following IP

administration. The percent of DADLE that is metabolized at 1 minute is

12.7 percent, and, at 15 minutes, it is 63.7 percent. Like LE, DADLE is

much more rapidly hydrolyzed in vivo (63.7 percent at 15 minutes) than in

plasma in vitro (16 percent at 150 minutes) (Martinez et al. 1988; Schulteis

et al., in press).

Because the in vivo metabolism of both enkephalins following IP injection

is much more rapid than in plasma in vitro, it is highly unlikely that plasma

enzymes alone account for the rapid hydrolysis of these peptides after

59
FIGURE 6. Changes in LE metabolism associated with conditioning

NOTE: Changes in percentage of LE metabolized between blood samples collected from rats through

an indwelling femoral artery cannula (mean absolute value of the difference between samples,

as corrected for age by analysis of covariance, ± SEM). Control group: difference in

enzyme activity between sample pairs when a single sample was divided in half for duplicate

assay. n=12. Experimental group: difference in enzyme activity in blood samples collected

immediately before and after the first conditioning trial, n=18. Analysis of covariance with

age as covariate: F(l.27)=10.61, p=.003.

SOURCE: Martinez and Weinberger (1988), copyright 1988, American Psychological Association.

peripheral administration. In accordance with this suggestion, we found that

bestatin (10 mg/kg), an inhibitor of aminopeptidase M, a major participant

60
in the hydrolysis of LE, inhibits LE hydrolysis more effectively when administered IP along with LE than when it is injected intra-arterially about

50 seconds before an IP injection of LE. This indicates that a major

portion of LE hydrolysis occurs as LE crosses membranes on its way from

the site of injection into the bloodstream.

These results have important implications for studies of the behavioral

effects of LB following its IP administration. First, although the injected

peptide, when administered at a behaviorally active dose of 3 μg/kg, is 95

to 98 percent metabolized at all time points after injection, the amount of

LE remaining intact in plasma is within the range of endogenous plasma

concentrations of LE (30 to 120 pg/ml plasma) (figure 7) (Martinez et al.

1988a; Martinez et al. 1988b). This suggests that a behaviorally active dose

of LE achieves a plasma LE concentration that is within a normal physiological range. Likewise, a dose of DADLE (0.6 μg/kg) that is approximately one-fifth the dose of LE on a molar basis yields plasma levels of intact

peptide that range from 2.4 to 5.1 times the levels achieved by LE across

the 15-minute sampling interval, consistent with the greater potency of

DADLE in conditioning studies (figure 7) (Rigter et al. 1980a; Rigter et al.

1980b). This indicates that the effects of LE on learning are not simply

due to the pharmacological effects of large doses of injected peptide, but

rather may mimic the action of endogenous peripheral enkephalin systems.

A second important consideration of the metabolism data described above is

the possible contribution of enkephalin metabolites to the observed behavioral effects of LE. This possibility is discussed in detail in the following

section.

Some LE Metabolites Impair Active Avoidance Conditioning

De Wied and coworkers were the first to suggest that metabolic fragments

of peptides may have biological activity (Burbach et al. 1983). Since LE is

rapidly degraded after release, the question of whether any LE degradation

products can also influence an animal’s behavior becomes important. We

examined the effects of a number of LE metabolites on conditioning in

mice. In an appetitively motivated Y-maze task, LE (300 μg/kg) but not its

metabolite des-tyr-LE (Gly-Gly-Phe-Leu (GGPL)) impairs retention (Linden

and Martinez 1986). Similarly, in the active avoidance task, LE

(100 μg/kg) impairs acquisition, while GGPL is without effect (Weinberger

and Martinez 1988). Since GGPL has less than one-thousandth the opioid

potency of LE, as measured in bioassays and binding assays (Hambrook

et al. 1976). these findings are to be expected if LE’s effects on conditioning are mediated through opioid receptors.

In contrast, three other potential LE metabolites impair acquisition in the

one-way active avoidance paradigm (table 1). The enkephalin metabolites

Tyr-Gly (TG), Tyr-Gly-Gly (TGG), and Tyr-Gly-Gly-Phe (TGGP) all impair

acquisition of the active avoidance response in mice (Janak et al. 1987;

61
Weinberger and Martinez 1988). Both TG and TGGP impair avoidance

conditioning at a dose equimolar to the behaviorally active 100 μg/kg dose

of LE; TGG is effective at a dose equimolar to the 30 μg/kg dose of LE.

FIGURE 7. Concentrations of unmetabolized LE and DADLE in plasma

as a function of time after IP injection

SOURCE: Developed from data presented in Martinez et al. 1988.

In addition, as the table indicates, the dose-response function for the

impairing effect is consistently U-shaped. Thus, three potential metabolites

of LE influence conditioning in a manner similar to that of the parent pep

tide. Neither GGPL nor tyrosine itself (Janak and Martinez in preparation)

has an effect on avoidance conditioning when tested over a wide dose

range. These results together indicate that the TG portion of the LE

molecule is the minimum sequence necessary for producing acquisition

impairment.

62
As table 1 illustrates, the avoidance impairments produced by TG and TGG

are not attributable to drug-induced changes in locomotor activity. Animals

that received the impairing dose of TG or TGG prior to testing for shockinduced locomotor activity in an open field did not differ from salinecontrol animals (Janak et al. 1987; Weinberger and Martinez 1988). However, animals that received the avoidance-impairing dose of TGGP showed a

decrease in shock-induced locomotor activity (Janak et al. 1987). Therefore,

the avoidance-impairing effects of TGGP may be secondary to the activity

decreases produced by the metabolite. Interestingly, both GGPL and free

tyrosine also produce a decrease in shock-induced locomotor activity in

mice but do not affect conditioning (Weinberger and Martinez 1988; Janak

and Martinez, in preparation).

TABLE 1. Effects of enkephalin metabolites on one-way active avoidance

conditioning in mice

Peptide Dose Range Maximally Effective Avoidance Decreased

(μg/kg) Dose (μg/kg) Impairment Locomotion

Tyr-Gly-Gly-Phe-Leu

([leu]enkephalin)

Tyr-Gly-Gly-Phe

Tyr-Gly-Gly

Tyr-Gly

Tyr

Gly-Gly-Phe-Leu

30-300 100

24-80 80

16-160 16

4.3-43 43

3.9-390 none

7.1-710 none

Yes

Yes

Yes

Yes

No

No

No

Yes

No

No

Yes

Yes

The avoidance impairing effects of some of these LE metabolites are

puzzling. As discussed previously, LE has strong affinity for and appears

to produce its avoidance impairment through the delta opioid receptor. In

contrast, TG, TGG, and TGGP have almost no opioid activity as measured

in a guinea pig ileum or mouse vas deferens bioassay (Dewey 1982). The

behaviorally active LE metabolites may, therefore, be exerting their effects

through some mechanism other than activation of opioid receptors. Such

effects are common in the literature, in that other short peptides are observed to produce opioid effects without seeming to activate directly opioid

receptors. For example, kyotorphin (Tyr-Arg) and kentsin (Thr-Pro-ArgLys) both produce naloxone-reversible analgesia (Takagi et al. 1979a;

Takagi et al. 1979b; Satoh et al. 1985; Fox et al. 1987), yet neither peptide

appears to occupy opioid receptors. These compounds are hypothesized to

exert their opioid effects by inducing release of endogenous enkephalins

(Takagi et al. 1979a; Satoh et al. 1985; Fox et al. 1987). An alternative

explanation is that the LE metabolites compete with their parent compound

for binding to degradative enzymes, as in the model proposed by LaBella et

al. (1985), thereby effectively increasing the levels of endogenous peptide.

TG, TGG, and TGGP also may increase circulating levels of endogenous

LE by binding to the regulatory proteins in plasma that appear to offer LE

63
some protection from enzymatic degradation (Venturelli et al. 1986).

Further investigation is necessary to differentiate among these possible

mechanisms.

Summary of LE Effects on Behavioral Plasticity

The above evidence suggests that LE may directly affect memory traces by

an action on the modulatory inputs to the neural substrate of memory storage, even though LE itself does not cross the BBB in sufficient quantities

to be of physiological significance. One major way to modulate the

strength of the memory trace is for the concentration of enkephalin to decrease or increase at its delta receptors that lie outside the BBB. A potential mechanism involved in regulating LE concentrations in the blood and,

hence, at its receptors is the enkephalin-hydrolyzing enzyme system in

plasma. This enzyme system appears to be able to alter its activity as a

result of learning.

ENKEPHALINS AND NEURAL PLASTICITY

Activity within the memory-modulating peripheral systems discussed above

must ultimately affect the memory trace. One site in the mammalian CNS

that is strongly implicated in memory function is the hippocampus. Synapses in the hippocampus are highly plastic, and the experimental manipulations that change synaptic strength may be quite long lasting. We are

investigating the involvement of opioid peptides in long-term potentiation

(LTP) (Bliss and Lomo 1973; Bliss and Lynch 1988), a synaptic phenomenon thought to underlie memory storage at the neural level. LTP can be

induced by repetitive, high-intensity stimulation of afferents to the hippocampus and dentate gyrus. An important aspect of LTP is that its induction

conforms to the idea of a Hebbian synapse. Generally, induction of LTP

requires a combination of pre- and postsynaptic activity. Hippocampal synapses will potentiate if, and only if, they are active at a time when the

regions of the dendrite where the synapses are located are strongly depolarized (Bliss and Lynch 1988).

Current evidence suggests that induction of LTP in both the CA1 region of

the hippocampus and the dentate gyrus involves activation of the N-methylD-aspartate (NMDA) receptor and the subsequent entry of calcium postsynaptically (Lynch et al. 1983). This receptor is a voltage-dependent subtype

of the glutamate receptor, and it requires both presynaptic transmitter release

and postsynaptic depolarization for its activation (Collingridge and Bliss

1985). It thus resembles a conjunctive mechanism of associative memory

postulated by Hebb 40 years ago (Hebb 1949), in that the efficacy of the

synapse is increased once the NMDA receptor is activated by repetitive

stimulation, presumably through the release of a magnesium block

(Collingridge and Bliss 1985).

64
LTP is also observed in the mossy fibers, a major projection to the CA3

region of the hippocampus. The mossy fibers contain a relatively high

content of proenkephalin and prodynorphin derivatives (Gall et al. 1981).

Release of these derivatives is Ca++dependent (Chavkin and Bloom 1985).

LTP of mossy-fiber responses, however, differs from LTP in other hippocampal regions, in that antagonists of NMDA receptors fail to block LTP in

the mossy-fiber pathway (Harris and Cotman 1986). This agrees with the

finding that the area where mossy fibers terminate has a low density of

NMDA receptors (Monaghan and Cotman 1985).

LTP of Mossy-Fiber-CA3 Responses May Be Dependent on Release of

Endogenous Opioid Peptides

The action of naloxone on LTP of both the mossy-fiber and commissuralCA3 response in the rat hippocampus in vivo was investigated. Under pentobarbital anesthesia, CA3 pyramidal cell responses were evoked both by

stimulation of the mossy fibers and by stimulation of the contralateral CA3

region to activate commissural fibers. A 1 μl volume of naloxone (1, 5,

or 10 mM) was applied via pressure ejection to the pyramidal layer. Naloxone, at all concentrations, did not attenuate field responses of either

the mossy-fiber or commissural pathway. However, LTP of the monosynaptic mossy-fiber response (induced by two l-second, 100-Hz stimulation

trains) was blocked by naloxone at concentrations of 5 and 10 mM. LTP

of the commissural response was unaffected by naloxone at these concentrations (figure 8) (Derrick and Martinez 1988). Importantly, (+)-naloxone

(10 mM) did not block mossy-fiber LTP, indicating that the effect of naloxone is stereospecific and, therefore, mediated by opioid receptors.

Exogenous application of enkephalin and its analogs produces excitation of

pyramidal cells, presumably due to an attenuation of recurrent inhibition

(Nicoll 1980; Zieglegansberger et al. 1979; Haas and Ryall 1980; Derrick

et al. 1987). The induction of LTP is facilitated when GABAergic inhibition is blocked (Wigstrom and Gustafsson 1985) and application of GABA

can prevent the induction of LTP in several hippocampal regions

(Scharfman and Sarvey 1985). Naloxone could effectively block the induction of LTP by antagonizing the disinhibitory effects of endogenously

released opioid peptides (Martin 1983; Segal 1988). In this view, opioid

peptides are necessary for mossy-fiber LTP, but they act indirectly, allowing

a sufficient level of postsynaptic depolarization for the induction of LTP via

actions on inhibitory mechanisms. GABAergic inhibitory mechanisms are

well-established postsynaptic processes (Alger and Nicoll 1982; Bliss and

Lynch 1988). If an influence of opioid peptides on GABAergic inhibitory

mechanisms is necessary for the induction of mossy-fiber LTP, it would

suggest that LTP induction is dependent on postsynaptic mechanisms, similar to the mechanism of induction observed in the dentate gyrus and CA1

region.

65
FIGURE 8. LTP of mossy-fiber and commissural responses following

application of 1.0 ul lactated Ringer’s or naloxone

*p<.01.

**p<.001.

NOTE: Each bar represents the average percent increase in population spike 20 minutes posttetanus.

(+) Naloxone was at a concentration of 10 mM. n=6 to 12 for each concentration.

SOURCE: Derrick and Martinez 1988.

However, a naloxone blockade of an opioid peptide-mediated disinhibition is

unlikely for several reasons. First, our studies indicate that LE and its

metabolically stable analog, DADLE, produce excitation of the mossy fiber

response without attenuating either feedback (figure 9) (Derrick et al. 1987)

or feedforward inhibition (figure 10) (Derrick et al. 1988). Second, if

opioid peptides play a role in the initiation of LTP solely via a disinhibitory

mechanism, then one would expect opioid antagonists to attenuate LTP in

other hippocampal regions where excitatory and, presumably, disinhibitory

effects of opioid peptides are observed. However, opioid antagonists do not

attenuate LTP in the CA1 field in vivo (Stringer et al. 1983; Linesman

et al. 1981).

66
FIGURE 9. The effect of DADLE (1.0 μl of 100 μM) on attenuation of

the mossy fiber-CA3 population spike amplitude in the

second (tart) response following paired-pulse stimulation

NOTE: Ordinate=percent of first population spike amplitude; n=5 for each group.

FIGURE 10. Change in paired-pulse measures of feedforward inhibition

following 1.0 μl of 100 μM DADLE

NOTE: First (conditioning) pulse was at an intensity that did not elicit a population spike, thereby

preventing activation of recurrent inhibitory mechanisms.

67
Opioid Peptides May Play a Direct Role in the Initiation of Mossy

Fiber-CA3 LTP

If a disinhibitory influence is not the mechanism of naloxone blockade of

mossy fiber-CA3 LTP, then how could opioid peptides serve in the expression of LTP in the mossy-fiber pathway? One possibility is that the

excitatory effect produced in the CA3 region by exogenous opioid peptides

and, presumably, by those that are released endogenously as well, may be

necessary for the induction of LTP in the mossy-fiber pathway. In this

view, the excitation produced by endogenously released opioid peptides

could allow a level of depolarization sufficient to induce LTP. Recently,

enkephalins were shown to block the M-current in CA3 pyramidal cells

(Siggins 1988). Through actions on such a mechanism, opioid peptides

could effectively amplify the postsynaptic response. Thus, a presynaptic

release of opioid peptides could be necessary to produce a level of postsynaptic depolarization sufficient to contribute to the induction of LTP in the

mossy fibers.

However, this mechanism needs the specificity of a Hebbian synapse.

Opioid peptides do not contribute to mossy-fiber responses during lowfrequency stimulation, as evidenced by the lack of an effect of opioid antagonists on single-pulse-evoked mossy fiber responses (Chavkin and Bloom

1985; Dalkara and Krnjevick 1984; Derrick and Martinez 1988). This is

similar to the effects observed with NMDA antagonists, which have little

effect on hippocampal-evoked responses during low-frequency stimulation

(Collingridge and Bliss 1985). The release of some peptide cotransmitters,

including opioid peptides, may be dependent on intense or repetitive presynaptic activity (Ishida 1970; Mitchell et al. 1987; Bramham et al. 1988).

The release of opioid peptides contingent on intense or repetitive presynaptic activity, such as is needed to induce LTP, coupled with an increase in

postsynaptic responsiveness resulting from opioid-peptide actions postsynaptically, may serve as a necessary condition for the induction of LTP at the

mossy fiber-CA3 synapse. In this way, mossy-fiber LTP may occur

through a unique, opioid-peptide-dependent mechanism of LTP induction

within the hippocampus. If the activity of the postsynaptic cell was

changed by the conjunction of mossy-fiber activation and the postsynaptic

action of opioid peptides, then this could be considered a Hebbian synapse.

Activation of the NMDA receptor is believed to induce LTP by allowing a

postsynaptic increase in calcium (Lynch et al. 1983). Similarly, an increase

in postsynaptic calcium would presumably be required for induction of LTP

in the mossy fibers. Opioid peptides could promote entry of calcium

through two possible mechanisms. First, activation of opioid receptors can

directly influence calcium conductance (North 1986) which can increase the

intracellular concentration of calcium (Henderson 1983). Second, opioid

receptor activation could lead to an increase in calcium via the second

messenger systems that underlie M-current blockade. A variety of peptides,

68
including opioid peptides, produce M-current blockade. These peptides

usually act via the receptor-stimulated metabolism of phosphoinositides (PI

turnover) (Nicoll 1982; Brown 1988). Activation of some opioid receptors

has recently been shown to stimulate PI turnover (Satoh et al. 1988). The

products of PI turnover can also rapidly release intracellular calcium from

nonmitochondrial stores (Nishizuka 1984). Thus, actions of opioid peptides

on this second messenger system could block the M-current and allow an

increase in intracellular calcium postsynaptically.

Mossy Fiber LTP May Represent a Fundamentally Distinct Form of

LTP in the Hippocampus

This distinct form of LTP induction observed in the mossy fibers suggests

that LTP in this system may represent a distinct form of synaptic plasticity

in the hippocampus. In line with this suggestion are the unique characteristics of mossy-fiber LTP seen in studies using pentobarbital-anesthetized rats.

Following tetanic stimulation of commissural afferents, the commissural-CA3

response shows a rapid increase of both the population spike and the field

EPSP. LTP of commissural responses is decremental, with a half-life of

about 70 minutes in vivo. In contrast, mossy-fiber LTP attains maximal

amplitude more slowly, reaches a smaller maximal amplitude, and does not

decrease over 3 hours posttetanus (Washington et al. 1988). These differences in the decay rate of LTP, in separate afferents to the same population

of pyramidal cells, induced with identical stimulation parameters, suggest

that distinct mechanisms may underlie both the induction and maintenance

of mossy-fiber LTP. These mechanisms are currently being investigated.

Mossy-fiber LTP is also differentially affected by other pharmacological

agents. Urethane, at anesthetic doses, was found to prevent the induction of

mossy-fiber but not commissural LTP (Washington et al. 1988). Others

report that pertussis toxin prevents LTP in the mossy fibers, while potentiation of Schaffer collateral-CAl synapses remains unimpaired (Ito et al.

1988). This toxin selectively inactivates certain G-proteins, and the results

suggest that G-proteins may be involved in the induction of mossy-fiber

LTP. Interestingly, anesthetics can also interfere with G-protein-receptor

coupling (Anthony et al. 1988). Thus, the mechanism of blockade of both

urethane and pertussis toxin may be the result of similar actions on specific

G-proteins.

In summary, induction of mossy-fiber LTP may be dependent on the release

of opioid peptides. The exact role of opioid peptides in the expression of

LTP is unknown, but they may contribute to mossy fiber LTP as a result of

actions on GABAergic inhibitory mechanisms or postsynaptic mechanisms

that augment depolarization. It seems unlikely that opioids affect LTP

through a release from inhibition in the mossy-fiber pathway, since opioids

do not affect either feedforward or feedback inhibition. The frequencydependent release and the subsequent postsynaptic effects of these peptides

69
may function as a Hebbian conjunctive mechanism. as do NMDA receptors

in other hippocampal regions.

CONCLUSION

The evidence presented here suggests that enkephalins are involved in two

different processes important to learning and memory. Enkephalins may

influence the memory trace that represents the actual memory in a manner

similar to their effects on plasticity in the hippocampus. Enkephalins may

also play an integral role in a hormonal modulatory system; this system

influences the modulatory input that regulates the strength of the memory

trace. The study of the mechanisms underlying both the memory trace

itself and its modulatory input may reveal some basic characteristics of how

mammalian nervous systems store information.

REFERENCES

Ader, R.; Friedman, S.B.; and Grota, L.J. Emotionality and adrenal cortical

function: Effects of strain, test and the 24-hour corticosterone rhythm.

Anim Behav 15:37-44, 1967.

Alger, B.E., and Nicoll, R.A. Pharmacological evidence for two kinds of

GABA receptor on rat hippocampal pyramidal cells studied in vitro.

J Physiol 328:125-141, 1982.

Anthony, B.L.; Dennison, R.L.; Narayanan, T.K.; and Aronstam, R.S. Halothane inhibition of acetylcholine-stimulated, pertussis-toxin sensitive

GTPase activity in the rat brain. Soc Neurosci Abs 14:811, 1988.

Belluzzi. J.D.. and Stein, L. Facilitation of long-term memory by brain

endorphins. In: Martinez, J., Jr; Jensen, R.A.; Messing, R.B.; Rigter, H.;

and McGaugh, J.L., eds. Endogenous Peptides and Learning and

Memory Processes. New York: Academic Press, 1981. pp. 291-303.

Bliss, T.V.P., and Lomo, T. Long-lasting potentiation of synaptic transmission in the dentate area of the anesthetized rabbit following stimulation of

the perforant path. J Physiol 232:357-374, 1973.

Bliss, T.V.P., and Lynch, M.A. Long-term potentiation of synaptic transmission in the hippocampus: Properties and mechanisms. In:

Landfield, P.W.. and Deadwyler, S.A., eds. Long-Term Potentiation:

From Biophysics to Behavior. New York: Alan R. Liss, Inc., 1988.

pp. 3-72.

Bramham, C.R.; Errington, M.L.; and Bliss, T.V. Naloxone blocks the

induction of long-term potentiation in the lateral but not in the medial

perforant pathway in the anesthetized rat. Brain Res 449:352-356, 1988.

Brown, D. M-currents: An update. Trends Neurosci 11:294-299, 1988.

Brown, D.R.; Robertson, M.J.; and Goldberg, L.I. Reversal of morphineinduced catalepsy in the rat by narcotic antagonists and their quaternary

derivatives, Neuropharmacology 22:317-321, 1983.

70
Burbach. J.P.H.; Kovacs, G.L.; De Wied, D.; Van Nispen, J.W.; and

Greven, H.M. A major metabolite of arginine vasopressin in the brain is

a highly potent neuropeptide. Science 221:1310-1312, 1983.

Calcagnetti, D.J.; Helmstetter, F.J.; and Fanselow, M.S. Quaterbary naltrexone reveals the central mediation of conditional opioid analgesia.

Pharmacol Biochem Behav 27:529-531, 1987.

Chaminade, M.; Fouts, A.S.; and Rossier, J. Co-release of enkephalins and

precursors with catecholamines from the perfused cat adrenal gland in

situ. J Physiol 353:157-169, 1984.

Chavkin, C., and Bloom, F.E. Opiate antagonists do not alter neuronal

responses to stimulation of opioid containing pathways in rat hippocampus. Neuropeptides 7:19-22, 1985.

Collingridge, G., and Bliss, T.V.P. NMDA receptors-their role in longterm potentiation. Trends Neurosci 7:288-293, 1985.

Corbett. A.D.; Gillan, M.G.C.; Kosterlitz, H.W.; McKnight, A.T.;

Paterson, S.J.; and Robson, L.E. Selectivities of opioid peptide analogues

as agonists and antagonists at the -receptor. Br J Pharmacol

83:271-279, 1984.

Corder, R.; Mason, D.F.J.; Perrett, D.; Lowry, P.J.; Clement-Jones, V.;

Linton, E.A.; Besser, G.M.; and Rees, L.H. Simultaneous release of

neurotensin, somatostatin, enkephalins, and catecholamines from perfused

cat adrenal glands. Neuropeptides 3:9-17, 1982.

Cornford, E.M.; Braun, L.D.; Crane, P.D.; and Olendorf, W.H. Blood-brain

barrier restriction of peptides and the low uptake of enkephalins.

Endocrinology 103:1297-1303, 1978.

Cotton, R.; Kosterlitz, H.W.; Paterson, S.J.; Rance, M.J.; and Traynor, J.R.

The use of [3H]-[d-pen2,d-pen5]enkephalin as a highly selective ligand

for the -binding site. Br J Pharmacol 84:927-932, 1985.

Dalkara. T., and Kmjevic, K. Does mossy fiber activity release an endogenous opiate in the CA2/3 pyramidal layer? Soc Neurosci Abs 10:1124,

1984.

Derrick, B.E., and Martinez, J.L., Jr. Naloxone blocks mossy fiber, but not

commissural or heterosynaptic, LTP in the rat CA3 region in vivo. Soc

Neurosci Abs 14:565, 1988.

Derrick, B.E., and Martinez, J.L., Jr. A unique, opioid-peptide dependent

form of long-term potentiation is found in the CA3 region of the rat

hippocampus. In: Cros, J., and Meunier, L.Cl., eds. Advances in the

Biosciences. Oxford: Pergamon Press, 1989.

Derrick, B.E.; Martinez, J.L., Jr.; and Cunningham, ST. Effect of enkephalins on the rat CA3 hippocampal region in vivo. Soc Neurosci Abs

13:767, 1987.

Dewey, W.L. Structure-activity relationships. In: Malick, J.B., and

Bell, R.M.S., eds. Endorphins: Chemistry, Physiology, Pharmacology,

and Clinical Relevance. New York: Marcel Dekker Inc., 1982.

pp. 23-56.

71
de Wied, D.; Bohus, B.; van Ree, J.M.; and Urban, I. Behavioral and

electrophysiological effects of peptides related to lipotropin (B-LPH).

J Pharmacol Exp Ther 201:570-580, 1978.

Dobrakovova, M., and Jurcovicova, J. Corticosterone and prolactin

responses to repeated handling and transfer of male rats. Exp Clin

Endocrinol 82:21-27, 1984.

Fox, D.A.; Ayres, E.A.; Wire, W.S.; Lui, G.; Yamamura, H.I.; and

Burks, T.F. Kentsin: Tetrapeptide from hamster embryos produces

naloxone-sensitive effects without binding to opioid receptors. Peptides

8:613-618, 1987.

Gall, C.M.; Brecha, H.J.; Chang, H.; and Kanten, H. Localization of

increased leucine-enkephalin immunoreactivity to identified axonal and

neuronal populations of the rat hippocampus. J Comp Neurol 198:335-

350, 1981.

Gallagher, M.; Kapp. B.S.; and Pascoe, J.P. Enkephalin analogue effects in

the amygdala central nucleus on conditioned heart rate. Pharmacol

Biochem Behav 17:217-222, 1982.

Gormley, J.J.; Morley, J.S.; Priestly, T.; Shaw, J.S.; Turnbull, M.J.; and

Wheeler, H. In vivo evaluation of the opioid delta receptor antagonist

ICI 154,129. Life Sci 31:1263-1266, 1982.

Haas, H.L., and Ryall, R.W. Is excitation by enkephalins of hippocampal

neurons in the rat due to presynaptic facilitation or to disinhibition?

J Physiol 308:315-330, 1980.

Hambrook, J.M.; Morgan, B.A.; Rance, M.J.; and Smith, C.F.C. Mode of

deactivation of the enkephalins by rat and human plasma and rat brain

homogenates. Nature 262:782-783, 1976.

Hanbauer, I.; Kelley. G.W.; Saiani, L.; and Yang, H.Y.T. [Met]5

enkephalin-like peptides of the adrenal medulla: Release by nerve stimulation and functional implications. Peptides 3:469-473, 1982.

Harris, E.W., and Cotman, C.W. Long-term potentiation of guinea pig

mossy fiber responses is not blocked by N-methyl-D-aspartate antagonists.

Neurosci Lett 70:132-137, 1986.

Hebb, D.O. The Organization of Behavior. New York: John Wiley and

Sons, Inc., 1949.

Heinrichs, S., and Martinez, J.L., Jr. Modification of place preference conditioning in mice by systemically administered leu-enkephalin. Behav

Brain Res 22:249-255, 1986.

Henderson, G. Electrophysiological analysis of opioid action in the central

nervous system. Br Med Bull 39(1):59-64, 1983.

Hruby, V.J. Design of conformationally constrained cyclic peptides with

high delta and mu opioid receptor specificities. In: Rapaka, R.S., and

Hawks, R.L., eds. Opioid Peptides: Molecular Pharmacology, Biosynthesis, and Analysis. National Institute on Drug Abuse Research Monograph 70. DHHS Pub. No. (ADM)86-1455. Washington, D.C., Supt. of

Docs., U.S. Govt. Print. Off., 1986. pp. 128-154.

72
Introini, I.B.; McGaugh, J.L.; and Baratti, C.M. Pharmacological evidence

of a central effect of naltrexone, morphine, and beta-endorphin and a

peripheral effect of met- and leu-enkephalin on retention of an inhibitory

response in mice. Behav Neural Biol 44:434-446, 1985.

Ishida, A. The oxytocin release and compound action potential evoked by

electrical stimulation of the isolated neurohypophysis of the rat.

Jap J Physiol 20:84-96, 1970.

Ito, I.; Okada, D.; and Sugiyama, H. Pertussis toxin suppresses long-term

potentiation of hippocampal mossy fiber synapse. Neurosci Left 90:181-

185, 1988.

Izquierdo, I., and Dias, R.D. Retrograde amnesia caused by met-, leu-, and

des-tyr-met-enkephalin in the rat and its reversal by naloxone. Neurosci

Lett 22:189-193, 1981.

Izquierdo, I.; Paiva, A.C.M.; and Elizabetsky, E. Post-training intraperitoneal administration of leu-enkephalin and beta-endorphin causes retrograde amnesia for two different tasks in rats. Behav Neural Biol 28:246-

250, 1980.

Janak, P.H.; Valedon, A.; Schulteis, G.; Derrick, B.E.; Fett, K.;

Weinberger, S.B.; and Martinez, J.L., Jr. Two enkephalin metabolites,

tyr-gly-gly-phe and tyr-gly, impair acquisition of an active avoidance

response in mice. Soc Neurosci Abs 13:85, 1987.

Kastin, A.J.; Nissen, C.; Schally, A.V.; and Coy, D.H. Blood-brain barrier,

half-time disappearance, and brain distribution for labelled enkephalin and

a potent analog. Brain Res Bull 1:583-589, 1976.

Kety, S.S. The biogenic amines in the central nervous system: Their possible roles in arousal, emotion, and learning. In: Schmitt, F.O. The

Neurosciences. New York: The Rockefeller University Press, 1970.

pp. 324-336.

Klein, R.L.; Wilson, S.P.; Dzielak, D.J.; Yang, W-H; and Viveros, O.H.

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles

from sympathetic nerve. Neuroscience 7:2255-2261, 1982.

Koob, G.F. Neuropeptides and memory. In: Iversen, L.L.; Iversen, S.D.;

and Snyder, S.H., eds. Handbook of Psychopharmacology, Vol 19. New

York: Plenum Press, 1987. pp. 531-573.

Kosterlitz, H.W., and Hughes, J. Isolated organs and cultured cells as

models for the study of narcotic action. In: Adler, M.W.; Manara, L.;

and Samamin, R., eds. Factors Affecting the Action of Narcotics. New

York: Raven Press, 1978. pp. 19-37.

Kosterlitz, H.W.; and Paterson, S.J. Types of opioid receptors: Relation to

antinociception. Philos Trans R Soc London 308:291-297, 1985.

LaBella, F.S.; Geiger, J.D.; and Glavin, G.B. Administered peptides inhibit

the degradation of endogenous peptides. The dilemma of distinguishing

direct from indirect effects. Peptides 6:645-660, 1985.

Linden, D., and Martinez, J.L., Jr. Leu-enkephalin impairs memory of an

appetitive maze response in mice. Behav Neurosci 100:33-38, 1986.

73
Linesman, M.A., and Corrigal, W.A. Are endogenous opiates involved in

potentiation of field potentials in the hippocampus of the rat? Neurosci

Lett 27:319-324, 1981.

Lynch, G.; Larson, J.; Kelso, S.; Barrionuevo; and Schottler, F. Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305:719-721, 1983.

Martin, M.R. Naloxone and long-term potentiation of hippocampal CA3

field potentials in vitro. Neuropeptides 4:45-50, 1983.

Martinez, J.L., Jr. Memory: Drugs and hormones. In: Martinez, J.L., Jr.,

and Kesner, R.P., eds. Learning and Memory: A Biological View. New

York: Academic Press, 1986. pp. 127-163.

Martinez, J.L., Jr.; Conner, P.; and Dana, R.C. Central versus peripheral

actions of leu-enkephalin on acquisition of a one-way avoidance response

in mice. Brain Res 327:37-43, 1985a.

Martinez, J.L., Jr., and de Graaf, J. Quaternary naloxone enhances acquisition of a discriminated y-maze and a one-way active avoidance task in

mice. Psychopharmacology 87:410-413, 1985.

Martinez, J.L., Jr.; de Graaf, J.; Chavkin, C.; and Dana, R.C. Leuenkephalin actions on avoidance conditioning are mediated by a peripheral

opioid receptor. Life Sci 37:2345-2353, 1985b.

Martinez, J.L., Jr.; Jensen, R.; Creager, R.; Veliquette, J.; Messing, R.;

McGaugh, J.; and Lynch, G. Selective effects of enkephalin on electrical

activity of the in vitro hippccampal slice. Behav Neurol Biol 26:128-131,

1979.

Martinez, J.L., Jr.; Olson, K.; and Hilston, C. Opposite effects of metenkephalin and leu-enkephalin on a discriminated shock-escape task.

Behav Neurosci 98:487-495, 1984.

Martinez, J.L,., Jr., and Rigter, H. Enkephalin actions on avoidance conditioning may be related to adrenal medullary function. Behav Brain Res

6:289-299, 1982.

Martinez, J.L., Jr.; Rigter, H.; Jensen, R.A.; Messing, R.B.; Vasquez, B.J.;

and McGaugh, J.L. Endorphin and enkephalin effects on avoidance conditioning: The other side of the pituitary-adrenal axis. In:

Martinez, J.L., Jr.; Jensen, R.A.; Messing, R.B.; Rigter, II.; and

McGaugh, J.L., eds. Endogenous Peptides and Learning and Memory

Processes. New York: Academic Press, 1981. pp. 305-324.

Martinez, J.L., Jr.; Schulteis, G.; Chisholm, L.; and Weinberger, S.B.

Plasma uptake and metabolism of [leu]enkephalin following intraperitoneal

administration in rats. Soc Neurosci Abs 14:397, 1988a.

Martinez, J.L., Jr., and Weinberger, S.B. Enkephalin hydrolysis is highly

correlated with escape performance in the rat. Behav Neurosci 102:404-

408, 1988.

Martinez, J.L., Jr.; Weinberger, S.B.; and Schulteis, G. Enkephalins and

learning and memory: A review of evidence for a site of action outside

the blood-brain barrier. Behav Neural Biol 49:192-221. 1988b.

74
McCarty, R., and Gold, P.E. Plasma catecholamines: Effects of footshock

level and hormonal modulators of memory storage. Horm Behav 15:168-

182, 1981.

McGaugh, J.L., and Martinez, J.L., Jr. Learning modulatory hormones: An

introduction to endogenous peptides and learning and memory processes.

In: Martinez, J.L., Jr.; Jensen, J.A.; Messing, R.B.; Rigter, H.; and

McGaugh, J.L., eds. Endogenous Peptides and Learning and Memory

Processes. New York: Academic Press, 1981. pp. 1-3.

Mendieta, E.A., and Martinez, J.L., Jr. Quaternary forms of naloxone and

naltrexone enhance acquisition of a one-way active avoidance response in

mice. Soc Neurosci Abs 14:1030, 1988.

Mitchell, C.L.; Grimes, L.; Hudson, P.M.; and Hong, J.S. Stimulation of

the perforant path alters hippocampal levels of opioid peptides, glutamine

and GABA. Brain Res 435:343-347, 1987.

Monaghan, D.T., and Cotman, C.W. Distribution of N-methyl-D-aspartatesensitive L-[3H] glutamate binding sites in rat brain. J Neurosci 5:2909-

2919, 1985.

Mosberg, H.I.; Hurst, R.; Hruby, V.J.; Gee, K.; Yamamura, H.I.;

Galligan, J.J.; and Burks, T.F. Bis-penicillamine enkephalins possess

highly improved specificity toward opioid receptors. Proc Natl Acad Sci

USA 80:5871-5874, 1983.

Nicoll, R.A. Neurotransmitters can say more than ‘yes’ or ‘no.’ Trends

Neurosci 5:369-374, 1982.

Nicoll, R.A.; Alger, B.E.; and Jahr, C.E. Enkephalin blocks inhibitory

pathways in the vertebrae CNS. Nature 287:22-25, 1980.

Nishizuka, Y. Turnover of inositol phospholipids and signal transduction.

Science 225:1365-1370, 1984.

North, R.A. Opioid receptor types and membrane ion channels. Trends

Neurosci 9:114-117, 1986.

Olson, G.A.; Olson, R.D.; Kastin, A.J.; Green, M.T.; Roig-Smith, R.;

Hill, C.W.; and Coy, D.H. Effects of an enkephalin analog on complex

learning in the rhesus monkey. Pharmacol Biochem Behav 11:341-345,

1979.

Olson, G.A.; Roig-Smith, R.; Mauk, M.D.; LaHoste, G.J.; Coy, D.H.;

Hill, C.W.; and Olson, R.D. Differential effects of neuropeptides on

short-term memory in primates. Peptides 2 (suppl. 1):131-136, 1981.

Paterson, S.J.; Robson, L.E.; and Kosterlitz, H.W. Classification of opioid

receptors. Br Med Bull 39:25-30, 1983.

Patterson, T.A.; Schulteis, G.; Alvardo, M.C.; Martinez, J.L., Jr.,;

Bennett, E.L.; and Rosenzweig, M.R. Influence of opioid peptides on

memory formation in the chick. Behav Neurosci 103:429-439, 1989.

Pardridge, W.M., and Mietus, L.J. Enkephalin and blood-brain barrier:

Studies of binding and degradation in isolated brain microvessels.

Endocrinology 109:1138-1143, 1981.

Pelto-Huikko, M.; Saliminen, T.; and Hervonen, A. Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat

adrenal medulla. Histochemistry 82:377-383, 1985.

75
Rigter, H. Attenuation of amnesia in rats by systemically administered

enkephalins. Science 200:83-85, 1978.

Rigter, H.; Dekker. I.; and Martinez, J.L., Jr. A comparison of the ability

of opioid peptides and opiates to affect avoidance conditioning in rats.

Regul Pep 2:317-322, 1981.

Rigter, H.; Hannan, T.J.; Messing, R.B.; Martinez, J.L, Jr.; Vasquez, B.J.;

Jensen, R.A.; Veliquette, J.; and McGaugh, J.L. Enkephalins interfere

with acquisition of an active avoidance response. Life Sci 26:337-345,

1980a.

Rigter, H.; Jensen, R.A.; Martinez, J.L., Jr.; Messing, R.B.; Vasquez, B.J.;

Liang, K.C.; and McGaugh, J.L. Enkephalin and fear-motivated behavior.

Proc Natl Acad Sci USA 77:3729-3732, 1980b.

Satoh, M.; Ueda, H.; Yoshihara, Y.; Misawa, H.; Fukushima, N.;

Katada, T.; Ui, M.; and Takagi, H. Kyotorphin receptors mediate phospholipase C stimulation via Gi

-protein. International Narcotic Research

Conference Abs, 1988. p. 83.

Satoh, M.; Wada, T.; Iwama, T.; and Takagi, H. Sites of analgesic actions

of kyotorphin and d-kyotorphin in the central nervous system of rats.

Neuropeptides 5:415-418, 1985.

Scharfman, H.E., and Sarvey, J.M. Postsynaptic firing during repetitive

stimulation is required for long-term potentiation in the hippocampus.

Brain Res 331:267-274, 1985.

Schulteis, G., and Martinez, J.L., Jr. Further studies on the role of opioid

delta receptors in the effects of [leu]enkephalin on active avoidance conditioning in mice. Soc Neurosci Abs 14:1029, 1988.

Schulteis, G.; Martinez, J.L., Jr.; and Hruby, V.J. Stimulation and antagonism of opioid delta receptors produce opposite effects on active avoidance conditioning in mice. Behav Neurosci 102:678-686, 1988.

Schulteis, G.; Weinberger, S.B.; and Martinez, J.L., Jr. Plasma uptake and

in vivo metabolism of [leu]enkephalin following its intraperitoneal administration to rats. Peptides, in press.

Schultzberg, M.; Lundberg, I.M.; Hokfelt, T.; Terenius, L.; Brandt, J.;

Elde, R.P.; and Goldstein, M. Enkephalin-like immunoreactivity in gland

cells and nerve terminals of the adrenal medulla. Neuroscience 3:1169-

1186, 1978.

Segal, M. Effects of mu opioid receptor activation in the rat hippocampal

slice. In: McGinty, J.F., and Friedman, D.P. Opioid in the Hippocampus. National Institute on Drug Abuse Research Monograph 82.

DHHS Pub. No. (ADM)88-1568. Washington, D.C.: Supt. of Docs.,

U.S. Govt. Print. Off., 1988. pp. 133-145.

Shaw, J.S.; Miller, L.; Turnbull, M.J.; Gormley, J.J.; and Morley, J.S.

Selective antagonists at the opiate delta-receptor. Life Sci 31: 1259-1262,

1982.

Siggins, G.R. Opioids reciprocally regulate the M-current in hippocampal

CA3 pyramidal neurons in vitro. Soc Neurosci Abs 14:464, 1988.

76
Stringer, J.L.; Greenfield, L.J.; Hackett, J.T.; and Guyenet, P.G. Blockade

of long-term potentiation by phencyclide and sigma opiates in the hippocampus in vivo and in vitro. Brain Res 280:127-138, 1983.

Takagi, H.; Satoh, M.; Hirohito, S.; Akaike, A.; Ueda, H.; Kawajiri, S.-I.;

Yamamoto, M.; and Amano, H. Identification of a new opioid peptide

from the bovine brain and its mechanism of action. In: Way, E.L., ed.

Endogenous and Exogenous Opiate Agonists and Antagonists. New York:

Pergamon Press, 1979a. pp. 201-204.

Takagi, H.; Shiomi, H.; Ueda, H.; and Amano, H. Morphine-like analgesia

by a new dipeptide, L-Tyrosyl-L-Arginine (kyotorphin) and its analogue.

Eur J Pharmacol 55:109-111, 1979b.

Venturelli, F.; Gilardi, E.; Roscetti, G.; Laugesen, G.; Barra, P.F.; and

Roda, L.G. Control mechanisms of peripheral enkephalin hydrolysis in

mammalian plasma. Comp Biochem Physiol 83:307-311, 1986.

Viveros, O.; Diliberto, E.J.; Hazum, E.; and Chang, K-J. Enkephalins as

possible adrenomedullary hormones: Storage, secretion, and regulation of

synthesis. In: Costa, E., and Trabucchi, M., eds. Advances in Biochemical Psychopharmacology. New York: Raven Press, 1980. pp. l14.

Washington, M.K.; Lieberman, D.N.; Derrick, B.E.; and Martinez, J.L., Jr.

Two forms of long-term potentiation in the CA3 region of the rat in vivo.

Soc Neurosci Abs 14:565, 1988.

Weinberger, S.B.; Gehrig, C.A.; and Martinez, J.L., Jr. [Leu]enkephalin and

its delta selective analog, D-pen2-[D-pen5]enkephalin, impair avoidance

conditioning in an automated shelf-jump task in rats. Soc Neurosci Abs

14: 1029, 1988.

Weinberger, S.B.; Gehrig, C.A.; and Martinez, J.L., Jr. D-pen2-[D-pen5]enkephalin, a delta opioid receptor-selective analog of [leu]enkaphalin,

impairs avoidance learning in an automated shelf-jump task in rats. Reg

Peptides, in press.

Weinberger, S.B., and Martinez, J.L., Jr. Differential effects on active

avoidance performance and locomotor activity of two major enkephalin

metabolites, Tyr-Gly-Gly and des-Tyr-[leulenkephalin. Life Sci 43:769-

776, 1988.

Wigstrom, H., and Gustafsson, B. Facilitated induction of hippocampal

long-lasting potentiation during blockade of inhibition. Nature 301:603-

604, 1985.

Yamamoto, M.; Kawamuki, H.; Satoh, M.; and Takagi, H. Kyotorphin

(L-TYR-AR) inhibits extinction of pole-jumping avoidance response in the

rat. Neurosci Lett 31:175-179, 1982.

Yang, H.Y.T.; Hexum, T.; and Costa, E. Opioid peptides in adrenal gland.

Life Sci 27:1119-1125, 1980.

Zieglegansberger, W.; French, E.D.; Siggins, G.R.; and Bloom, F.E. Opioid

peptides may excite hippocampal pyramidal neurons by inhibiting adjacent

inhibitory interneurons. Science 205:415-416, 1979.

77
Zlokovic, B.V.; Begley, D.J.; and Chain-Eliash, D.G. Blood-brain barrier

permeability to leucine-enkephalin, d-alanine-d-leucine-enkephalin and

their N-terminal amino acid (tyrosine). Brain Res 336:125-132, 1985.

Zlokovic, B.V.; Lipovac, M.N.; Begley, D.J.; Davason, II.; and Rakic, L.J.

Transport of leucine-enkephalin across the blood-brain barrier in the

perfused guinea-pig brain. J Neurochem 49:310-315, 1987.

ACKNOWLEDGMENTS

Portions of this work were supported by U.S. Public Health Service grants

DA 04195 and DA 04795, grants 1 F31 DA 05334-01 and 1 F32 DA

05313-01 from the National Institute on Drug Abuse, and the Rennie Fund.

AUTHORS

Joe I,. Martinez, Jr., Ph.D.

Patricia H. Janak

Susan B. Weinberger. Ph.D.

Gery Schulteis

Brian E. Derrick

Department of Psychology

University of California

Berkeley. CA 94720

78
The Effects of Delta-9-THC on

Mechanisms of Learning and

Memory

Sam A. Deadwyler, Charles J. Heyser, Ron C. Michaelis,

and Robert E. Hampson

INTRODUCTION

One of the most important concerns regarding drug abuse by the individual

in society is the potential impairment in learning and performance while

under the influence of these agents. The added threat of long-term exposure to these substances suggests that drug-induced altered neurobiological

mechanisms may not only change cognitive and behavioral processes when

the drug is present, but also may have persistent effects on these mechanisms; effects that outlast the duration of drug exposure. It is, therefore,

important to ascertain the action of abused substances on known neurobiological mechanisms of learning and memory in order to evaluate both cognitive and performance deficits that accrue following short- and long-term

exposure to these substances.

Modem neurobiological conceptualizations of learning processes have used

information-processing models to approximate the manner in which the brain

encodes and retrieves experiences (Hopfield 1984). This approach has been

applied to animal models of learning and memory. To the extent that such

models achieve a high degree of representativeness of human information

processing, they can serve as a neurobiological base for manipulations of

neural processes and as a determination of neurophysiological correlates

(Donchin et al. 1986). Such processes, however, are not easily identified.

In the past, one limitation regarding the appropriateness of animal models

for human information processing has been the inability to construct the

task for the animal to test the same cognitive function(s) that can be

assessed in humans. Recently, however, there have been important breakthroughs in this capability (Kesner and Beers 1988; Olton 1984) that suggest that certain cognitive functions associated with specific brain regions in

animal models have similar functions in humans.

The hippocampus has been one of the brain structures most consistently

implicated in the neurobiological basis of learning and memory (Thompson

79
et al. 1983). There is abundant evidence suggesting that this structure is

critically involved in many aspects of the spatial and temporal coding of

sensory information (Eichenbaum and Cohen 1988). Both human and animal models have recently yielded convergent information showing the requirement of an intact hippocampus to encode and retrieve task relevant

sensory information (Winocur 1984). With the current emphasis on behavioral tasks that measure specific types of memory, the precise nature of the

memory deficits caused by disruption of the hippocampus and related structures is becoming better characterized (Olton, in press; Squire and Cohen

1984; Mishkin and Petri 1983). An important goal for eventually understanding the manner in which drugs of abuse influence memory processes is

knowledge of how the hippocampus processes sensory information and

which aspects of hippocampal function are altered by exposure to these

substances.

Understanding how drugs of abuse alter performance and cognition in

humans may rely greatly on knowledge of how these drugs influence hippocampal mechanisms of learning and memory. If it can be shown that

disruptions in performance brought about by drug exposure are related in a

dose-dependent manner to alterations in hippocampal function, it is likely

that the actions of the abused substance can then be directly related to other

forms of hippocampal disruption.

EFFECTS OF DELTA-9-THC ON DETECTION OF SENSORY

EVENTS

There are several aspects of learning and memory processes that could be

altered by drugs abused in society. Delta-9-THC, the psychoactive ingredient in marijuana, has a long history of being associated with memory

impairments in humans (Miller and Branconnier 1983). Delineation of the

exact manner in which a substance like delta-9-THC affects learning and

performance is, therefore, an important consideration and a first step in

determining the neurobiological basis of the memory-disruptive effects of

this agent. Such endeavors require determination of the possible influence

of delta-9-THC pharmacology on critical stages of information processing

and retrieval (Dewey 1986). As a first step, it is important to determine

whether delta-9-THC disrupts memory and performance by distracting the

animal from attending to the critical features of the environment.

Animals were trained to detect a very short duration (250 milliseconds) tone

stimulus that varied in intensity. Tone intensity ranged across seven different levels, with the midpoint anchored each day at the behavioral detection

threshold (defined as 50 percent responding). This paradigm generated a

series of input-output curves ranging from zero correct at the lowest intensity (no tone) to 96 percent correct responses to the highest tone intensity

(figure 1). In addition, it was possible to determine the relationship of

hippocampal activity to behavioral detection of tone stimuli. In control

80
sessions, sensory-evoked potentials were present in the hippocampus only on

those trials in which the tone stimulus was detected (responded to) behaviorally but not when the stimulus was undetected. When the tone was responded to, a long (onset) latency (70-millisecond) component (N2) of the

well-characterized averaged stimulus-evoked potential was registered in the

dentate gyrus of the hippocampus (Deadwyler et al. 1981; Deadwyler et al.

1985). The acute effects of delta-9-THC and recovery from that exposure

were examined on the behavioral aspects of tone detection in this task.

FIGURE 1. Behavioral performance curves and hippocampal correlates of

auditory signal detection

NOTE: Upper: Hippocampal sensory-evoked potentials OM AEPs from two different animals averaged over tone level 3 (below) and sorted on the basis of behaviorally defected (responded to)

and nondetected stimuli. The upper trace shows averages from detect trials only in which a

typical sensory-evoked potential recorded from the outer molecular layer of the dentate gyrus

is present. The bottom trace indicates the absence of sensory-evoked activity on nondetect

trials at the same tone level (3) on trials in which the tone was not detected behaviorally.

Each trace is the result of over 50 averaged single-trial potentials. The dotted trace represents

the standard error over all averaged trials. Bottom: Behavioral performance curves for each

of the seven tone-intensity levels for each animal. Level 3 is designated as threshold (T) on

the basis of a 50-percent response criterion. Note, however, that tone level 2 was detected 30

percent of the time, and levels 4 to 6 were detected more than 50 percent of the time. Bars

represent ± SEMs over all sessions.

81
Delta-9-THC had a marked effect on behavioral performance in this task.

Figure 2 shows the effects of delta-9-THC for each of four different dose

levels. The 0.5 mg/kg dose was not effective in altering sensory detection

thresholds, while doses of 1.0, 1.5, and 2.0 mg/kg systematically increased

detection threshold and decreased performance accuracy. Importantly, these

changes in detection were not accompanied by alterations in the latency to

respond to the tone stimulus on detection trials at any dose level. The

effects of delta-9-TIlC were, therefore, not the result of a direct motor

impairment that incapacitated responding. Since the performance curves

were shifted to the right as a function of increasing dose of administration,

the decrease in behavioral sensitivity appeared to be linearly related to

increasing systemic levels of delta-9-THC. At the higher doses of delta-9-

THC (1.5 mg/kg), there was no recovery of the tone detection behavior

within 2 to 4 hours after injection (figure 2). Preliminary evidence showed

that the change in detection threshold was accompanied by a decrease in

amplitude and appearance of hippocampal sensory-evoked potentials under

the influence of delta-9-TllC.

FIGURE 2. Dose-response effects of delta-9-THC on signal detection

performance during predrug, postdrug, and recovery

sessions

NOTE: Mean percent correct responses for each dose level are shown averaged over all tone-intensity

levels. Note that significant recovery did not occur following 2 to 4 hours, the 1.5 and 2.0

mg/kg doses. Bars indicate SEMs.

82
These findings show that delta-9-THC raised the threshold for detecting

auditory stimuli in a dose-dependent manner. The deficit in tone detection

produced by delta-9-THC can be described as due to a decrease in recognition, since there were no changes in latency to respond. This leads to the

question of whether or not delta-9-THC selectively influenced the ability of

animals to discriminate as a function of dose levels that produce the recognition deficit.

EFFECTS OF DELTA-9-THC ON TONE DISCRIMINATION

PERFORMANCE IN RATS

In previous studies, delta-PTHC produced a dose-dependent disruption of

auditory discrimination performance in rats. The deficit was manifested as

a decreased responding to the positive (i.e., reinforced) tone in a successive

(two tone) discrimination paradigm. This deficit was not accompanied by

an increase in responding to the negative (unreinforced) tone (Campbell

et al. 1986a). The decrease in discrimination performance in this paradigm

was accompanied by a significant dose-dependent increase in behavioral

latency (figure 3). Even though the task can be performed at about 90

percent efficiency in animals with hippocampal lesions, this behavioral paradigm is sufficient to evoke considerable task-related changes in extracellularly recorded hippocampal synaptic and cellular activity (Deadwyler et al.

1981; Deadwyler 1985). Hippocampal electrical correlates of performance

accuracy were disrupted during behavioral impairment by acute injections of

delta-9-THC. An intraperitoneal dose of 0.5 mg/kg of delta-9-THC

produced minimal alterations in behavior and in the components of sensoryevoked synaptic processes, as well as in granule cell discharges to the conditioned tone stimulus. As in the tone detection task, a dose of 2.0 mg/kg

produced a marked (60 percent) disruption in behavioral performance and in

hippocampal tone-evoked electrical activity (figures 3, 4, and 5), while

0.5 mg/kg had little effect. Baseline (predrug) levels of discrimination performance and hippocampal electrical activity recovered within 2 to 4 hours

after a single acute injection (Campbell et al. 1986a; Campbell et al. 1986b)

for all dose levels.

The changes manifested in hippocampal electrical correlates during the twotone discrimination task were similar over the same dose range as the

behavioral deficits. However, a marked specificity of the delta-9-THC

influence on hippocampal processes was shown by the differential nature of

the effect on the amplitudes of two separate components of the wellcharacterized tone-evoked potential (figure 4). Recently, it has been

determined how such changes in sensory-evoked potential amplitudes were

produced by delta-9-THC. The effects were the result of changes in the

sequential dependency of trial-to-trial amplitude fluctuations in the

hippocampal sensory-evoked potentials (Deadwylcr et al. 1985). Fluctualions in amplitude of these components are predictable in the sense that

they are determined by the preceding sequence of trials and not by the

83
FIGURE 3. Dose-response effects of delta-9-THC on dual tone discrimination performance for predrug, postdrug, and recovery

sessions as shown in figure 2

NOTE: Mean percent responses to the positive conditioned stimulus (CS+) tone are shown. Bars

indicate SEMs.

actual tone stimulus (positive or negative) presented on any given trial

(West et al. 1982). Thus, runs of positive or negative tones produce

respective decreases or increases in amplitude of the early (N1) component

of the sensory-evoked potential relative to its (intermediate) amplitude on

single-alternating trials (figure 6). Double-alternating trials produce maximal

and minimal fluctuations in N1, presumably because the sequence appears to

oscillate between “runs” of positive or negative trials (i.e., two like trials in

a row). This sequential dependency suggests that information is continually

processed on a trial-by-trial basis and that the temporal context in which a

particular trial occurs determines the magnitude of its effect on hippocampal

cellular and synaptic activity (Foster et al. 1988).

Since delta-9-THC had a pronounced dose-dependent influence on hippocampal electrical correlates, it is likely that the disruption resulted from a

change in the sequential dependency of the electrical events. This was

examined using previous methods of sorting and combining single trial data.

84
FIGURE 4. Effects of delta-9-THC on hippocampal sensory-evoked

potentials recorded daring performance of dual tone

discrimination task as shown in figure 3

NOTE: Upper panel shows marginal and maximal effects related to the lowest (0.5 mg/kg) and

highest (2.0 mg/kg) doses of delta-9-THC. Graph at the top shows mean amplitude changes

measured over all animals and doses for the three types of sessions. Bars are ± SEMs.

Delta-9-THC (1.0 mg/kg dose) altered the sequential fluctuations in the

early component (N1) of the sensory-evoked potential. This component,

which reflects transmission of sensory information from the entorhinal cortex to the dentate gyrus via the perforant path (Deadwyler et al. 1981;

Foster et al. 1988), exhibited two major changes from the normal pattern of

trial-to-trial fluctuation: (1) the degree of amplitude change during singlealternating trial sequences was drastically exaggerated, and (2) there was a

85
FIGURE 5. Effects of delta-9-THC on tone-tvoked dentate granule cell

(g-cell) activity recorded in the dentate gyrus of the

hippocampal formation

NOTE: Poststimulus histograms are shown for predrug, postdrug, and recovery sessions at two dose

levels indicating decreased g-cell discharge that recovered differentially as a function of dose

level. In each case, an isolated g-cell was recorded during a 100-trial session. Poststimulus

histograms are normalized across trials, sessions, and animals. Dotted tracings above histogram indicate SEM-of-unit discharges. Calibration: 0.2 spikes/bin.

shift in the pattern of N1 amplitude fluctuation such that N1 amplitude

switches appropriate to changes in particular sequences did not occur on the

same trial as in control sessions (figure 6). The shift in amplitude during

single-altemation trials was due to a change from intermediate-amplitude N1

potentials on unrewarded stimulus (CS-) trials to virtually no response on

those same types of trials under the influence of delta-9-THC (1.0 mg/kg).

This occurred without any significant change in N1 amplitude on CS+ trials

during either single- or double-alternation trial sequences (figure 6).

Although the general pattern of amplitude fluctuation for other types of

sequences remained similar to that in the control condition, there was a onetrial “lag” in the large fluctuations across different sequences with respect to

predrug sessions. This is illustrated in figure 6, where the pattern of

change for each sequence type is shifted slightly to the right by one trial.

Since the pattern and not the amplitude of these potentials was altered after

delta-9-THC, it is likely that the drug action was specific to informationprocessing mechanisms in the hippocampus.

The change in pattern of evoked potential amplitude fluctuation may have

reflected a decreased mnemonic capacity to track changes in trial sequences

produced by delta-9-THC. This was assessed by examining the serial

dependence of N1 amplitude as a function of the number of trials preceding

the current trial (Deadwyler et al. 1985). This analysis showed that under

control conditions the most recent trial (position 1) exerted the largest

86
FIGURE 6. Effects of delta-9-THC (1.0 mg/kg) on trial-to-trial

fluctuations in N1 amplitude provoked by specific types of

trial sequences

NOTE: Mean amplitude of the N1 component (see figure 4) is plotted for potentials in which the trial

evoking the potential was preceded by five trials of the type indicated on the axis marked

trial sequence. Note that single-alternating trial sequences were most affected by delta-9-THC

(post-THC), causing maximal-to-minimal amplitude changes that do not normally occur for

type-of-sequence in predrug control sessions (pre-THC). Means are averaged over animals,

trials. and sessions.

influence on the amplitude of N1, while the next four serial positions in

the sequence were much less influential, showing only about a 10- to 12-

percent contribution for each successive position. Figure 7 illustrates the

serial position curve for N1 amplitude in the predrug and 1.0 mg/kg-delta-9-

THC condition. The contribution of the most recent and the most remote

(i.e., the one furthest removed in time) trials were significantly altered by

injections of delta-9-THC. The manifestation of these changes in serial

position effect is verified by the altered pattern of sequential influences discussed above (figure 6). These findings suggest that the mechanics of

serial processing of sensory events in the hippocampus were disrupted by

delta-9-THC. Given this observation, it was important to determine whether

or not this disruption would be manifested in tasks in which the retrieval of

trial-specific information is required.

87
FIGURE 7. Influence of delta-9-THC trial-position effect in the prior

sequence on N1 amplitude

NOTE: The most influential trial averaged over all sequences was most recent (i.e., the trial immediately preceding the trial on which amplitude was assessed). The remaining positions contributed relatively equal amounts but were much less influential. Delta-9-THC (1.0 mg/kg)

decreased the percentage contribution of the most recent trial (position 1), but increased the

contribution of the more remote trials (positions 4 and 5) in the sequence.

EFFECTS OF DELTA-9-THC ON RETENTION IN A DELAYEDMATCH-TO-SAMPLE (DMTS) TASK

The demonstration that the same dose range of delta-9-THC systematically

increased the detection threshold for tones presented at different intensities

and decreased responding in the presence of the positive tone in a successive discrimination paradigm supports previous suggestions of delta-9-THC’s

capacity to alter the perception of stimulus features as demonstrated in

human studies (Hollister 1974; Pertwee 1983). Previous investigations in

humans have shown that the most consistent effect of exposure to delta-9-

THC on performance is the disruption of short-term memory tasks requiring

retention of item-specific information (Miller and Branconnier 1983). The

subtle but significant change in sequential dependency of tone-evoked electrical activity produced by delta-9-THC, however, implicates the disruption

of retention as well as perceptual processes.

88
This possibility was investigated by testing animals in a DMTS task that

required retention of spatial information over a l- to 31-second delay interval. Performance in this task is critically dependent on the length of the

delay interval and decreases from 92 percent at l- to 5-second delays to

70 percent at the 25- to 31-second delay period. If delta-9-THC produced

an impairment in retention of the DMTS task under the influence of delta9-THC should be selectively reduced at long, but not at short, delay

intervals.

Figure 8 shows the effects of delta-9-THC on DMTS performance. There

was a marked dose-by-delay effect, which was manifested across the 0.5- to

2.0-mg/kg dose range. The 2.0-mg/kg dose of delta-9-THC disrupted performance at the longer delay intervals; however, performance during l- to

5-second delay intervals was not significantly altered. Thus, the deficit at

longer delay intervals was not due to a generalized effect of the delta-9-

THC on the spatial discrimination performance. Numerous studies have

established that a deficit in DMTS performance of the type demonstrated

here is the hallmark of hippocampal disruption (Rawlins 1985; Eichenbaum

and Cohen 1988). The results of the DMTS task further implicate the

hippocampus as a target of delta-9-THC action.

DELTA-9-THC AND HIPPOCAMPAL CORRELATES OF LEARNING

AND MEMORY

In the three types of studies, the dose-dependent behavioral effects of

delta-9-THC were associated with behavioral deficits characteristic of hippocampal damage. In addition, hippocampal electrophysiological correlates

were altered over the same dose range of delta-9-THC as the behavior.

The signal detection results suggest that, at higher doses (above 0.5 mg/kg

in the rat) of delta-9-THC, behavioral impairments may arise from the lack

of registration of the sensory information in the hippocampus (Wilkison and

Pontzer 1987). In this connection, it is curious that recovery from acute

exposure to THC was slower in the signal detection task than in the twotone discrimination task at the same dose levels. Thus, delta-9-THC may

have progressively more severe influence on tasks requiring a high degree

of vigilance than on tasks where the stimulus termination is response contingent (discrimination and DMTS tasks). The disruptive effects of delta-9-

THC in the tone discrimination task were manifested as a decrease in

responsiveness to the positive tone stimulus (Campbell et al. 1986a) but

not as an increase in responding to the negative stimulus. Given the tone

detection results, an increase in the detection threshold of the positive

stimulus may have been partially responsible for these results. However,

since recovery on the tone discrimination task was more rapid than in

the signal detection task over the same dose range, the effects of delta-9-

THC differed with respect to the behavioral requirements of the task, even

though the presentation of sensory stimuli in the two tasks was quite similar. Performance in all three tasks was not affected by cannabidiol, the

89
nonpsychoactive derivative of marijuana, even at twice the highest dose of

delta-9-THC (Martin 1986)

FIGURE 8. Effects of delta-PTHC on DMTS performance at four

different dose levels

NOTE: There is a progressive increase in effects of delta-9-THC at the longer delay intervals. Performance was not affected at the 1- to 5-second delay intervals, indicating no gross disruption

in the two-lever spatial discrimination capacity.

It is also important to note that the dose range shown to be effective in all

three of the behavioral paradigms described was sufficient to produce a full

range of disruption in behavior and hippocampal neural correlates. The

degree to which the various behaviors were sensitive to delta-9-THC is

reflected in the rank ordering of the severity of deficits at each dose level:

the signal detection task was the most sensitive, the tone discrimination task

was next, and the DMTS task was the least affected at each of the four

dose levels. This ranking agrees, in general, with the recent reports of

delta-9-THC action in the monkey on similar behavioral tasks (Schulze et

al. 1988). In terms of the tasks that have been shown to be the most dependent on the integrity of the hippocampus, however, the DMTS deficit

was the most similar to that following a hippocampal lesion (Gaffan et al.

1984; Etherington et al. 1987; Rawlins 1985).

90
CONCLUSION

In conclusion, it can be stated that delta-9-THC can differentially alter

behavioral processes requiring information processing by the hippocampus,

including the detection and processing of conditioned sensory stimuli, as

well as the retrieval of trial-specific information. Recent reports have

shown the hippocampus to be a target for neuropathological changes following long-term exposure to delta-9-THC (Landfield et al. 1988; Scallet et al.

1987). Tasks that require a higher degree of vigilance, such as the tone

detection task, exhibited a more marked influence at intermediate- and highdose levels (1.0 to 2.0 mg/kg). A similar relationship was also reported in

the recent studies on monkeys (Schulze et al. 1988). This same dose, however, did not affect recall of recently acquired information over brief time

intervals in the DMTS task. In the latter case, delta-9-THC differentially

altered responding a! longer delay intervals. Thus, delta-9-THC appears to

be a potent centrally acting agent that has critical consequences for the

maintenance of optimal performance in tasks requiring either the detection

and/or the recall of sensory information. One of the actions of this compound appears to be directed toward disruption of hippocampal neural

processes involved in the performance of such tasks.

REFERENCES

Campbell, K.A.; Foster, T.C.; Hampson, R.E.; and Deadwyler, S.A. Delta9-tetrahydrocannabinol differentially affects sensory-evoked potentials in

the rat dentate gyrus. J Pharmacol Exp Ther 239:936-940, 1986a.

Campbell, K.A.; Foster, T.C.; Hampson, R.E.; and Deadwyler, S.A. Effects

of delta-9-tetrahydrocannabinol on sensory evoked discharges of granule

cells in the dentate gyrus of behaving rats. J Pharmacol Exp Ther

239:941-945, 1986b.

Deadwyler, S.A. Involvement of hippocampal systems in learning and

memory. In: Weinberger, N.J.; McGaugh, J.L.; and Lynch, G., eds.

Memory Systems and the Brain. New York: Guilford Press, 1985.

pp. 1-49.

Deadwyler, S.A.; Foster, T.C.; and Hampson, R.E. Processing of sensory

information in the hippocampus. Crit Rev Clin Neurobiol 2:335-355,

1987.

Deadwyler, S.A.; West, M.O.; Christian, E.P.; Hampson, R.E.; and Foster,

T.C. Sequence-related changes in sensory-evoked potentials in the dentate

gyms: A mechanism for item-specific short-term information storage in

the hippocampus. Behav Neural Biol 44:201-212, 1985.

Deadwyler, S.A.; West, M.O.; and Robinson, J.H. Entorhinal and septal

inputs differentially control sensory-evoked responses in the rat dentate

gyrus. Science 211:1181-1183, 1981.

Dewey, W.L. Cannabinoid pharmacology. Pharmacol Rev 38:151-178,

1986.

9 1
Donchin, E.; Karis, D.; Bashore, T.; Coles, M.G.H.; and Gratton, G. Cognitive psychophysiology and human information processing. In: Coles,

M.G.H.; Donchin, E.; and Porges, S.W., eds. Psychophysiology:

Systems, Processes, and Applications. New York: Guilford Press, 1986.

pp. 244-266.

Eichenbaum, H., and Cohen, N.J. Representation in the hippocampus:

What do hippocampal neurons encode? TINS 11:244-248, 1988.

Etherington, R.; Mittleman, G.; and Robbins, T.W. Comparative effects of

nucleus basalis and fimbria-fornix lesions on delayed matching and alternation tests of memory. Neurosci Res 1:135-143, 1987.

Foster, T.C.; Hampson, R.E.; West, M.O.; and Deadwyler, S.A. Control of

sensory activation of granule cells in the fascia dentata by extrinsic

afferents: Septal and entorhinal inputs. J Neurosci 8:3869-3878, 1988.

Gaffan, D.; Gaffan, E.A.; and Harrison, S. Effects of fornix transection on

spontaneous and trained non-matching by monkeys. Q J Exp Psychol [B]

36:285-303, 1984.

Hollister, L.E. Structure-activity relationships in man of cannabis constituents, and homologs and metabolites of delta-9-tetrahydrocannabinol.

Pharmacology 11:3-11, 1974.

Hopfield, J.J. Neurons with graded response have collective computational

properties like those of two-state neurons. Proc Natl Acad Sci USA

81:3088-3092, 1984.

Kesner, R.P., and Beers, D.R. Dissociation of data-based and expectancybased memory following hippocampal lesions in rats. Behav Neural Biol

50:46-60, 1988.

Landfield, P.W., Cadwallader, L.B., and Vinsant, S. Quantitative changes in

hippocampal structure following long-term exposure to delta-9-tetrahydrocannabinol: Possible mediation by glucocorticoid systems. Brain Res

443:47-62, 1988.

Martin, B.R. Cellular effects of cannabinoids. Pharmacol Rev 38:45-74,

1986.

Miller, L.L., and Branconnier, R.J. Cannabis: Effects on memory and the

cholinergic limbic system. Psychol Bull 93:441-456, 1983.

Mishkin, M., and Petri, H.L. Memories and habits: Some implications for

the analysis of learning and retention. In: Squire, L.R.; Lynch, G.; and

Weinberger, N., eds. Neuropsychology of Memory. New York: Guilford

Press, 1983. pp. 65-77.

Olton, D.S. Animal models of human amnesias. In: Squire, L.R., and

Butters, N., eds. Neuropsychology of Memory. New York: Guilford

Press, 1984. pp. 367-373.

Olton, D.S. Inferring psychological dissociations from experimental dissociations: The temporal context of episodic memory. In: Varieties of

Memory and Consciousness: Essays of Endel Tulving. New York:

Lawrence Erlbaum Associates, in press.

Pertwee, R.G. Cannabis. In: Graham-Smith, D.G., and Cower, P.J., eds.

Psychopharmacology, Part I. Preclinical Psychopharmacology.

Princeton: Excerpta Medica, 1983. pp. 377-397.

92
Rawlins, J.N.P. Associations across time: The hippocampus as a temporary memory store. Behav Brain Sci 8:479-528, 1985.

Scallet, A.C.; Uemura, E.; Andrews, A.; Ali, S.F.; McMillan, D.E.;

Paule, M.G.; Brown, R.M.; and Slikker, W., Jr. Morphometric studies of

the rat hippocampus following chronic delta-9-tetrahydrocannabinol (THC).

Brain Res 436:193-198, 1987.

Schulze, G.E.; McMillan, D.E.; Bailey, J.R.; Scallet, A.; Ali, S.F.;

Slikker, W., Jr.; and Paule, M.G. Acute effects of delta-9-tetrahydrocannabinol in rhesus monkeys as measured by performance in a battery of

complex operant tests. J Pharmacol Exp Ther 245:178-186, 1988.

Squire, L.R., and Cohen, N.J. Human memory and amnesia. In: Lynch,

G.; McGaugh, J.L.; and Weinberger, N.M., eds. Neurobiology of Learning and Memory. New York: Guilford Press, 1984. pp. 3-64.

Thompson, R.F.; Berger, T.W.; and Madden, J., IV. Cellular processes of

learning and memory in the mammalian CNS. Annu Rev Neurosci 6:447-

491, 1983.

West, M.O.; Christian, E.; Robinson, J.H.; and Deadwyler, S.A. Evoked

potentials in the dentate gyrus reflect the retention of past sensory events.

Neurosci Lett 28:319-324, 1982.

Wilkison, D.M., Pontzer, N.J. The actions of THC on the intact hippocampus: A comparison of dentate and CA1 responses. Brain Res Bull

19:63-67, 1987.

Winocur, G. Memory localization in the brain. In: Squire, L.R., and

Butters, N., eds. Neuropsychology of Memory. New York: Guilford

Press, 1984. pp. 122-133.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health grants

DA 03502, DA 04441, and DA 00119.

AUTHORS

Sam A. Deadwyler, Ph.D., Professor of Physiology

Charles J. Heyser, Research Assistant

Ron C. Michaelis, Ph.D., Research Associate

Robert E. Hampson, Ph.D., Research Associate

Department of Physiology and Pharmacology

Bowman Gray School of Medicine of

Wake Forest University

300 South Hawthorne Road

Winston-Salem, NC 27103

93
Calcium-Mediated Events in

Associative Learning

John F. Disterhoft

INTRODUCTION

This chapter will summarize a series of experiments analyzing the cellular

bases of rabbit eyeblink conditioning with a combination of in vivo, in vitro,

and neuropharmacological approaches. The hippocampus was chosen as a

starting point. Previous experiments analyzing the involvement of the

hippocampus in associative learning have generally analyzed the effects of

dorsal hippocampal lesions on the human’s or animal’s ability to acquire or

to retain a learned behavioral task (O’Keefe and Nadel 1978; Squire 1987).

A second major approach has utilized extracellular recording of single or

multiple neurons in the behaving animal during or after learning (Segal

1973; Berger et al. 1980). These approaches have demonstrated a profound

involvement of the hippocampus in many associative learning tasks.

The experiments that will be described have added a new approach to those

two basic techniques. Our purpose is to analyze the cellular events that

underlie the increased excitability reported to occur in hippocampal neurons

during and after associative learning. In addition, we wish to evaluate

cellular alterations that we know a priori are local to the hippocampus and

are not secondary reflections in this region of alterations located elsewhere

on the conditioned reflex arc. This localization issue is a severe problem in

attempting to localize neural substrates of learning with lesions or recording

in the intact animal.

Therefore, an in vitro approach has been used in combination with in vivo

experiments. Hippocampal neurons in brain slices from trained animals

were evaluated biophysically (Disterhoft et al. 1984). We knew beforehand

that any conditioning-specific alterations observed in the hippocampal slice

had to be localized there. They cannot be interpreted as secondary reflections of changes projected to the hippocampus from some other brain

region-neurons in the slice are separated from their normal interconnections

with other brain regions.

Drain slice experiments have delineated the importance of conditioningspecific reductions in slow calcium-mediated potassium currents during

94
learning (Disterhoft et al. 1988a). These results encouraged us to test the

effect of nimodipine, a calcium channel blocker, on acquisition of eyeblink

conditioning. This chapter will conclude by describing the facilitation in

learning eyeblink conditioned responses observed in rabbits treated with this

dihydropyridine (Deyo et al. 1989). The facilitation is particularly interesting, because it is most clearcut in aging rabbits.

EYEBLINK CONDITIONING IN RABBITS

Behavioral training was done with standard procedures for rabbit eyeblink

or nictitating membrane conditioning (Disterhoft et al. 1977). Experiments

were controlled, behavioral data stored online, and data processed and displayed on- or offline by computer. Subjects were young adult male albino

rabbits, Oryctolagus cuniculus, weighing 1.5 kg at the start of training

(except, of course, in the aging studies). The pure tone conditioned stimulus (CS) was a 2,000 Hz pure tone presented at 85 db in a sound-attenuated

chamber. In delay conditioning, the 400-millisecond tone CS was paired

and coterminated with a 150-millisecond air puff or periorbital shock unconditioned stimulus (UCS) just sufficient to cause a reliable blink unconditioned response (UCR). Pseudoconditioned control rabbits received

explicitly unpaired presentations of the CS and UCS in a random fashion.

Trace conditioning combined a 100-millisecond, 6,000 Hz tone pip CS

followed after 300 milliseconds with a 150-millisecond corneal air puff.

Pseudoconditioning control training again consisted of explicitly unpaired

presentations of the CS and UCS. In the nimodipine experiments, the trace

interval was 500 milliseconds. An example of the type of behavioral

responses observed on individual trials early and late in trace conditioning

as well as the acquisition curve for one group of eight rabbits is shown in

figure 1. Trace conditioning (even with only a 300-millisecond trace interval) has been found to be much more difficult for the rabbits to acquire

than delay conditioning. For example, almost all rabbits trained for 3 days

in the delay task reached 80 percent of the total CRs on the third training

day. However, it takes, on average, 10 training sessions for rabbits to reach

an 50-percent behavioral criterion in the trace paradigm with a 500-millisecond trace interval (Moyer et al., in press).

SINGLE NEURON RECORDING

Berger and Thompson and their colleagues have described a striking neural

modeling of the eyeblink conditioned response (CR) in CA1 and CA3 pyramidal neurons in both multiple and single unit recordings (Berger et al.

1980; Berger et al. 1983). Recording has begun of single neurons in CA1

hippocampus of trace-conditioned rabbits (Akase et al. 1988). A variety of

responses have been seen in the complex spike cells of CA1 hippocampus

(presumed pyramidal cells). Almost all neurons are functionally

95
FIGURE 1. Trace eyeblink conditioning using a 300-millisecond

trace interval

NOTE: Examples of eyeblink responses on individual trials early in training (left) and during

asymptotic acquisition. The center panel shows an acquisition curve for the last 5 days of

training for a group of eight rabbits. Note that trace eyeblink conditioning, even with only a

300-millisecond trace interval. is a considerably more difficult task for the albino rabbit. This

group of animals took an average of 7.5 days to teach an 80 percent CR performance

criterion; a comparable group trained in the delay task required 2 to 3 days.

96
modulated during the trials. Some cells show clear modulation similar to

the type described by Berger and colleagues, except with an additional

sensory response to the tone CS (figure 2). Others show clear inhibitory

components in their response profiles, which is especially marked during the

tone CS air puff UCS interstimulus interval and may be followed by enhanced responses to the air puff UCS (figure 2). This type of inhibition

during the trace interval may be important in the control of CR temporal

characteristics disrupted in hippocampal-lesioned rabbits trained in the trace

paradigm (Graves and Solomon 1985; Moyer et al., in press). The distinctive sensory response to the tone in the trace paradigm is unique to this

situation; it was not reported previously in studies of delay conditioning.

Perhaps this response reflects a registration of the onset of the CS that will

be useful in the temporal patterning of the CR after the appropriate trace

interval.

FIGURE 2. CA1 pyramidal neuron activity during asymptotic trace

eyeblink conditioning

NOTE: Averaged behavioral response is shown above. poststimulus time histogram during the same

time period is shown below for each panel. Both neurons showed components of “neural

modeling” in their responses. The neuron on the left showed a noticeable sensory response. to

the tone CS. That on the right showed an inhibitory response, especially during the trace

interval, followed by enhanced firing during the air puff UCS presentation.

SOURCE: Figure developed from data presented in Akase et al. 1988.

97
BRAIN SLICE EXPERIMENTS

Initial Observations

A similar procedure has been used in the several replications of the brain

slice experiment that have been done to date (Disterhoft et al. 1986; Coulter

et al. 1989). Animals first were trained to behavioral criterion or received

pseudoconditioning. Naive controls were also used. Twenty-four hours

later. slices were made and maintained with standard procedures. Next,

cells were impaled and their membrane properties were studied. The

studies that will be summarized concern CA1 pyramidal neurons and dentate

gyrus granule cells, two of the major cell types in the classic trisynaptic

hippocampal circuit (figure 3) (Andersen et al. 1971).

The major conditioning-specific effect observed in CA1 neurons recorded in

slices from trained rabbits was a reduction in the afterhyperpolarization

(AHP) response observed after action potentials were elicited with intracellular current pulses. This response was smallest in trained animals and

approximately the same in the pseudoconditioned and naive groups (Disterhoft et al. 1986; Coulter et al. 1989). The AHP duration, as well as the

amplitude, was decreased after conditioning. Another important feature of

the AHP decrease was that it was present in the conditioned group of cells

in the absence of any alteration in spike height, resting potential, or input

resistance from the other two groups. Since the AHP increases as the number of spikes increases (it is additive with increasing numbers of spikes)

(Hotson and Prince 1980), the conditioning effect was more marked as the

number of spikes in the burst became larger. Finally, the AHP reduction

was present in cells from trained animals, even when sodium-dependent

synaptic transmission was blocked by bathing the slices in tetrodotoxin

(TTX) and tetracthylammonium chloride (TEA) (Coulter et. al. 1989).

Functionally, the AHP is presumed to shut the hippocampal pyramidal cells

off during and after bursts of action potentials (Hotson and Prince 1980).

A reduction in AHP after conditioning would make the cells more excitable.

As mentioned above, in vivo recording shows that hippocampal pyramidal

cells are more responsive to the tone CS after conditioning (figure 2). A

reduction in the AHP could certainly cause, or contribute to, this increased

excitability.

Acquisition

Next, a series of experiments was performed to determine when, during the

course of CR acquisition, the decrease in AHP size occurs (Disterhoft et al.

1988b). In all of the work discussed thus far, rabbits were trained for three

80-trial sessions in the short-delay paradigm to insure that the animals were

very well conditioned. In the acquisition studies, rabbits were trained for

98
two 80-trial sessions. Some rabbits were very well trained at this point,

while others showed almost no CRs.

FIGURE 3. A schematic drawing of a hippocampal slice

NOTE: Camera lucida drawings of Gogi-stained individual cells have been placed as they are actually

situated in the brain slice. Examples of the cell types studied in the brain slice experiments

discussed here, CA1 pyramidal neurons and dentate gyros granule cells, are presented.

SOURCE: Thompson and Disterhoft, unpublished observations.

99
Behavioral acquisition curves for the rabbits were used to separate them into

two groups—low and high acquisition (figure 4). Note that the low group

showed about 10 percent CRs on the second day of training. The high

group showed 80 to 90 percent CRs after the same number of training

trials. The AHP was markedly reduced in both amplitude and duration in

the high CR group as compared to comparable measurements taken in the

low group (figure 5). Again, these shifts in AHP amplitudes and durations

occurred in the absence of alterations in other biophysical indices such as

spike height, membrane resting potential, or input resistance.

FIGURE 4. Learning curves for the high- and low-acquisition groups

NOTE: Note that each session consisted of 80 trials, therefore, trials 81 to 160 were in the second

training session. On each trial, a CR was an eyeblink that occurred during the CS-US

interval.

SOURCE: Disterhoft et al. 1988b, copyright 1988, Elsevier.

When histograms were made of the distribution of AHP amplitudes in the

two acquisition groups, a clear shift in the distribution of maximum AHPs

was seen between the low and high groups. This observation was consistent with what was observed in earlier comparisons made between cells from

conditioned, pseudoconditioned, and naive rabbits (Disterhoft et al. 1986).

It is clear that large numbers of cells show increased excitability after

conditioning. But not all cells are changed; there is an overlap in the

distributions. This is consistent with the single neuron data that have been

100
reported in vivo. Berger and his colleagues reported that about 60 percent

of neurons showed enhanced firing in the CS-UCS interval in trained rabbits

(Berger et al. 1983).

FIGURE 5. Computer-averaged traces from neurons in the high- and lowacquisition groups

NOTE: Four action potentials were elicited by intracellular current injection, and the AHP response

averaged five times. The frequency response of the analysis system (500 Hz) was adequate

for accurate analysis of the afterhyperpolarization response during the 1.7 seconds after the

depolarizing pulse but did not follow the action potentials elicited during the pulse.

SOURCE: Disterhoft et al. 1988b. copyright 1988, Elsevier.

101
The important things about this study are (1) that it links the decreased

AHP after conditioning more closely to behavioral performance, and (2) it

is further evidence that the decreased AHP is not some aberration of the

conditioning paradigm, All animals in the acquisition study received 160

CS-UCS pairings. Only those that learned, as evidenced by the number of

CRs they showed, had CA1 pyramidal cells with reduced AHPS. This links

the NIP reduction closely to CR acquisition and actual performance of the

response. The acquisition group serves as another type of “control” in

addition to the pseudoconditioned and naive groups.

Trace Conditioning

It is well known that hippocampal lesions do not affect the acquisition of

short-delay conditioned eyeblink responses (Schmaltz and Theios 1972;

Solomon and Moore 1975; Akase et al. 1989). There is no doubt that the

AHP alterations that have been observed reflect conditioning-specific alterations that are local to the hippocampus. They are present in hippocampal

slices separated from their normal afferent and efferent connections and

even in slices where all sodium spike-dependent synaptic activity is absent

(Coulter et al. 1989). It has been shown that hippocampectomy does retard

retention even in the short-delay paradigm, if rabbits arc not overtrained

(Akase et al. 1989). However, we wished to insure that the biophysical

alterations that have been seen are intimately related to the behavior that the

rabbit was learning. We also wanted to determine if the changes observed

in rabbits learning a task that may more clearly involve the hippocampus

(as indicated by the effects of hippocampectomy) were the same as those

that occurred after the short-delay paradigm. Therefore, examination has

begun of the conditioning-specific changes in ionic currents that might be

seen during trace conditioning, a paradigm whose acquisition Solomon,

Thompson, and their colleagues have shown is blocked by hippocampal lesions (Solomon et al. 1986). Lesion data with trace conditioning indicate

that hippocampectomy markedly retards extinction of trace conditioning with

a relatively short, 300-millisecond trace interval (Moyer et al., in press).

When the longer 500-millisecond trace interval studied by Solomon et al.

(1986) was used, hippocampectomized rabbits were unable to acquire the

trace-conditioned response, even after 25 training sessions (figure 6).

An initial survey has now been completed of CA1 pyramidal neurons

studied in hippocampal slices after trace conditioning (de Jonge et al., in

press). It has been found that both the AHP amplitude and area are reduced in a conditioning-specific manner (figure 7). The effects of conditioning on both measurements were larger as the number of action potentials

in the intracellularly elicited burst increased from one to four. Again, as in

the studies of the biophysical alterations seen after short-delay conditioning,

no differences were seen among the behavioral groups in terms of other

indices that might be expected to affect the AHP measurements directly,

102
i.e., resting potential, input resistance, action potential height, or amount of

current required to elicit the action potentials.

FIGURE 6. Learning curves for rabbits in three surgical conditions

(hippocampectomized, neocortical, sham) when trained in

the trace-conditioning paradigm with a 500-millisecond

trace interval

NOTE: The curves are “Vincentized” according to the slowest learner in each group. The major

point is that no hippocampectomized rabbit acquired the response even after 25 training

sessions. A mean number of 10 acquisition sessions was required by the other two groups to

reach 80 percent CRs in a training session.

SOURCE: Figure developed from data presented in Moyer et al. 1989.

The role of CA1 pyramidal neurons in mediating eyeblink conditioned

responses has been stressed. It is the CA1 and CA3 pyramidal neurons that

exhibit the striking “neural modeling” of the CR as learning occurs (Berger

et al. 1983). Multiunit recordings that have been made from the dentate

gyrus granule neurons during eyeblink conditioning suggest that this cell

type is involved more in changing responsiveness to the sensory significance

of the CS during learning (Berger and Weisz 1987). Dentate neurons show

103
FIGURE 7. AHP amplitude and area for CA1 pyramidal neurons in the

three behavioral groups

NOTE: The average responses were significantly smaller for both amplitude and area at all levels.

Note that the response reduction became larger as the number of action potentials in the burst

became larger.

SOURCE: Figure developed from data presented in de Jonge et al. 1988

104
an enhanced response to the presentation of the CS very early in training.

The response does not mimic the shape of the CR, as the pyramidal cell

response does, nor does the amplitude of the response increase markedly as

behavioral learning proceeds, as is the case for pyramidal neurons (Weisz

et al. 1982).

Given this interesting functional differentiation, which has been demonstrated for two adjacent cell types with in vivo recording, the next test was

whether dentate granule neurons did or did not demonstrate a similar AHP

reduction, as was the case for CA1 pyramidal neurons (Black et al. 1988).

First, it was confirmed that dentate granule neurons have a substantial AHP

response (figure 8) as was reported previously (Durand and Carlen 1984;

Fricke and Prince 1984). More important, after thoroughly examining about

50 dentate gyrus granule cells, no evidence was found for conditioningspecific alterations in the AHP response (figure 9). Again, there was no

difference among groups in any of the other biophysical indices examined,

including membrane resting potential, spike height, input resistance, or current required to elicit action potentials.

These data clearly demonstrate an anatomical differentiation in the AHP reductions occurring within the hippocampus. They also show an interesting

convergence with the functional data gathered with unit recording techniques

in the conscious animal. It would appear that the AHP reductions could be

an ionic change underlying the increased excitability reflected in the neural

modeling of the behavioral response. Since the evidence is quite good that

the CA1 pyramidal neuron AHP reduction is local and postsynaptic, the

lack of such a conditioning-specific change in dentate gyrus granule neurons

may indicate that the increased firing these cells show in vivo is not local.

The enhanced responsiveness to the tone CS may reflect altered responsivity

in entorhinal cortex neurons, which project to dentate gyrus granule neurons

via the perforant path and have been shown to increase firing during conditioning (Berger et al. 1980). Finally, the results in the dentate gyrus region

lead to the hypothesis that CA3 pyramidal neurons will demonstrate a

conditioning-specific reduction in the slow AHP, since these cells demonstrate the neural modeling of the CR, as do CA1 pyramidal neurons. This

hypothesis is presently being tested.

Temporal Analysis of AHP Changes

Alterations in the slow AHP response have been emphasized to this point.

But it has become obvious that there are several potassium currents that

contribute to the action potential repolarization and whose reduction could

cause enhanced excitability in hippocampal neurons (Adams and Galvan

1986; Schwindt et al. 1988). There are two calcium-mediated potassium

currents, IC and IAHP. IC is a relatively fast current lasting milliseconds that

is active on the lower half of the action potential downward trajectory

(Storm 1987). IAHP is a relatively slowly activating current that is

105
FIGURE 8. The functional response of a dentate granule neuron

NOTE: Note the typically high input resistance (100 to a .2 na hyperpolarizing pulse) and the

relatively large and long-lasting AHP after four action potentials (8 mv) in this neuron.

SOURCE: Figure developed from data presented in Black et al. 1988.

evident for hundreds of milliseconds to seconds after a train of action potentials (Lancaster and Adams 1986; Storm 1987). There is also a medium

AHP, evident in the 50 to 100 milliseconds after the action potential train,

which is calcium independent (Gustaffson et al. 1982; Storm 1987). Finally, there are the fast, voltage-dependent potassium current IA that is active

in the upper half of the downward trajectory of the action potential and the

slower, delayed rectifier, a potassium current that is also active in the lower

portion of the downward portion of the action potential (Storm 1987).

We were interested in determining whether the alteration in the calciummediated potassium currents was specific to a particular portion of the AHP

response or was a more generalized phenomenon. We also wanted to

explore the comparability between the cellular mechanisms underlying the

excitability changes that have been observed in the hippocampus after

classical conditioning and those that have been reported in invertebrates.

Alkon and his colleagues have observed reductions in both the fast,

106
FIGURE 9. AHP amplitude after four action potentials in dentate

gyrus granule neurons recorded from the three behavioral

groups

NOTE: No statistically detectable difference in AHP amplitude was observed.

SOURCE: Figure developed from data presented in Black et al. 1988.

voltage-activated potassium current IA and in the somewhat slower, calciummediated potassium current IC after associative conditioning in Hermissenda

(Alkon et al. 1982; Alkon 1984). Kandel and his colleagues have reported

reductions in a voltage-activated potassium current, apparently the serotoninsensitive IKS, in Aplysia after sensitization (Klein and Kandel 1980). It is of

interest to determine if the same pattern of changes occurs in the mammalian brain during associative learning, particularly as attempts are made

to unravel the subcellular and molecular chain of events mediating the

alterations.

Therefore, the timecourse of the AHP reductions observed in CA1 pyramidal neurons was looked at in some detail. First, the timecourse of the AHP

reduction was examined after a train of four action potentials (figure 10).

The AHP reduction was present in the conditioned group as early as

50 milliseconds after the action potential train and remained throughout the

entire 750 milliseconds that were analyzed in detail. This was an indication

that both the medium and slow AHP responses were reduced in a

conditioning-specific fashion. This detailed analysis clearly confirmed the

reduction in the calcium-mediated slow AHP. Second, the upward and

107
FIGURE 10. An analysis of the average AHP amplitude in the 750

milliseconds after the four action potential train

NOTE: The response for each neuron was averaged over five trials, then the average for each

behavioral condition was derived. The AHP amplitude was smallest in the trace-conditioned

group at each 50-millisecond time point examined. This indicated that both the medium and

slow AHP responses were reduced in the trace eyeblink conditioning group.

SOURCE: de Jonge et al.. in press

downward trajectory of the action potential was examined, as well as its

width, for evidence of any alteration due to conditioning. Because of the

contribution of IA, IC, and the delayed rectifier to the action potential repolarization, an alteration in any of these currents should have been evident in

the action potential waveform (Storm 1967). Unlike the case for the medium and slow AHP responses, there was no alteration evident in the action

potential in any of the behavioral groups (figure 11). This lack of effect

was found in both the CA1 pyramidal neuron and the dentate gyrus granule

cell populations. It should be noted that this experiment was not a

definitive evaluation of the possibility of alterations in the early currents. A

more detailed examination using pharmacological techniques to isolate the

various currents and/or single electrode voltage clamp studies will be

required to make a definitive statement. At this point, it can be said that

the learning-induced AHP reduction appears to be confined to the medium

and slow AHP responses.

108
FIGURE 11. A computer-generated display of a single action potential in

a dentate granule neuron to demonstrate how the detailed

analysis of the action potential was done

NOTE: The rising and falling slopes, the action potential width, and the action potential height were

determined for each neuron five times and averaged. A d-millisecond pulse was used to elicit

the action potential. The data were gathered at 10-μsecond intervals, yielding sufficient accuracy for this analysis. The action potential width and falling slope measurements were particularly sensitive to possible alterations in the fast currents, IA, IC, and the delayed rectifier.

No differences were seen between the behavioral groups in either CA1 pyramidal or dentate

gyms granule neurons with this detailed analysis.

SOURCE: Figure developed fmm data presented in Black et al. 1988.

NIMODIPINE FACILITATES EYEBLINK CONDITIONING IN

AGING RABBITS

The final set of experiments that will be described involves an evaluation of

the effect of intravenous administration of nimodipine on the acquisition of

the trace eyeblink conditioned response in aging rabbits. Nimodipine is a

dihydropyridine known to be a calcium channel blocker (Scriabine et al.

1985). It is being tested in humans for use in controlling the consequences

of stroke in the period immediately after the cerebrovascular event. What

interested us was the preliminary report that learning ability was enhanced

in aging humans with chronic cerebrovascular disease to whom nimodipine

was administered (Bono et al. 1985). We were intrigued by the possibility

that this effect could be a result of a reduction of calcium-mediated

109
potassium currents as a result of direct blockade of the calcium current

itself. Some evidence exists for nimodipine blockade of calcium currents in

hippocampal neurons in vitro (Docherty and Brown 1986; Gahwiler and

Brown 1987). Eyeblink conditioning in the rabbit is a good preparation in

which to evaluate a pharmacological agent that may counteract learning

deficits during aging. Eyeblink conditioning was initially developed as an

experimentally controlled behavioral test in the human; its parameters are

rather similar in humans and rabbits (Gormezano 1966). Aging also slows

behavioral acquisition similarly in both species (Woodruff-Pak and

Thompson 1985).

It was found that intravenous administration of nimodipine (1 μg/kg/minute)

markedly facilitated the acquisition of eyeblink conditioning in aging rabbits

(retired breeders averaging 36.3 months old) when compared to vehicle controls (mean age 37.7 months) (figure 12) (Deyo et al. 1989). As expected

from previous studies (Graves and Solomon 1985; Woodruff-Pak and

Thompson 1985), the aging vehicle-control rabbits learned significantly

slower than did the young adult (3 months) controls. It was also found that

young adult rabbits receiving nimodipine learned faster than young vehicle

controls, at about the same performance levels as the aging nimodipine rabbits. Nimodipine had no effect on the size of CRs that did occur nor on

the size of unconditioned eyeblink responses to the air puff. There was no

effect of nimodipine in pseudoconditioned rabbits; i.e., there was no increased sensory responsivity as a result of drug application. These data

suggested that the nimodipine facilitated whatever central pathway was involved in mediating the learned association itself, rather than facilitating

responsivity in a generalized fashion.

We do not yet have a definitive understanding of the mechanism for the facilitation of learning that has been observed. There seem to be two obvious

possibilities. First, as mentioned above, nimodipine may be reducing AHP

responses directly in brain regions critical for mediating the tone-air puff

association via a reduction of calcium currents. There is some evidence for

nimodipine effects on calcium currents in the hippocampus (Docherty and

Brown 1986; Gahwiler and Brown 1987). Possible reductions of the AHP

response, which have not yet been examined, are currently being evaluated.

Consistent with the brain slice work summarized above (Disterhoft et al.

1986; de Jonge et al., in press), it is suggested that nimodipine could then

increase cellular excitability directly in a fashion appropriate to facilitate

acquisition. The second major possibility could be that nimodipine is

enhancing cerebral blood flow and acting as a vasodilator. Many of the

“nootropic” substances that have been described to facilitate learning

apparently act via this mechanism (Hock 1987). Direct blockade of central

nervous system calcium channels is the hypothetical mechanism that seems

most theoretically appealing at this point.

110
FIGURE 12. The mean number of trials (plus the standard error) to reach

behavioral criterion (8 CRs in a block of 10 trials) in each

treatment group in the nimodipine study

SOURCE: Deyo et al. 1989, copyright 1989, AAAS.

CONCLUSION

An ionic mechanism that would appear to underly associative learning in

the mammalian brain has been demonstrated in vitro. The AHP response in

hippocampal cells is primarily caused by a calcium-mediated potassium current. Functionally, this AHP is presumed to shut the hippocampal pyramidal cells off during bursts. A reduction in the AHP could certainly cause,

or contribute to, the increased excitability (responsivity to the CS) seen in

hippocampal pyramidal neurons after conditioning. It was observed that the

slow, but not the fast, AHP response was reduced after conditioning. This

demonstrates an important specificity in the ionic current that is modulated

during learning: IAHP, but apparently not IC or the delayed rectifier, was

reduced. It has also been seen that there is anatomical localizability, even

within the hippocampus, in the AHP reduction. CA1 pyramidal neurons

111
demonstrate this postsynaptic reduction, while dentate granule neurons do

not. The AHP reduction is similar in trace and eyeblink conditioning.

Finally, evidence exists that a calcium channel blocker, nimodipine, facilitates the acquisition of the eyeblink conditioned response. This facilitation

is especially marked in the aging rabbit. It is appealing to speculate that

this learning enhancement may be mediated by directly causing an AHP reduction with drug administration. If so, studies of the cellular mechanisms

of learning may have practical application considerably sooner than anticipated. All the studies we have done thus far emphasize an important role

for calcium flux in mediating associative learning in the mammalian brain.

REFERENCES

Adams, P.R., and Galvan, M. Voltage-dependent currents of vertebrate

neurons and their role in membrane excitability. In: Delgado-Escueta,

A.V.; Ward, A.A., Jr.; Woodbury, D.M.; and Porter, R.J., eds. Advances

in Neurology. Vol. 44. New York: Raven Press, 1986. pp. 137-170.

Akase, E.; Alkon, D.L.; and Disterhoft, J.F. Hippocampal lesions impair

memory of short-delay conditioned eye blink in rabbit. Behav Neurosci

103:935-943, 1989.

Akase, E.; Deyo, R.A.; and Disterhoft, J.F. Activity of single hippocampal

CA1 pyramidal neurons during trace eye-blink conditioning. Soc

Neurosci Abst 14:394, 1988.

Alkon, D.L. Calcium-mediated reduction of ionic currents. A biophysical

memory trace. Science 226:1037-1045, 1984.

Alkon, D.L.; Lederhandler, J.; and Shoukimas, J.J. Primary changes of

membrane currents during retention of associative learning. Science

215:693-695, 1982.

Andersen, P.; Bliss, T.V.P.; and Skrede, K.K. Lamellar organization of

hippocampal excitatory pathways. Exp Brain Res 13:222-238, 1971.

Berger, T.W.; Clark, G.A.; and Thompson, R.F. Neuronal plasticity

recorded from limbic system structures during conditioning. Physiol

Psychol 8:155-167, 1980.

Berger, T.W.; Rinaldi, P.C.; Weisz, D.J.; and Thompson, R.F. Single-unit

analysis of different hippocampal cell types during classical conditioning

of rabbit nictitating membrane response. J Neurophysiol 50:1197-1219,

1983.

Berger, T.W., and Weisz, D.J. Single unit analysis of hippocampal pyramidal and granule cells and their role in classical conditioning of the rabbit

nictitating membrane response. In: Gormezano, I.; Prokasy, W.F.; and

Thompson, R.F., eds. Classical Conditioning. 3rd edition. Hillsdale, NJ:

Lawrence Erlbaum Associates, 1987. pp. 217-253.

Black, J.P.; Deyo, R.A.; and Disterhoft, J.F. Electrophysiological characteristics of dentate gyrus granule cells following classical trace conditioning.

Soc Neurosci Abs 14:394, 1988.

112
Bono, G.; Sinforiani, E.; Trucco, M.; Cavallini, A.; Acuto, G.C.; and

Nappi, G. Nimodipine in cCVD patients: Clinical and neuropsychological results of a double-blind cross-over study. In: Betz, E.; Deck, K.;

and Hoffmeister, F., eds. Nimodipine—Pharmacological and Clinical

Properties. Stuttgart: F.K. Schattauer Verlag, 1985. pp. 275-285.

Coulter, D.A.; Lo Turco, J.J.; Kubota, M.; Disterhoft, J.F.; Moore, J.W.;

and Alkon, D.L. Classical conditioning reduces the amplitude and duration of the calcium-dependent afterhyperpolarization in rabbit hippocampal

pyramidal cells. J Neurophysiol 61:971-981, 1989.

de Jonge, M.C.; Black, J.P.; Deyo, R.A.; and Disterhoft, J.F. Learninginduced afterhyperpolarization reductions in hippocampus are specific for

cell type and potassium conductance. Exp Brain Res, in press.

de Jonge, M.C.; Deyo, R.A.; Black, T.P.; and Disterhoft, J.F. Changes in

electrophysiological characteristics of hippocampal CA1 neurons after trace

eye-blink conditioning. Soc Neurosci Abst 14:394, 1988.

Deyo, R.A.; Straube, K.T.; and Distcrhoft, J.F. Nimodipine facilitates

associative learning in aging rabbits. Science 243:809-811, 1989.

Disterhoft, J.F.; Coulter, D.A.; and Alkon, D.L. Conditioning-specific

membrane changes of rabbit hippocampal neurons measured in vitro.

Proc Nat1 Acad Sci USA 83:2722-2737, 1986.

Disterhoft, J.F., Coulter, D.A., and Alkon, D.L. Conditioning-specific biophysical alterations in rabbit hippocampus. In: Woody, C.D.;

Alkon, D.L.; and McGaugh, J.L., eds. Cellular Mechanisms of Conditioning and Behavioral Plasticity. New York: Plenum Publishing, 1988a.

pp. 89-104.

Disterhoft, J.F.; Golden, D.T.; Read, H.R.; Coulter, D.A.; and Alkon, D.L.

AHP reductions in rabbit hippocampal neurons during conditioning are

correlated with acquisition of the learned response. Brain Res 462:118-

125, 1988b.

Disterhoft, J.F.; Kwan, H.H.; and Lo, W.D. Nictitating membrane conditioning to tone in the immobilized albino rabbit. Brain Res 137:127-143,

1977.

Docherty, R.J., and Brown, D.A. Interaction of 1,4-dihydropyridines with

somatic Ca currents in hippocampal CA1 neurones of the guinea pig in

vitro. Neurosci Lett 70:110-115, 1986.

Durand, D., and Carlen, P.L,. Decreased neuronal inhibition in vitro after

long-term administration of ethanol. Science 224:1359-1361, 1984.

Fricke, R.A., and Prince, D.A. Electrophysiology of dentate gyrus granule

cells. J Neurophysiol 51:195-209, 1984.

Gahwiler, B.H., and Brown, D.A. Effects of dihydropyridines on calcium

currents in CA3 pyramidal cells in slice cultures of rat hippocampus.

Neurosci 20:731-738, 1987.

Gormezano, I. Classical conditioning. In: Sidowski, J.B., ed. Experimental Methods and Instrumentation in Psychology. New York:

McGraw-Hill, 1966. pp. 385-420.

113
Graves, C.A., and Solomon, P.R. Age-related disruption of trace but not

delay classical conditioning of the rabbit’s nictitating membrane response.

Behav Neurosci 99:88-96, 1985.

Gustaffson, B.; Galvan, M.; Grafe, P.; and Wigstrom, H. A transient outward current in a mammalian central neuron blocked by 4-aminopyridine.

Nature 299:252-254, 1982.

Hock, F.J. Drug influences on learning and memory in aged animals and

humans. Neuropsychobiology 17:145-160, 1987.

Hotson, J.R., and Prince, D.A. A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons. J Neurophysiol 43:409-

419, 1980.

Klein, M., and Kandel, E.R. Mechanism of calcium current modulation

underlying presynaptic facilitation and behavioral sensitization in Aplysia.

Proc Natl Acad Sci USA 77:6912-6916, 1980.

Lancaster, B., and Adams, P.R. Calcium-dependent current generating the

afterhyperpolarization of hippocampal neurons. J Neurophysiol 55:1268-

1282, 1986.

Moyer, J.R., Jr.; Deyo, R.A.; and Disterhoft, J.F. Hippocampectomy

prevents trace eye-blink conditioning in rabbits. Sac Neurosci Abst

15:888, 1989.

Moyer, J.R., Jr.; Deyo, R.A.; and Disterhoft, J.F. Hippocampal lesions

impair trace eye-blink conditioning in rabbits. Behav Neurosci, in press.

Nicoll, R.A. The coupling of neurotransmitter receptors to ion channels in

the brain. Science 241:545-551, 1988.

Numann, R.E.; Wadman, W.J.; and Wong, R.K.S. Outward currents of

single hippocampal cells obtained from the adult guinea-pig. J Physiol

393:331-353, 1987.

O’Keefe, J., and Nadel, L. The Hippocampus as a Cognitive Map.

London: Oxford, 1978.

Schmaltz, L.W., and Theios, J. Acquisition and extinction of a classically

conditioned response in hippocampectomized rabbits (Oryctalagus

cuniculus). J Comp Physiol Psychol 79:328-333, 1972.

Schwindt, P.C.; Spain, W.J.; Foehring, R.C.; Stafstrom, C.E.; Chubb, M.C.;

and Crill, W.E. Multiple potassium conductances and their functions in

neurons from cat sensorimotor cortex in vivo. J Neurophysiol 59:424-

449, 1988.

Scriabine, A.; Battye, R.; Hoffmeister, F.; Kazda, S.; Towart, R.;

Garthoff, B.; Schluter, G.; Ramsch, K.-D.; and Scherling, D. Nimodipine.

In: Scriabine, A., ed. New Drugs Annual: Cardiovascular Drugs.

Vol 3. New York: Raven, 1985. pp. 197-218.

Segal, M. Flow of conditioned responses in the limbic telencephalic system

of the rat. J Neurophysiol 36:840-854, 1973.

Solomon, P.R., and Moore, J.W. Latent inhibition and stimulus generalization of the classically conditioned nictitating membrane response in rabbits

(Oryctolagus cuniculus) following dorsal hippocampal ablations. J Comp

Physiol Psychol 89:1192-1203, 1975.

114
Solomon, P.R.; Vander Schaff, E.; Thompson, R.F.; and Weisz, D.J.

Hippocampus and trace conditioning of the rabbit’s classically conditioned

nictitating membrane response. Behav Neurosci 100:729-744, 1986.

Squire, L. Memory and Brain. New York: Oxford, 1987.

Storm, J.F. Action potential repolarization and a fast after-hyperpolarization

in rat hippocampal pyramidal cells. J Physiol 385:733-750, 1987.

Thompson, L.T., and Disterhoft, J.F. Neuronal morphology in the hippocampal system of the rabbit (Oryctolagus cuniculus). Submitted for

publication.

Weisz, D.J.; Clark, G.A.; Thompson, R.F.; and Solomon, P.R. Activity of

dentate gyrus during NM conditioning in rabbit. In: Woody, C.D., ed.

Conditioning-Representation of involved Neural Functions. New York:

Plenum, 1982. pp. 131-145.

Woodruff-Pak, D.S., and Thompson, R.F. Classical conditioning of the

eyelid response in rabbits as a model system for the study of brain

mechanisms of learning and memory in aging. Exp Aging Res 11:109-

122, 1985.

ACKNOWLEDGMENTS

Supported by U.S. Public Health Service grant NIH R01 NS 23482, ONR

Contract N00014-88-K-0399, the Whitehall Foundation, the American

Federation of Aging Research, and a gift from the Pharmaceutical Division

of the Miles Institute. The contributions of collaborators Eisuke Akase,

Joel P. Black, Maarten C. de Jonge, Richard A. Deyo, James R. Moyer, Jr.,

Karen T. Straube, and L. Tres Thompson in gathering and summarizing data

were critical to the experiments described here.

AUTHOR

John F. Disterhoft, Ph.D.

Associate Professor

Department of Cell Biology and Anatomy

Northwestern University Medical School

303 East Chicago Avenue

Chicago, IL 60611

115
Hippocampal Theories and the

Information/Computation

Perspective

William B. Levy

INTRODUCTION

Through the years, there seems to be no end of theories about the function

of the hippocampus. Such functions include olfactory processing, recent

memory, memory consolidation, response inhibition, selective attention (see

Douglas 1967 for review), spatial mapping (O’Keefe and Nadel 1978), stimulus mismatch operations, temporal and sequence learning (Schmajuk 1987),

storage of declarative knowledge, and working memory. Squire (1982) reviewed many of the psychological theories of hippocampal function. Worse

still, the functioning rat’s hippocampus may not do what H.M.’s hippocampus does (compare Scoville and Milner 1957 to above citations).

The point is that, after 30 plus years of serious experimental and intellectual

assaults, we have a theory of hippocampal function that is only a little

better than when we started. Certainly none of these theories is very sharp

nor is there any particular trend for such theories becoming precise statements of hippocampal function. Even more bothersome is the fact that

none of these theories relates function to the rather special anatomy of the

hippocampus or to what we have learned or even to what we will learn

about the cellular physiology of the hippocampus. Clearly this old approach

to understanding hippocampal function alone will not do.

As a reductionistic neuroscientist, I would like to understand how groups of

cells function to form an integrated system. This approach generally tries

to relate cellular physiology and anatomy to system physiology. Within the

context of hippocampal function, such an approach seeks to understand the

input-output characteristics of the hippocampus as a system from its parts

and to relate this system’s behavior to the behavior of other brain regions

and to brain functions. If such an abstract approach is properly framed,

certain questions will be natural, while other questions will be easily

avoided. Consider the four examples that follow.

116
First, we should not have to know the function of the entorhinal cortex

(EC), the major extrinsic input to the hippocampus, or the Hubel and

Wiesel-like feature-detecting properties of entorhinal neurons before we can

study the hippocampus. Otherwise, by a natural regression, we would have

to know the feature codings of all the brain regions that precede the hippocampus in sensory processing before we formulate a theory of hippocampal

function. This approach certainly would postpone research on the hippocampus for a long time.

Second, we should be able to compare input-output functions between areas

and between species. For example, how do primary visual cortex (area 17)

and the hippocampus differ in the way they process signals? How do the

rat and human hippocampi differ in their signal-processing abilities?

Third, a proper framework should stimulate quantitative as well as qualitative statements of function. Thus, when there are thousands of stimulating

and recording electrodes in the future, there will be predictions worth

testing.

Finally, independently obtained results from various brain regions should

easily fit together. That is, if a proper language of input-output functions is

used consistently for different regions, then we should be able to plug various parts of the brain together after studying individual systems and obtain

a prediction of total brain function.

Therefore, the idea underlying the proposed approach is to study the abstract input-output transformations of systems like the hippocampus at the

level of cells and groups of cells over physiologically relevant ranges of

parameters. Even this is not enough of a framework, however, to gain an

understanding of the function of the hippocampus or of any other brain

region. We have to include, in our framework, questions of some central

but general issue of brain function itself. The issue should guarantee

relevance to the entire organism and must be identifiable in a consistent

way in isolated brain regions.

But what is it that is special about the brain’s function?

The function of the brain is to control the environment and the organism

so that the organism survives and produces fertile offspring, thereby ensuring the survival of the species. This control is accomplished in large part

by the brain’s abilities to predict (1) future states of the environment,

(2) how current and future states of the environment will affect the organism, and (3) how the organism’s interactions with the environment will

alter the environment.

Central to all of this predicting is our ability to find, store, and use the

predictiveness—the regularities-that exist in the environment. Successful

117
prediction is possible, because there are spatiotemporal regularities and

physical constraints in the environment. We will refer to these regularities

and constraints as “redundancies” for four reasons: (1) the formal mathematical measures suggested by this term; (2) the ease of identifying this

formalized redundancy with what we call correlations, concepts, associations,

regularities, and constraints; (3) the formalization applies both to the environment and to individual brain regions; and (4) the meaning of the word

redundancy, at least at its extremes in these mathematical formulations,

agrees with the meaning of the word in conversation. Thus, successful prediction of the environment depends upon environmental redundancy.

Stored redundancies can be used to form predictions. When a new signal

arrives, it is transformed to a less redundant signal based on past associations. These transformed signals are called predictive representations and

are synonymous with recodings and with coded representations and are, in a

mathematical sense, predictions.

It is, then, the storage of redundancy at synapses and predictive representation by reduction of signal redundancy that finally frames the abstract

approach to understanding brain function. This issue gives our abstract

approach to the brain and its regions that extra something that takes it

beyond simple input-output questions to a general problem central to brain

function that is applicable to almost any brain region as well as to the

environment itself.

APPLICATION OF THE APPROACH TO THE HIPPOCAMPUS

As a general issue, the reduction of redundancy is particularly easy to

apply when there is only one class of high-information-carrying inputs.

As an example, let’s consider the dentate gyrus (DG)-CA3 system of the

hippocampus.

Given this approach, the theory of DG-CA3 function is, loosely stated, as

follows. The layer-II stellatc cells of the EC provide information to the

neurons of the DG-CA3 system. These neurons then reduce the signal

redundancy while maintaining a distinctiveness of coding that allows for

minimal loss of information. That is, the DG-CA3 system recodes the EC

afferent information before sending it to the hippocampal CA1 region. The

recoding is based on synaptically encoded associations, i.e., environmental

redundancies, that have been learned via associative synaptic modification.

This recoding is helpful to CA1, or to any other brain area, because of the

severely restricted size and procesing speed of CA1 (and of every other

brain region for that matter; see below). Figure 1 schematically illustrates

these simplifications.

118
FIGURE 1. Recoding a signal to remove redundancy

Note that there is only one class of inputs in figure 1. This simplification

comes from a basic idea in information theory (Shannon and Weaver 1949)

that information capacity increases with both the number of channels and

the maximum frequency of each channel. In this view, the EC afferents to

the DG-CA3 system carry essentially all of the information to this part of

the hippocampus because of their large numbers and their high-frequency

firing rates. This is not to say that the monoaminergic and cholinergic

afferents are unimportant-indeed, I am as certain as anyone that the hippocampus would not function properly without them-but that their function is

not to carry high-density information to the hippocampus. This job is left

for entorhinal afferents. Note that this view does not take away from the

importance of monoaminergic or cholinergic inputs. To gain a better understanding, consider a metaphor of a TV set with two wires going in: the

power cord, identified with the nonspecific inputs, and the antenna wire,

identified with the entorhinal afferents. The antenna wire carries the information, but cut the power cord and nothing works.

Since there is no other high-density, extrahippocampal information source

interacting with the entorhinal input, the EC signal is interacting only with

itself: but to what end? Certainly the EC is the beginning of the funnel

that brings together signals from all the diverse sensory systems. The DGCA3 helps in this funneling, so obviously a recoding is going on, but why?

Action potentials are sure, so there is no reason to insert redundancy. Textbooks to the contrary, there are no relay areas that are not processors. To

merely pass on a signal not only wastes time and processing units, but it

risks the almost certain injection of noise into the signals. On the other

hand, reducing signal redundancy will help CA1, whatever its task. By the

way, CA1 desperately needs help, because the number of cells in CA1 is

woefully inadequate to create, much less compare, all possible hypotheses.

Now, note that we concern ourselves not with the environment that the entire organism experiences but with the environment that the hippocampus

itself experiences. This is a major simplification of the framework compared to the old approach. That is, aside from hormonal influences, the

environment of the hippocampus is no more and no less than the information provided by its afferents.

119
Thus, we no longer must identify cells by their feature-detection properties

relative to the environment external to the organism. Instead, we determine

the redundancy reduction at each stage of processing, though we always

have the option of speculating on the redundancy of the environment and

the feature-detection codings that are, in fact, predictive representations.

It is not that environmental redundancy is an uninteresting question; it is

just that it requires solving a more difficult problem than understanding the

brain itself, i.e., understanding the redundancy or organization of the entire

world. Also, it implicitly evokes an almost dogmatic belief that we know

what we see and that what we consciously see are the proper dimensions of

the world. I am suspicious that we no more know the component features

of what we see than we know the component features of what we smell.

A LITTLE MORE ABOUT REDUNDANCY

The regularities of the world are, through learning and heredity, stored in

the connections—indeed as the quantitative connections—between neurons.

Thus, we should understand that the associations that are learned and stored

at synapses are synonymous with the regularities, correlations, and constraints, i.e., the redundancies, of the environment. Information and coding

theories allow us to abstract and quantify the predictability of the environment using the idea of redundancy.

More precisely, redundancy is the amount of statistical dependence in a

group of signals. When a linear measure of redundancy is desired, the

mathematical formulations are quite explicit (Watanabe 1969).

The importance of predictive reduction of redundancy deserves comment.

The reasons are both practical and philosophical. Practically, the brain is

too small and too slow. This may seem strange, since we have always

tried to impress the lay public and our students with the notion of how big

the brain is—more than 1010 cells, more than 1014 synapses—but consider

how many possible games of chess there are, and the brain is small in

comparison. Consider how many possible configurations of the environment

there are, each one you might encounter and need to represent, and the

brain is minuscule by comparison.

Because of the overwhelming numbers of possible patterns in the world, it

is critical to remove as much redundancy as possible from the representations of the environment. In addition, reducing redundancies will speed up

computations and decrease storage requirements. Furthermore, when redundancies are removed from representations, there are some safe, fast, and

easy ways to process representations and make predictions. Even these

improvements will not be enough, however, and neural computations will

usually have to “cheat” to compute fast enough for real time interactions.

By cheating I mean make predictions, or equivalently do constraint

120
satisfaction, without examining each individually possible configuration of

the environment. Such incomplete examination risks error. By removing

redundancies, the average size of this error can be minimized.

The final argument may appeal only to the more philosophically minded but

has application far beyond the hippocampus. First, note that the configurations of the environment that reach our sensory receptors are in fact signals.

Then, the redundancy in these special signals is all there is to learn about

the environment. In signals with no redundancy or in an environment with

no redundancy, there is nothing to learn. Obviously, then, if we can recode

the environment by removing and storing the redundancies (or use the predictive redundancies we have inherited) while maintaining pattern distinctiveness, we understand all there is to know about these signals with regard

to themselves. Thus, identifying and storing redundancy is what learning

and, therefore, much of synaptic modification is all about.

AN EXAMPLE OF REDUNDANCY IN OUR DAY-TO-DAY LIFE

Imagine this scene-you are at home, reading a book, waiting to be picked

up and driven to the airport. A horn sounds from outside. Honk! You

get up and go to the door to leave. Note how the honk predicts the car

outside. Now suppose we recreate this scene again except you are not

reading but sitting at the window. Just at the same time the horn sounds,

you see the arrival of the expected car. Note that the sound alone was

enough to predict the car so that, in this second case, hearing the horn and

seeing the car are to a large extent redundant in the information that your

ride has arrived.

AN EXAMPLE OF NEURAL REDUNDANCY

In figure 2, we consider a very simple nervous system with two afferent

lines of signals.

An event is an active cell; PROB ( ) stands for probability of.

(A) There is zero redundancy if there is total independence; e.g.,

PROB (EVENT 1 AND EVENT 2) = PROB (EVENT 1) . PROB (EVENT 2)

(B) There is redundancy if there is not statistical independence, e.g.,

PROB (EVENT 1 AND EVENT 2) PROB (EVENT 1) . PROB (EVENT 2)

FIGURE 2. Examples of neural redundancy

121
If the probability of joint activity, i.e., the probability of both cells being

active, is equal to the product of the individual probabilities of cell activity,

as in (A) in figure 2, then there is statistical independence and no redundancy in the signals. If the equality does not hold, as in (B) in figure 2,

then there is redundancy in the system. When there is no redundancy, conditional prediction is a waste of time, because there are no useful correlations or associations. When there is redundancy, we have something to

gain from predictions about activity at one cell conditional on the activity of

the other cell.

This idea of redundancy vs. no redundancy can be refined to formal

measurements quantifying gradations that vary between no redundancy and

total redundancy. One such measurement is an entropy called mutual

information.

Using mutual information, we now state the theory more precisely:

The object of the DG-CA3 transformation is to minimize the

mutual information of the CA3 output signals with respect to

themselves, while maximizing the mutual information between

EC input signals and CA3 output signals.

What we are proposing is that subsystems of the brain like the hippocampus

face a dual optimization problem that involves (1) minimizing the mutual

information of CA3 cell activities with regard to themselves, while, at the

same time, (2) maximizing the mutual information of the layer-II EC input

relative to the CA3 output. Part (1) is the optimization that reduces signal

redundancy. The transformation performed by the dentate gyrus and CA3

recodes the EC representation of whatever into a CA3 representation of this

same whatever. Notably the CA3 representation is less internally redundant

than the EC representation. Part (2) is the optimization that maintains pattern distinctiveness. That is, those patterns that are easily separable or distinguishable in the EC representation are still easily distinguishable by their

CA3 representations.

Just to show that this dual optimization problem is well formulated, we

point out that there actually is a unique solution to this problem if the

world is Gaussian (of course it is not!). For zero mean Gaussian environments, the Karhunen-Loeve transformation is the optimal mapping that

uniquely solves this dual optimization (Watanabe 1969). This transformation codes the environment so that all output lines considered pairwise are

independent and no higher order interactions exist. Because the transformation is invertible, the input-output mutual information is maximal. A more

general solution suitable for optimally transforming mote complex environments is desirable.

122
CONCLUSION

Finally, we should realize that this new framework can happily exist with

the old game. The following are examples of this coexistence.

1.

2.

Spatial sequences, such as encountered by small animals running

through the maze of the underbrush and through drain pipes, require

bringing together codings of rather diverse spatial relations and

sequences. These spatial and spatiotemporal patterns are likely represented differently by the different senses (e.g., visual and olfactory

codings must be very different initially). Recoding for an efficient

compatible melding of these diverse dimensions seems essential to using

all the cues quickly and accurately. Recoding to achieve redundancy

reduction in DG-CA3 and other limbic structures may be critical to the

degree of success or failure in such an environment.

Stimulus mismatch is a trigger for selective attention and an attempt to

recode our stored, predictive redundancies. Mismatch activates certain

nonspecific inputs to the hippocampus that in turn facilitate associative

synaptic modification in the DG-CA3. This facilitation of modification

in a mismatch situation is an opportunity to store redundancies that

more accurately coincide with those of the environment.

3. Short-term memory incorporates a redundancy reduction process. This

process saves space later when memories are transferred from shortterm to long-term storage.

HISTORICAL NOTES

The ideas expressed here have appeared at various times in various places.

Included below are the authors who have been most influential on me or

who, discovered in retrospect, presented relatively clear expositions of their

ideas.

The ideas here are strongly related to those of S. Watanabe (1969) relating

pattern recognition and inference to reduction of entropy.

The theme of redundancy reduction was quite explicitly introduced to neuroscience by Barlow (1959) and is still occasionally found as an issue in the

visual system.

W.R. Ashby (1956) and Richard Bellman (1967) clearly expressed the idea

that the nervous system or any other device was too small and too slow

compared to the possibilities of the environment.

Marr (1970) mentions the issue of redundancy reduction in terms of the

overwhelming encoding problems faced by the brain.

123
The unsupervised concept formation systems of various authors, including

Anderson (1968), Amari (1977), Kohonen (1984), and Marr (1970) strongly

resemble the problem and the system considered here. However, predictive

reduction of redundancy is a more general issue than concept formation as

it is usually presented. That is, the redundancy issue includes concept formation (as Marr points out). However, predictive redundancy reduction is

less restrictive, and the mathematics are much better developed.

The equivalency between total understanding and recoding for minimum

redundancy is almost a direct quote from the work by Rissanen (1986),

while the germ of the idea appears in the writings of several philosophers.

J.A. Gray (1982) has emphasized the importance of prediction and related it

to the limbic system.

The ideas of this chapter appeared previously in abstract form (Levy 1985).

REFERENCES

Amari, S.-I. Neural theory of association and concept-formation. Biol

Cybern 26:175-185, 1977.

Anderson, T.A. A memory storage model utilizing spatial correlation functions. Kybernetik 5:113-119, 1968.

Ashby, W.R. Design for an intelligence-amplifier. In: Shannon, C.E., and

McCarthy, J., eds. Automata Studies. Princeton: Princeton University

Press, 1956. pp. 215-234.

Barlow, H.B. Sensory mechanisms, the reduction of redundancy, and intelligence. In: Mechanisation of Thought Processes. London: Her

Majesty’s Stationery Office, 1959. pp. 537-559.

Bellman, R. Adaptive Control Processes: A Guided Tour. Princeton:

Princeton University Press, 1961. 255 pp.

Douglas, R.J. The hippocampus and behavior. Psychol Bull 67:416-442,

1967.

Gray, J.A. The Neuropsychology of Anxiety: An Enquiry Into the Functions of the Septo-Hippocampal System. New York: Oxford University

Press, 1982. 548 pp.

Kohonen, T. Correlation matrix memories. IEEE Trans Computers

C-21:353-359, 1972.

Kohonen, T. Self-Organization and Associative Memory. Berlin: SpringerVerlag, 1984. 255 pp.

Levy, W.B. An information/computation theory of hippocampal function.

Soc Neurosci Abs 11:493, 1985.

Marr, D. A theory for cerebral neocortex. Proc R Soc Lond B 176:161-

234, 1970.

O’Keefe, J., and Nadel, L. The Hippocampus as a Cognitive Map.

Oxford: Oxford University Press, 1978. 570 pp.

124
Rissanen, J. Stochastic complexity and modeling. Ann Stat 14:1080-1100,

1986.

Schmajuk. N.A. The hippocampus and the control of information storage in

the brain. In: Arbib, M., and Amari, S.I., eds. Competition and

Cooperation in Neural Nets. New York: Springer, 1987.

Scoville, W.B., and Milner, B. Loss of recent memory after bilateral hippocampal lesions. J Neurosurg Psychiat 20:11-21, 1957.

Shannon, C.E., and Weaver, W. The Mathematical Theory of Communication. Chicago: University of Illinois Press, 1949. 125 pp.

Squire, L. The neuropsychology of human memory. Ann Rev Neurosci

5:241-273, 1982.

Watanabe, S. Knowing and Guessing. A Quantitative Study of Inference

and Information. New York: John Wiley, 1969. 592 pp.

ACKNOWLEDGMENTS

This work was supported by the U.S. Public Health Service grant

NS 15488 from the National Institutes of Health and the RSDA grant

MII 00622 from the National Institute of Mental Health to

William B. Levy.

AUTHOR

William B. Levy, Ph.D

Research Associate Professor

Department of Neurosurgery

University of Virginia Health Sciences Center

Box 420

Charlottesville, VA 22908

125
Pharmacological and Anatomical

Analysis of Fear Conditioning

Michael Davis

INTRODUCTION

A major challenge in neuroscience is understanding the biological substrates

of learning and memory. This will eventually involve a detailed cellular

and biochemical description of the events in the nervous system that result

in a relatively permanent change in neural transmission—a change that

allows a formerly neutral stimulus to produce or affect some behavioral

response. The most definitive work on the cellular and biochemical analysis of learning and memory has been carried out in invertebrate nervous

systems (Alkon 1979; Carew 1984; Castellucci et al. 1970; Crow and Alkon

1980; Hawkins et al. 1983; Walters and Byrne 1985). A major advance in

the analysis of these questions was choosing a simple reflex behavior that

could be modified by experience and then determining the neural circuit that

mediated the behavior being measured. Once this was done, it was possible

to isolate where different types of plasticity occurred and then to determine

how these changes were brought about at the cellular level.

Comparably detailed biochemical analyses of learning and memory that relate directly to behavioral output have not been possible in vertebrate nervous systems. In large part, this is because it has been very difficult to

isolate where plastic changes take place in complex vertebrate nervous systems. Thus, at this stage, it is important to develop, in complex vertebrates,

simple models of learning and memory. Such models can be used to isolate loci within the nervous system where plastic changes take place that

allow a conditioned stimulus to induce or affect behavior. The short-latency

acoustic startle reflex enhanced by prior classical fear conditioning may be

an especially promising model system with which to carry out this type of

analysis.

The purpose of this chapter is to describe the fear-potentiated startle paradigm and the advantages it provides for a neuroanatomical analysis of fear

conditioning. The neural pathways involved in the startle reflex will be

described. The role of the amygdala in fear-potentiated startle and its possible connections to the startle pathway and critical visual structures that

carry information about the conditioned stimulus will be reviewed. Finally,

126
the importance of the central nucleus of the amygdala and its efferent projections to several brainstem target areas for the expression of fear will be

outlined.

THE FEAR-POTENTIATED STARTLE PARADIGM

Brown et al. (1951) demonstrated that the amplitude of the acoustic startle

reflex in the rat can be augmented by presenting the eliciting auditory

startle stimulus in the presence of a cue (e.g., a light) that has previously

been paired with a shock. This phenomenon has been termed the “fearpotentiated startle effect” and has been replicated using either an auditory or

visual conditioned stimulus, when startle has been elicited by either a loud

sound or an airpuff (Davis 1986).

In this test, a central state of fear is considered to be the conditioned

response (McAllister and McAllister 1971). Conditioned fear is operationally defined by elevated startle amplitude in the presence of a cue previously

paired with a shock (figure 1). Thus, the conditioned stimulus does not

elicit startle. Furthermore, the startle-eliciting stimulus is never paired with

a shock. Instead, the conditioned stimulus is paired with a shock, and

startle is elicited by another stimulus either in the presence or absence of

the conditioned stimulus. Fear-potentiated startle is said to occur if startle

is greater when elicited in the presence of the conditioned stimulus. Potentiated startle only occurs following paired, rather than unpaired or “random,”

presentations of the conditioned stimulus and the shock, which indicates that

it is a valid measure of classical conditioning (Davis and Astrachan 1978).

Discriminations between visual and auditory conditioned stimuli (Hitchcook

and Davis 1986) or between auditory cues that differ in duration (Siegel

1967) have also been demonstrated with potentiated startle. Generalization

decrements resulting from a change in the frequency of a pure tone conditioned stimulus between training and testing have also been reported (Siegel

1967). Increased startle, in the presence of the conditioned stimulus, still

occurs very reliably at least 1 month after original training, making it

appropriate for the study of long-term memory as well (Cassella and Davis

1985).

TEMPORAL SPECIFICITY OF FEAR-POTENTIATED STARTLE

Recent data indicate that fear-potentiated startle shows a good deal of temporal specificity (Davis et al. 1989). To test this, separate groups of rats

were given 10 pairings of a light and a shock on each of 3 successive days.

In one group, the light-shock interval (conditioned stimulus-unconditioned

stimulus (CS-US) interval) was 200 milliseconds (i.e., a 700-millisecond

light terminating with a 500-millisecond shock); in the other group, the

light-shock interval was 51,200 milliseconds. A third group, the random

group, had lights and shocks presented in a nonsystematic fashion. Two

days later, all groups were tested identically by eliciting startle in the

127
TRAINING: LIGHT and SHOCK PAIRED

TESTING:

NOISE-ALONE

TRIALS

NORMAL STARTLE (in dark)

LIGHT-NOISE

TRIALS

POTENTIATED STARTLE (in light)

FIGURE 1. Cartoon depicting the fear-potentiated startle paradigm

NOTE? Bring training, a neutral stimulus (conditioned stimulus) such as a light is consistently paired

with a footshock. During testing, startle is elicited by an auditory stimulus (e.g., a 100-dB

burst of white noise) in the presence (light-noise trial type) or absence (noise-alone trial type)

of the conditioned stimulus. The positions and postures pictured may not represent the actual

behavior of the animals.

128
absence of the light (noise-alone trials) or 25, 50, 100, 200, 400, 800,

3,200, 12,800, or 51,200 milliseconds after onset of the light. The 10 to

20 occurrences of each of the 10 different test trial types were presented in

a balanced, irregular order across a 1.5-hour test session.

Unconditioned Effect of Light on Startle

The left panel of figure 2 shows the mean amplitude startle response at

each of the various test intervals for each of the three groups. The solid

line was drawn between the average values of the noise-alone trials for the

three groups to facilitate visualizing the effects of the light in the different

groups. In the random group, startle was markedly depressed from about

25 to 200 milliseconds after light onset (prepulse inhibition) and then

slightly elevated at the long test intervals. These data were consistent with

earlier reports showing that the presentation of a visual stimulus in untrained animals leads to prepulse inhibition at short test intervals and facilitation at long test intervals (Ison and Hammond 1971).

FIGURE 2. Temporal specificity of fear-potentiated startle

NOTE The left panel shows the mean amplitude startle response at each of the various light-noise

test intervals (25, 50, 100, 200, 400, 800, 3,200, 12,800, or 51,200 milliseconds plotted on a

log scale) for animals trained with either a 200-millisecond (open squares) light-shock interval,

a 51,200-millisecond (closed squares) light-shock interval, or a random relationship between

lights and shocks (triangles). The solid line was drawn between the average values of the

noise-alone trials for the three groups, which did not differ significantly from each other. The

right panel shows these same data transformed in the following way. First, the startle level

on the noisealone trials was subtracted from the startle level on each of the light-noise trials

for every animal, allowing startle at each light-noise test interval to be expressed relative to

the individual noise-alone baseline in that group. Next, using these difference scores, the data

from the random group at each test interval were subtracted from the data of each group at

every test interval, thus factoring out unconditioned effects of the light (i.e., prepulse inhibition at short intervals, facilitation at long intervals) so as to visualize the effects of prior

conditioning.

129
Conditioned Effects of Light on Startle After Training With Short vs.

Long CS-US Intervals

In the 200- and 51,200-millisecond groups, there was also a decrease in

startle at the 25-, 50-, and 100-millisecond test intervals. This indicates that

the light still produced prepulse inhibition, even in groups that had the light

previously paired with a shock. In the 200-millisecond group, there was a

sharp increase in startle from 50 to 400 milliseconds, which declined back

to the level of the random group at the later test intervals. In contrast, in

the 51,200-millisecond group, the largest increase in startle occurred much

later, at the 12,800- and 51,200-millisecond test intervals.

To more fully visualize the conditioned changes in startle at various lightnoise test intervals after previous training at different light-shock intervals, it

is necessary to factor out the complex unconditioned effects of the light on

startle seen in naive animals or the random group. To accomplish this, the

following data transformations were made. First, the startle level on the

noise-alone trials was subtracted from the startle level on each of the lightnoise trials for every animal. In this way, changes in startle at each of the

light-noise test intervals, relative to the individual noise-alone baseline for

that animal, could be compared after various light-shock training intervals.

Using these difference scores, the data from the random group at each test

interval were then subtracted from the data of each group at each test interval. This was done to factor out the unconditioned effects of light on

startle at the various test intervals (i.e., prepulse inhibition, facilitation at

long test intervals) to visualize differences based on prior conditioning.

The right panel of figure 2 shows the transformed data for the 200- and

51,200-millisecond groups derived from the subtractions listed above. Any

points above the zero line represent potentiated startle relative to an animal’s individual baseline and the random group. This panel shows quite

clearly that potentiated startle was maximal about 200 milliseconds after

light onset in rats trained with a 200-millisecond light-shock interval, with

no potentiation at long test intervals. In contrast, potentiated startle was

maximal about 51,200 milliseconds after training with a 51,200-millisecond

light-shock interval, with little or no potentiation at short test intervals.

To analyze the degree of temporal specificity of potentiated startle, other

groups of 10 rats were each given light-shock intervals of either 0, 25, 50,

100, 800, 3,200, or 12,800 milliseconds. They were tested 2 days later at

each of the several light-noise intervals, as described above. Figure 3

shows the transformed data for each of nine different training groups.

Again, data points above the zero line indicate increased startle at that test

interval relative to the random group. Figure 3 shows three important findings. First, potentiated startle was obtained over a very wide range of lightshock training intervals, from 25 to 51,200 milliseconds. Secondly, the test

interval of maximum potentiation within a group tended (although certainly

130
FIGURE 3. Mean change in startle amplitude at each of the various lightnoise test intervals after the nine different light-shock

training intervals

NOTE: The data have been transformed for each group as described in figure 2.

131
not in every group) to match the CS-US interval used in training. For

example, with a 200-millisecond training interval, potentiation was maximal

about 200 to 400 milliseconds after light onset but was completely gone at

the long test intervals. In contrast, with a 51,200-millisecond training

interval, potentiation was greatest at the 12,800- and 51,200-millisecond test

intervals but was very weak at the 200- to 400-millisecond test intervals.

Finally, the magnitude of potentiation tended to grow with longer training

intervals. Thus, at the 0-millisecond training interval (i.e., simultaneous

light and shock onset), no data points were above the line, indicating no

potentiation at any interval. With a 200-millisemnd training interval, the

maximal change was about 30 units, whereas, with a 51,200-millisecond

training interval, the maximal change was about 40 to 45 units.

These data indicate, therefore, that fear-potentiated startle shows considerable temporal specificity, since potentiated startle tends to be maximal at the

test interval that matches the training interval. Other studies showed that

temporal specificity tended to “sharpen” with an increasing number of training trials. Remarkably, a temporal pattern of potentiated startle similar to

that shown in figure 2 for the 51,200-millisecond group could be obtained

after a single training trial when the long, 51,200-millisecond light-shock

interval was used. Taken together, these data indicate that the conditioned

stimulus in fear conditioning is not simply the presence or absence of the

light, but instead is the temporal pattern of events that are paired with the

US in training.

THE PHARMACOLOGY OF FEAR-POTENTIATED STARTLE

Advantages of Fear-Potentiated Startle for Studying the Pharmacology

of Fear or Anxiety

Fear-potentiated startle offers a number of advantages for analyzing how

drugs affect fear or anxiety. First, potentiated startle is defined as a withinsubjects difference in startle amplitude in the presence (light-noise trials) vs.

the absence (noise-alone trials) of the visual conditioned stimulus. This

makes it a sensitive measure, because it reduces problems caused by

between-subjects variability in startle. Second, it allows an evaluation of

specific effects (reduction of startle on light-noise trials) vs. nonspecific

effects (reduction of startle on noise-alone trials), so that qualitative as well

as quantitative drug profiles can be compared. Third, different intensities of

the auditory stimulus can be used within the same test session to elicit

startle. This allows potentiated startle to be assessed at several points on

the measurement scale, circumventing problems of interpretation that can

arise when markedly different parts of the measurement scale are involved

(e.g., rate-dependent drug effect in operant paradigms; percent figures used

with very different baselines). Fourth, no shocks are given in testing.

Thus, drug effects observed in testing cannot be explained in terms of

changes in sensitivity to shock. Fifth, the separation between training and

132
testing sessions allows one to evaluate whether a drug alters original learning or performance, and tests for state-dependent learning can be easily

evaluated (Davis 1979a). Sixth, potentiated startle does not involve any

obvious operant. Thus, the animal is not required to make or withhold a

voluntary response to indicate fear or lack of fear, and, consequently, druginduced effects that might be expected to alter operant performance (e.g.,

rate-dependent, motivational, disinhibitory motor effects) are circumvented.

In addition, most animal tests of fear or anxiety involve a suppression of

ongoing behavior in the presence of a fear stimulus (e.g., suppression of bar

pressing in the conditioned emotional response test or the operant conflict

test; suppression of licking in the lick-suppression test; suppression of

movement measured by freezing; suppression of normal activity in the

social interaction test). Hence, certain treatments (e.g., decreases in

serotonin transmission) might appear anxiolytic in these tests if they interacted with neural systems common to each of these tests (e.g., response

inhibition), even though they might not reduce anxiety clinically. Because

fear in the potentiated startle paradigm is reflected by enhanced response

output, it may provide an important alternative test with which to analyze

potential anxiolytic compounds.

Effects of Different Drugs on Fear-Potentiated Startle

Table 1 shows that a variety of drugs that reduce fear or anxiety in humans

decrease potentiated startle in rats. Clonidine, morphine, diazepam, and

buspirone, which differ considerably in their mechanism of action, all block

potentiated startle. In most cases, these treatments do not depress startle

levels on the noise-alone trials (figures 4 and 5), although clonidine

(figure 6) does have marked depressant effects on both types of trials. In

addition, yohimbine and piperoxane (figure 6), which induce anxiety in

normal people and exaggerate it in anxious people (Chamey et al. 1984;

Goldenberg et al. 1947; Holmberg and Gershon 1961; Soffer 1954), actually

increase potentiated startle in rats. Thus, at very low doses, these drugs increase startle amplitude on the light-noise trials without having any effect

on startle on the noise-alone trials; this only occurs in rats conditioned to

fear the light (Davis et al. 1979).

On the other hand, table 1 shows that a variety of treatments that alter

serotonin (5-HT) transmission do not affect potentiated startle. This is

important because treatments that decrease 5-HT transmission have an

anxiolytic profile in several animal tests of fear or anxiety (e.g., operantconflict test, lick-suppression test, and social interaction test), perhaps by

interfering with response inhibition (Soubrie 1986). These effects may

represent false positives in these tests, because treatments like p-chlorophenylalanine do not appear to be anxiolytic when tested clinically (Shopsin

et al. 1976). In addition, although the anxiolytic effects of buspirone have

been suggested to be mediated through the serotonin system, the ability of

buspirone to selectively decrease fear-potentiated startle does not seem to be

133
TABLE 1. Effects of different treatments that alter different

neurotransmitters on fear-potentiated startle

Treatment Effects on Dose Range

Potentiated Startle (mg/kg) Reference

Block

Sodium amytal 10-40

Diazepam 0.3-2.5

Flurazepam

Morphine

Alcohol

Nicotine*

2.5-20

2.5-10

7.5-22.5 cc/kg

0.4 mg/kg

Buspirone

Gepirone

Clonidine

Do Not Block

Cinanserin

Cyproheptadine

Ipsapirone

8-0H-DPAT

p-chloroamphetamine

p-chlorophenylalanine

Naloxone

Ro-15-1788

Raphe lesions

1-PP (buspirone

metabolite)

Imipramine (chronic

or acute)

WB-4101

Propranolol*

Increase

Piperoxane

Yohimbine

0.6-10

0.6-10

0.01-0.04

10

5

5-20

2.5-10

5

400x2

2.0

1.0

---

0.5-40

5-10

1.0

20

0.25-1.0

0.125-.25

Chi 1965

Davis 1979a

Berg and Davis 1984

Davis 1979a

Davis 1979b

Miller and Barry 1960

Sorenson and Wilkinson

1983

Kehne et al. 1988

Kehne et al. 1988

Davis et al. 1979

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Davis et al. 1988

Kehne et al. 1988

Cassella and Davis 1985

Davis et al. 1979

Davis et al. 1979

Davis et al. 1979

Davis et al. 1979

*Partial blockade.

attributable to its actions at either pre- or postsynaptic 5-HT receptors

(Davis et al. 1988). Thus, other drugs such as ipsapirone or 8-OH-DPAT,

134
FIGURE 4. Mean amplitude startle on noise-alone trials (dark bars) or

light-noise trials (open bars) following IP injection 15

minutes earlier of saline or morphine (0.6, 2.5, 10 mg/kg)

NOTE: Rata were given 45 light-shock pairings (1,000-millisecond light, 500-millisecond, 0.25-mA

shock, 500-millisecond light-shock interval) on each of 2 days. One day later, a total of 40

noise-alone and 40 light-noise trials (20 at 105 dB and 20 at 110 dB) were given in an irregular order at a 30-second interval. Data were collapsed across the two stimulus intensities

used to elicit startle.

SOURCE Adapted from Davis 1979b.

which bind to 5-HT1A-binding sites as buspirone and gepirone do, do not

block fear-potentiated startle. Moreover, raphe lesions by themselves do

not block fear-potentiated startle, nor do they prevent buspirone from having

its usual anxiolytic effect in this test. Currently, the effects of drugs that

135
FIGURE 5. Mean amplitude startle on noise-alone trials (dark bars) or

light-noise trials (open bars) 5 minutes after SC injection

of saline or buspirone (0.6, 1.25, 2.5, 5.0 mg/kg)

NOTE: Rats were given 10 light-shock pairings (3,700-millisecond light, 500-millisecond later, 0.6-mA

shock, 3,200-millisecond light-shock interval) on each of 2 days. Two days later a total of 30

Norse-alone and 30 light-noise trials (10 at 90, 95, or 105 dH) were given in an irregular

order at a 30-second interval. Data were collapsed across the three stimulus intensities used

to elicit startle.

SOURCE: Kehne et al. 1988, Copyright 1988, Springer-Verlag.

act on dopamine, vs. dopamine, receptors on fear-potcntiated startle that

may be relevant to the actions of drugs like buspirone (Taylor et al. 1982)

are being tested.

THE ACOUSTIC STARTLE PATHWAY

A major advantage of the potentiated startle paradigm is that it measures

fear by a change in a simple reflex. Hence, with potentiated startle, fear is

expressed through some neural circuit that is activated by the conditioned

stimulus and ultimately impinges on the startle circuit. These two neural

136
FIGURE 6. Mean amplitude startle on noise-alone trials (dark bars) or

light-noise trials (open bars) 5 minutes after IP injection of

saline, clonidne (20 pg/kg), piperoxane (1.0 mg/kg), or

yohimbine (0.25 mg/kg)

NOTE: Rats were given 45 light-shock pairings (1,000-millisecond light, 500-millisecond later,

0.25-mA shock, 500-millisecond light-shock interval) on each of 2 days. One day later, a

total of 40 noise-alone and 40 light-noise trials (20 at 105 dB and 20 at 110 dB) were given

in an irregular order at a 30-second interval. Data were collapsed across the two stimulus

intensities used to elicit startle.

SOURCE: Adapted from Davis et al. 1977.

pathways are being delineated to see how they interconnect to mediate

potentiated startle.

In the rat, the latency of acoustic startle is 6 milliseconds recorded electromyographically in the foreleg and 8 milliseconds in the hindleg (Ison et al.

1973). This is an extraordinarily short latency and indicates that only a few

synapses can be involved. Figure 7 illustrates the nuclei and fiber tracts

137
believed to mediate the acoustic startle response in the rat (Davis et al.

1982).

The posteroventral cochlear nucleus (VCN) appears to be the first synapse

in the primary acoustic startle circuit. Bilateral lesions of the VCN, but not

the neighboring dorsal cochlear nuclei, abolish acoustic startle. In awake

rats, bilateral, single-pulse stimulation of the VCN (1-millisecond pulse

width, 10 to 25 μA) elicits startlelike responses with a latency of 7.0 to

7.5 milliseconds (figure 8).

The next synapse appears to occur in the ventral nucleus of the lateral lemniscus (VLL), which is known to receive direct projections from the VCN.

Bilateral lesions of the VLL eliminate acoustic startle, and electrical stimulation of this nucleus elicits startlelike responses with an average latency of

about 6.0 milliseconds (figure 8).

The next synapse may occur in a ventromedial region of the nucleus reticularis pontis caudalis (RPC). Bilateral lesions of this area abolish acoustic

startle. Electrical stimulation of points within the RPC elicits startlelike

responses with an average latency of about 5 milliseconds. Cell bodies in

the RPC send their axons to all levels of the spinal cord (SC) by way of

reticulospinal tract. This tract courses near or through the medial longitudinal fasciculus (MLF) on the midline and then bifurcates to form the ventral funiculi in the SC. Complete lesions of the MLF eliminate acoustic

startle, and electrical stimulation on the midline through the MLF elicits leg

movements with a latency of about 4.0 to 4.5 milliseconds (figure 8).

Fibers from the reticulospinal tract synapse in the spinal cord, forming the

final synapse before the neuromuscular junction. Direct monosynaptic connections onto motorneurons, as well as indirect ones through an interneuron

in the cord, are possible. To date, it has not been determined whether

spinal interneurons are involved.

More recently, it has been found that infusion of ibotenic acid, which

destroys cell bodies without damaging fibers of passage (Schwarcz et al.

1979; Zaczek and Coyle 1982), into either the VCN, VII, or RPC also

eliminates acoustic startle (Cassella and Davis 1986).

DETERMINING THE POINT WITHIN THE STARTLE PATHWAY

WHERE FEAR ALTERS NEURAL TRANSMISSION

Having delineated the startle reflex circuit involved in fear-potentiated

startle, the next task was to determine the point within the startle circuit

where the visual conditioned stimulus modulates transmission following conditioning. To do this, startlelike responses were elicited electrically from

various points along the startle pathway before and after presentation of a

light that was either paired or not paired with a shock in different groups of

138
FIGURE 7. Schematic diagram of a primary acoustic startle circuit

consist& of the VCN, VLL, and RPC

NOTE: Other abbreviations used are: A, aqueduct; CNIC, central nucleus of the inferior colliculus;

CU, cuneate nucleus; DCN, dorsal cochlear nucleus; DP, decussation of pyramids; DR, dorsal

raphe nucleus; ENIC, external nucleus of the inferior colliculus; HRP, horseradish peroxidase;

IO, inferior olive; LL, lateral lemniscus; LM, medial lemniscus; LV, lateral vestibular nucleus;

MLF, medial longitudinal fasciculus; MTB, medial nucleus of the trapezoid body; MV, medial

vestibular nucleus; nVII, nucleus of the seventh nerve; P, pyramids; RGI, nucleus reticularis

gigantocellularis; RPO, nucleus reticularis pontis oralis; RST, reticulospinal tract; RSTm

medial reticulospinal tract; SO, superior olive; TSV, spinal tract of the fifth nerve; VAS,

ventral acoustic stria; VII, seventh nerve.

SOURCE: Davis et al. 1982, Copyright 1982, Society for Neuroscience

139
FIGURE 8. Electromyographic recording from the quadriceps femoris

muscle complex of the “startle” response elicited by

electrical stimulation in the SC, MLF, RPC, LL, VCN, or

by a tone

SOURCE: Davis et al. 1982, Copyright 1982, Society for Neuroscience

140
rats (Berg and Davis 1985). Startle elicited by electrical stimulation at or

before the point in the startle circuit where the light modulates transmission

should show potentiation, whereas startle elicited beyond this point should

not (see footnote to figure 9 for the rationale of this experiment). In this

experiment, separate groups of rats were implanted, at least 24 hours prior

to training, with bilateral monopolar electrodes (0.25 mm diameter, 0.5 mm

uninsulated tip) in either the VCN, VLL, or RPC. In addition, other groups

were implanted with electrodes aimed at lateral as well as medial aspects of

the ventral acoustic stria (VAS), the fibers that connect the VCN to the

VLL. Following recovery, all groups were trained to be fearful of a light

by pairing it with a footshock.

FIGURE 9. Schematic diagram of modulation of electrically elicited

startle by presentation of a light previously paired with a

shock

NOTE: The point of electrical elicitation of startle is indicated by the “stim” symbol. The model predicts that if the light ultimately modulates neural transmission at the RPC, then startle elicited

electrically in the VCN or VLL will be enhanced by the light, since the signal produced by

electrical stimulation must travel through the area where the light modulates neural transmission. In contrast, startle elicited electrically in the RPC beyond the area of modulation or

startle elicited electrically in spinal motor neurons will not be enhanced by the light, since the

signal produced by electrical stimulation will bypass the area where the light modulates neural

transmission.

Figure 10 shows that potentiation of electrically elicited startle was equivalent to potentiation of acoustic startle at all locations up through the VLL.

141
FIGURE 10. Magnitude of fear-potentiated startle effect when startle was

elicited either acoustically (white bars) or electrically

(black bars) in different groups of rats that had electrodes

implanted in the VCN, VAS, VLL, or RPC

NOTE: The white bars represent the degree of potentiation of startle elicited acoustically in tats with

electrodes implanted into different parts of the startle pathway. The black bars represent the

degree of potentiation of startle elicited electrically through electrodes implanted in different

parts of the startle pathway. Degree of potentiation is expressed as the mean difference in

startle amplitude in the presence vs. absence of the visual conditioned stimulus.

SOURCE: Berg and Davis 1985, Copyright 1985, American Psychological Association.

In contrast, startle elicited from the RPC was not potentiated. The difference in potentiation of electrically and acoustically elicited startle at sites

beyond the VLL cannot be attributed to the extent of effective conditioning,

because potentiation of startle elicited acoustically was just as great in the

RPC group as it was in the VCN, VAS, and VLL groups. Furthermore, the

potentiation of electrically elicited startle was specific to an explicit pairing

142
of light and shock during training. Animals with electrodes implanted in

the VCN and trained with a random temporal relation between light and

shock showed no sign of potentiation of either electrically or acoustically

elicited startle. Finally, other studies showed that drugs like diazepam,

which are known to block fear-potentiated startle elicited acoustically, also

block fear-potentiated startle elicited electrically through the VCN (figure

11) (Berg and Davis 1984). Taken together, these data indicate that the

VLL or the RPC is the point in the startle pathway where a visual conditioned stimulus ultimately modulates transmission following conditioning so

as to affect the startle reflex.

THE ROLE OF THE AMYGDALA IN FEAR-POTENTIATED

STARTLE

Lesions of the Central Nucleus of the Amygdala Block Fear-Potentiated

Startle

It was hypothesized that lesions of the amygdala, a structure long implicated

in fear, should block potentiated startle, because potentiated startle is a

measure of fear. In fact, lesions of the central nucleus of the amygdala

following fear conditioning were shown to completely eliminate potentiated

startle (figure 12) (Hitchcock and Davis 1986; Hitchcock and Davis 1987).

In contrast, transection of the cerebellar peduncles or lesions of the red

nucleus had no effect on potentiated startle. Another experiment using a

visual prepulse test indicated that the blockade of potentiated startle

observed in animals with lesions of the amygdala could not be attributed to

visual impairment. A third experiment indicated that the absence of potentiation in the amygdala-lesioned animals did not simply result from a lowered

startle-level ceiling, because the amygdala-lesioned animals could show

increased startle with increased stimulus intensity and with administration of

strychnine, a drug that reliably increases startle (Kehne et al. 1981). Taken

together, the results of these experiments support the hypothesis that the

amygdala is involved in potentiated startle—a measure of conditioned fear.

Enhancement of Acoustic Startle By Electrical Stimulation of the

Amygdala

At the present time, it is not clear how the amygdala participates in fearpotentiated startle. It is possible that the light, after being paired with

shock, activates the amygdala, which would then increase startle. Shortlatency visual-evoked potentials have been recorded in the amygdala

(Pollock et al. 1976; Sanghera et al. 1979), and electrical stimulation of the

amygdala has been reported to produce fearlike behaviors in animals

(Applegate et al. 1983; Gloor 1960), to mimic conditioned and unconditioned cardiac effects in rabbit heart-rate conditioning (Kapp et al. 1982),

and to elicit feelings of anxiety in humans (Chapman et al. 1954). Consistent with this, it was recently found that low-level electrical stimulation of

143
FIGURE 11. Mean amplitude of startle elicited by electrical stimulation of

the VCN in the presence (white bars) or absence (black

bars) of the light after administration of various doses of

diazepam or its vehicle

SOURCE: Berg and Davis 1984, Copyright 1984, Pergamon Press.

the amygdala (e.g., 40 to 400 μA, 25-millisecond trains of 0.1-millisecond

square-wave cathodal pulses) markedly increases acoustic startle amplitude

(Rosen and Davis 1988). This excitatory effect has occurred in every rat

tested so far in which electrodes had been placed in the central, intercalated,

or medial nucleus of the amygdala or in the area just medial to the amygdaloid complex. Stimulation of the area just medial to the amygdala had

the lowest threshold for increasing acoustic startle. This area coincides with

the initial part of the ventral amygdalofugal pathway as it begins its projection to the lower brainstem (Krettek and Price 1978; Schwaber et al.

144
FIGURE 12. Mean amplitude startle on the light-noise or noise-alone

trials in rats in which the cerebellum was surgically

transected from the brainstem or in rats with electrolytic

lesions of the red nucleus or the central nucleus of the

amygdala

NOTE: Sham animals for the cerebellar experiment had the transection knife inserted under the

cerebellum hut the fiber pathways were not cut. Sham animals for the other experiments had

the electrodes lowered into the brain. but no current was passed.

SOURCE: Hitchcock and Davis 1986, Copyright 1986, American Psychological Association

1982; Post and Mai 1980). Low-level electrical stimulation at this site

would be expected to activate a large number of fibers projecting to the

brainstem, since they are highly concentrated at this part of the pathway.

In contrast, stimulation of the amygdala nuclei themselves, where the

neurons of these fibers originate, would require higher currents (i.e., more

current spread) to activate the same number of brainstem projections,

because the neurons are dispersed throughout the nuclei. Further studies

involving stimulation of the area just medial to the amygdala where the

ventral amygdalofugal pathway begins, in combination with lesions of cell

bodies (e.g., ibotenic acid lesions) in the central, medial, intercalated, or

basolateral nucleus, would help clarify whether enhancement of startle by

stimulation in this pathway actually results from activation of the axons

originating from these amygdaloid nuclei.

Electrical stimulation of the amygdala did not produce any signs of behavioral activation except for an enhancement of startle at these stimulation

145
currents and durations, indicating that startle is an extremely sensitive index

of amygdala stimulation. Moreover, the duration of stimulation is well

below that used to produce kindling in rats (Handforth 1984) so that the

effects on startle are not associated with convulsions.

The excitatory effect on startle occurs within a few milliseconds from the

onset of amygdala stimulation, This rapidity of action means that the

increase in startle is not secondary to autonomic or hormonal changes that

might be produced by amygdala stimulation, because these would have a

much longer latency. In addition, electrical stimulation of the amygdala

alone does not elicit startle, even at high currents. Finally, electrical

stimulation of several other nearby brain areas such as the endopiriform

nucleus, fundus striati, internal capsule, or some sites in the basolateral

nucleus of the amygdala do not increase startle.

The Role of Different Amygdala Efferent Projections in FearPotentiated Startle

As mentioned earlier, lesions of the central nucleus of the amygdala block

fear-potentiated startle (figure 12). Recently, the sensitive Phaseolus

vulgaris-leucoagglutinin (PHA-L) anterograde tracing technique (Gerfen and

Sawehenko 1984) and lesion studies have been used to delineate possible

connections between the amygdala and the startle pathway in relation to

fear-potentiated startle (Hitchcock and Davis, in press). The central nucleus

of the amygdala projects to a variety of brain regions via two major efferent pathways, the stria terminalis and the ventral amygdalofugal pathway.

Lesions of the stria terminalis itself, or of the bed nucleus of the stria

terminalis, a major projection area of this pathway, do not block potentiated

startle. Knife cuts of the rostral part of the ventral amygdalofugal pathway,

which would interrupt its projections to the rostral lateral hypothalamus and

substantia innominata, also fail to block potentiated startle. On the other

hand, lesions of the caudal part of the ventral amygdalofugal pathway, at

the point where it passes through the subthalamic area and cerebral peduncles, completely block potentiated startle. Interestingly, Jarrell et al. (1986)

found that lesions of this area also block heart-rate conditioning, another

measure of fear conditioning. Finally, lesions of the substantia nigra, which

receives central nucleus projections as well as fibers of passage from the

central nucleus of the amygdala to more caudal brainstem regions, also

block potentiated startle. This blockade does not seem to involve dopamine

cells in the zona compacta, since infusion of the dopamine neurotoxin

6-0HDA into the substantia nigra did not block potentiated startle despite a

more than 90 percent depletion of dopamine in the caudate nucleus.

These lesion experiments indicate that the pathway from the central nucleus

of the amygdala to the startle circuit travels through the caudal part of the

ventral amygdalofugal pathway, which is known to project directly to many

parts of the pons, medulla, and probably the SC (Krettek and Price 1978;

146
Mizuno et al. 1985; Post and Mai 1980; Price and Amaral 1981; Sandrew

et al. 1986; Schwaber et al. 1982). In fact, Inagaki et al. (1983) have

reported direct connections between the central nucleus of the amygdala and

the exact part of the RPC (an area just dorsal to the superior olive) that is

critical for startle. This projection has been verified through the use of

both anterograde tracing techniques.

Relationship Between Fear-Potentiated Startle and Startle Increased By

Electrical Stimulation of the Amygdala

It is not clear whether startle enhanced by electrical stimulation of the

central nucleus of the amygdala is related to fear-potentiated startle.

Several experiments could be performed to evaluate this hypothesis. For

example, lesions of points along the ventral amygdalofugal pathway that

block fear-potentiated startle should also block startle enhanced by stimulation of the amygdala. In fact, in a preliminary experiment, lesions of the

caudal ventral amygdalofugal pathway at the level of the subthalamic

nucleus and substantia nigra blocked the enhancement of startle normally

produced by electrical stimulation of the central nucleus of the amygdala.

Relationship of the Amygdala to the Visual Structures Involved in

Fear-Potentiated Startle

Thus far, there is no direct information linking the amygdala to any visual

structures that appear critical for fear-potentiated startle. The central

nucleus of the amygdala is known to receive input from the lateral nucleus

of the amygdala, which receives afferents from the insular cortex (Turner

and Zimmer 1984). The insular cortex receives visual information

(Turner and Zimmer 1984) through a pathway probably involving the lateral

geniculate nucleus, visual cortex, and visual association cortex. Thus, it is

possible that the visual conditioned stimulus would activate the central nucleus of the amygdala after being relayed through these structures. Hitchcock et al. (unpublished observations) have found that lesions of the lateral

geniculate nucleus and insular cortex block fear-potentiated startle. In

contrast, lesions of superficial layers of the superior colliculus, the pretectal

area, the parietal cortex, or the dorsal lateral lemniscus did not block

potentiated startle (Tischler and Davis 1983). It was also reported in that

study that lesions of deep and intermediate layers of the superior colliculus

blocked fear-potentiated startle. Recently, this result was replicated with

testing 6 to 7 days after lesions of deep and intermediate layers of the

superior colliculus (Hitchcock and Davis, unpublished observations). At this

time, however, baseline levels of startle (i.e., on the noise-alone trials) were

markedly elevated after lesions of the superior colliculus. In fact, when

these animals were retested using a very weak, 75-dB startle stimulus, they

each showed increased startle in the presence of the light. Moreover,

lesions of deep and intermediate layers of the superior colliculus did not

prevent electrical stimulation of the amygdala from enhancing startle

147
(Hitchcock et al., unpublished observations). Hence, it is now believed that

deep and intermediate layers of the superior colliculus, which project directly to the VLL (Henkel and Edwards 1978; Henkel 1981), tonically inhibit

acoustic startle. This effect may interfere with the measurement of fearpotentiated startle unless special test conditions are arranged. However, the

superior colliculus does not appear to be either an obligatory visual relay in

startle potentiated by a visual-conditioned fear stimulus or part of the pathway connecting the central nucleus of the amygdala to the startle circuit.

Moreover, Hitchcock and Davis (unpublished observations) have recently

found that complex removal of the visual cortex does not block fearpotentiated startle. Further studies will try to determine the role of connections between the lateral geniculate nuclei and the perirhinal cortex in fearpotentiated startle.

ENHANCEMENT OF STARTLE BY FOOTSHOCKS

Effects of Footshocks on Acoustic Startle

Fear-potentiated startle is defined by an increase in startle in the presence of

a cue previously paired with shock. One might expect, therefore, that shock

itself should increase startle amplitude, so that fear-potentiated startle would

reflect the familiar finding that the conditioned response mimics the unconditioned response. Curiously, however, Brown et al. (1956) reported that

acoustic startle was actually depressed when elicited from 15 to 60 seconds

after a footshock, although longer intervals were not tested. Recently,

Fanselow and colleagues (Fanselow 1981; Fanselow 1982; Fanselow 1984;

Fanselow and Bolles 1979) have shown that shock leads to an increase in

freezing, a traditional measure of fear in rats. Interestingly, however, freezing does not occur immediately after the shock, but develops gradually over

several minutes. Since the magnitude of fear-potentiated startle correlates

highly with the amount of freezing measured in the same experimental situation (Leaton and Borszcz 1985), it was reasoned that startle should increase for several minutes following footshock with a timecourse similar to

that of freezing reported by Fanselow. Consistent with this expectation,

figure 13 shows that a series of 10 shocks (0.6 mA, 0.5 seconds in duration

presented at a rate of 1 shock per second) markedly increased startle measured over a 20-minute period after the shocks. Interestingly, this effect did

not occur immediately, but developed over several minutes, similar to the

timecourse of freezing seen by Fanselow. However, unlike Fanselow’s results where freezing appears to be context specific, several laboratory experiments have led to the conclusion that this increase in startle does not result

from a rapid conditioning to the context. Therefore, it is thought that the

increase in startle seen after footshocks may represent the unconditioned

effects of the shock on startle, and that this becomes conditioned to a light

that is consistently paired with that shock.

148
FIGURE 13. Enhancement of startle by footshock

NOTE Mean amplitude startle response prior to and following a series of five 0.6-mA, 500-

millisecond footshocks (indicated by the arrow) presented once per second (solid) or no intervening shock (open). Forty 105-dB noise bursts were presented at a 30-second interval both

before and after the shocks.

The Role of the Amygdala in Footshock-Induced Enhancement of

Startle

Because footshock is both aversive and fear producing, it is possible that

footshock activates the amygdala, which then leads to an elevation of startle

via connections between the amygdala and the startle pathway. In fact,

figure 14 shows that lesions of the central nucleus of the amygdala prevent

footshocks from enhancing startle (Hitchcock et al. 1989). Other studies

showed that this effect could not be attributed to a lesion-induced change in

149
shock sensitivity. Moreover, lesions of the immediately adjacent lateral

amygdala nucleus did not block shock sensitization. Finally, lesions of the

caudal ventral amygdalofugal pathway, which connects the central nucleus

of the amygdala to the startle circuit, also completely blocked shock

sensitization.

FIGURE 14. Enhancement of startle by footshock in lesioned or

unoperated animals

NOTE: Mean amplitude startle response over successive 2-minute periods preceding and following

presentation (arrow) of 10 0.6-mA footshocks (solid) or no footshocks (open) in unoperated

(left panel), sham-lesioned (center panel), and amygdala-central-nucleus-lesioned animals (right

panel).

SOURCE: Hitchcock et al. 1989, Copyright 1989, American Psychological Association.

THE ROLE OF THE AMYGDALA IN INNATE AND

CONDITIONED FEAR

A variety of animal models have been used to infer a central state of fear

or anxiety. In some models, fear is inferred when an animal freezes, thus

interrupting some ongoing behavior such as pressing a bar or interacting

socially with other animals. In other models, fear is measured by changes

in autonomic activity such as heart rate, blood pressure, or respiration. Fear

can also be measured by a change in simple reflexes or a change in facial

expressions and mouth movements. Thus, fear appears to produce a complex pattern of behaviors that are highly correlated with each other.

150
Anatomical Connections Between the Amygdala and Brain Areas

Involved in Fear

Reflecting suggestions of several previous reviews (Gloor 1960; Kapp and

Pascoe 1984; Kapp et al. 1984; Sarter and Markowitsch 1985), figure 15

summarizes work done in many different laboratories indicating that the

central nucleus of the amygdala has direct projections to a variety of brainstem areas that might be expected to be involved in many of the signs of

fear. Thus the central nucleus of the amygdala projects to a region of the

central grey (Beitz 1982; Post and Mai 1980) that has been implicated in

fear in a number of behavioral tests (Iwata et al. 1986; Liebman et al.

1970). Direct projections to the dorsal motor nucleus of the vagus

(Hopkins and Holstege 1978; Schwaber et al. 1982; Takeuchi et al. 1983;

Veening et al. 1984) may be involved in several autonomic measures of

fear, since the vagus nerve controls many different autonomic functions.

FIGURE 15. Schematic diagram of connections of the central nucleus of

the amygdala to target areas probably involved in the

pattern of behaviors typically associated with fear

Projections of the central nucleus of the amygdala to the parabrachial

nucleus (Krettek and Price 1978; Price and Amaral 1981; Takeuchi et al.

1982) may be involved in respiratory changes during fear, since electrical

stimulation of the parabrachial nucleus is known to alter respiratory rate

(Cohen 1971; Bertrand and Hugelin 1971). Direct projections to the trigeminal (Post and Mai 1980) and perhaps the facial motor nuclei may mediate

151
some of the facial expressions of fear. As outlined earlier, projections of

the amygdala to the RPC (Inagaki et al. 1983) probably are involved in fear

potentiation of the startle reflex. Finally, projections from the central

nucleus of the amygdala to the ventral tegmental area (Phillipson 1979) may

mediate stress-induced changes in dopamine turnover in the frontal cortex

(Thierry et al. 1976). Moreover, this projection might also link the central

nucleus of the amygdala to the locus coeruleus (Deutch et al. 1986), which

itself has been implicated in fear and anxiety (Redmond 1977) or increased

vigilance and attention (Aston-Jones and Bloom 1981).

Fear Produced by Electrical Stimulation of the Amygdala

Significantly, it has also been shown that electrical stimulation of the central

nucleus of the amygdala can produce a complex pattern of behavioral and

autonomic changes that, taken together, constitute a state that closely

resembles a state of fear. Thus, electrical stimulation of the central nucleus

of the amygdala produces a cessation of ongoing behavior (Applegate et al.

1983; Gloor 1960). In fact, cessation of ongoing behavior is the critical

measure of fear or anxiety in several animal models such as the operant

conflict test (Geller and Seifter 1960). the conditioned emotional response

(Estes and Skinner 1941), the social interaction test (File 1980), and freezing itself (Fanselow and Bolles 1979). Stimulation of the amygdala can

also alter heart rate (Applegate et al. 1983; Kapp et al. 1982) and blood

pressure (Morgenson and Calaresu 1973), both of which are measures used

to study cardiovascular conditioning. Electrical stimulation of the central

nucleus of the amygdala also alters respiration (Applegate et al. 1983;

Harper et al. 1984) a prominent symptom of fear, especially in panic disorders. Electrical stimulation of the amygdala also elicits jaw movements

(Applegate et al. 1983; Gloor 1960; Ohta 1984), which often accompany the

fear response. Amygdala stimulation can also produce gastric ulceration

(Henke 1980a; Henke 1980b; Innes and Tansy 1980; Sen and Anand 1957),

which may result from chronic fear or stress. As outlined earlier, electrical

stimulation of specific parts of the amygdala increases the acoustic startle

reflex, which is elevated during fear. Finally, it has been reported in

humans that electrical stimulation of the amygdala elicits feellngs of fear or

anxiety as well as autonomic reactions indicative of fear (Chapman et al.

1954; Gloor et al. 1981).

Taken together, the highly correlated set of behaviors seen during fear may

result from activation of a single area of the brain (the central nucleus of

the amygdala), which then projects to a variety of target areas that are critical for each of the specific signs of fear. Moreover, it must be assumed

that all of these connections arc already formed in an adult organism, since

electrical stimulation produces these effects in the absence of prior explicit

fear conditioning. Given this innate wiring diagram, it would seem most

parsimonious to assume that a neutral stimulus will elicit a state of fear

when that stimulus comes to activate the amygdala after being paired with

152
an aversive stimulus. Thus, plasticity following fear conditioning probably

results from a change prior to or in the amygdala rather than a change in

its efferent target areas.

The Role of the Amygdala in Fear Elicited by a Conditioned Stimulus

Consistent with this interpretation, several studies have shown that a neutral

stimulus paired with aversive stimulation will now alter neural firing in the

amygdala, especially in the central nucleus of the amygdala (Henke 1983;

Pascoe and Kapp 1985). Moreover, lesions of the central nucleus are

known to eliminate or attenuate conditioned changes measured by a cessation of ongoing behavior such as freezing (Iwata et al. 1986); reduced bar

pressing in the operant conflict test (Shibata et al. 1986); or the conditioned

emotional response paradigm (Kellicut and Schwartzbaum 1963; Spevack et

al. 1975). Lesions of the central nucleus also block conditioned changes in

heart rate (Cohen 1975; Gentile et al. 1986; Kapp et al. 1979), blood pressure (Iwata et al. 1986) or ulceration induced by immobilization stress

(Henke 1980a; Henke 1980b). Data outlined earlier indicate that lesions of

the central nucleus of the amygdala block fear-potentiated startle (Hitchcock

and Davis 1986). Lesions of the amygdala are known to block several

measures of innate fear in different species (Blanchard and Blanchard 1972;

Ursin et al. 1981). This, along with a large literature implicating the amygdala in many other measures of fear such as active and passive avoidance

(Kaada 1972; Sarter and Markowitsch 1985; Ursin et al. 1981) and evaluation and memory of emotionally significant sensory stimuli (Bennett et al.

1985; Bresnahan and Routtenberg 1972; Ellis and Kesner 1983; Gallagher

and Kapp 1978; Gallagher and Kapp 1981; Gold et al. 1975; Handwerker et

al. 1974; Kesner 1982; Liang et al. 1985; Liang et al. 1986; Mishkin and

Aggleton 1981), compellingly indicates a crucial role of the amygdala in

fear.

SUMMARY AND CONCLUSIONS

The potentiated startle paradigm measures conditioned fear by an increase in

the amplitude of a simple reflex (the acoustic startle reflex) in the presence

of a cue previously paired with shock. This paradigm offers a number of

advantages as an alternative to most animal tests of fear or anxiety, since it

involves no operant and is reflected by an enhancement rather than a suppression of ongoing behavior. Lesion and electrical stimulation studies on

fear-potentiated startle and startle increased by electrical stimulation of the

amygdala are being used to define the neural pathways necessary for a visual conditioned stimulus to alter the acoustic startle reflex. The current

working hypothesis is that the conditioned stimulus activates the central

nucleus of the amygdala through a pathway involving the lateral geniculate

nucleus and insular cortex. The central nucleus of the amygdala may then

project directly to the acoustic startle pathway, modulating the startle

response. More work has to be done to define conclusively the relevant

153
neural pathways involved in fear-potentiated startle. Nonetheless, by

combining behavioral, anatomical, physiological, and pharmacological

approaches, it will be possible to determine each step along the pathway

that mediates the ability of a stimulus signaling fear to alter behavior.

Once the exact structures are delineated, it should be possible to determine

the neurotransmitters that are released during a state of fear and how this

chemical information is relayed along these pathways to affect behavior.

Eventually, this approach should help to determine where plastic changes

take place along these pathways to mediate the conditioned effects that are

being measured and the biochemical processes that are involved.

REFERENCES

Alkon, D.L. Voltage-dependent calcium and potassium ion conductances:

A contingency mechanism for an associative learning model. Science

205:810-816, 1979.

Applegate, C.D.; Kapp, B.S.; Underwood, M.D.; and McNall, C.L. Autonomic and somatomotor effects of amygdala central nucleus stimulation in

awake rabbits. Physiol Behav 31:353-360, 1983.

Aston-Jones, G., and Bloom, F.E. Norepinephrine-containing locus coeruleus neurons in behaving rats exhibit pronounced responses to nonnoxious environmental stimuli. J Neurosci 1:887-900, 1981.

Beitz, A.J. The organization of afferent projections to the midbrain

periaqueductal gray of the rat. Neuroscience 7:133-159, 1982.

Bennett, C.; Liang, K.C.; and McGaugh, J.L. Depletion of adrenal catecholamines alters the amnestic effect of amygdala stimulation. Behav

Brain Res 15:83-91, 1985.

Berg, W.K., and Davis, M. Diazepam blocks fear-enhanced startle elicited

electrically from the brainstem. Physiol Behav 32:333-336, 1984.

Berg, W.K., and Davis, M. Associative learning modifies startle reflexes at

the lateral lemniscus. Behav Neurosci 99:191-199, 1985.

Bertrand, S., and Hugelin, A. Respiratory synchronizing function of the

nucleus parabrachialis medialis: Pneumotaxic mechanisms. J Neurophysiol 34:180-207, 1971.

Blanchard, D.C., and Blanchard, R.J. Innate and conditioned reactions to

threat in rats with amygdaloid lesions. J Comp Physiol Psychol 81:281-

290, 1972.

Bresnaham, E., and Routtenberg, A. Memory disruption by unilateral low

level, subseizure stimulation of the medial amygdaloid nucleus. Physiol

Behav 9:513-525, 1972.

Brown, J.S.; Kalish. H.I.; and Farber, I.E. Conditioned fear as revealed by

magnitude of startle response to an auditory stimulus. J Exp Psychol

41:317-328, 1951.

Brown, J.S.; Meryman, J.W.; and Marzocco, E.R. Sound-induced startle

response as a function of the time since shock. J Comp Physiol Psychol

49:190-194, 1956.

154
Carew, T.J. An introduction to cellular approaches used in the analysis of

habituation and sensitization in Aplysia. In: Peeke, H.V.S., and

Petrinovich, L., eds. Habituation, Sensitization, and Behavior. New

York: Academic Press, 1984. pp. 205-249.

Cassella, J.V., and Davis, M. Fear-enhanced acoustic startle is not attenuated by acute or chronic imipramine treatment in rats. Psychopharmacology 87:278-282, 1985.

Cassella, J.V., and Davis, M. Neural structures mediating acoustic and tactile startle reflexes and the acoustically-elicited pinna response in rats:

Electrolytic and ibotenic acid studies. Abst Soc Neurosci 12:1273, 1986.

Castellucci, V.; Pinsker, H.; Kupfermann, I.; and Kandel, E.R. Neuronal

mechanisms of habituation and dishabituation of the gill-withdrawal reflex

in Aplysia. Science 167:1745-1748, 1970.

Chapman, W.P.; Schroeder, H.R.; Guyer, G.; Brazier, M.A.B.; Fager, C.;

Poppen, J.L.; Solomon, H.C.; and Yakolev, P.I. Physiological evidence

concerning the importance of the amygdaloid nuclear region in the integration of circulating function and emotion in man. Science 129:949-950,

1954.

Charney, D.S.; Heninger, G.R.; and Breier, A. Noradrenergic function in

panic anxiety. Arch Gen Psychiatry 41:751-763, 1984.

Chi, C.C. The effect of amobarbital sodium on conditioned fear as measured by the potentiated startle response in rats. Psychopharmacologia

7:115-122, 1965.

Cohen, D.H. Involvement of the avian amygdalar homologue (archistriatum

posterior and mediale) in defensively conditioned heart rate change.

J Comp Neurol 160:13-36, 1975.

Cohen, M.I. Switching of the respiratory phases and evoked phrenic

responses produced by rostra1 pontine electrical stimulation. J Physiol

(Lond) 217:133-158, 1971.

Crow, T.J., and Alkon, D.L. Associative behavior modification in

Hermissenda: Cellular correlates. Science 209:412-414, 1980.

Davis, M. Diazepam and flurazepam: Effects on conditioned fear as

measured with the potentiated startle paradigm. Psychopharmacology

62:1-7, 1979a.

Davis, M. Morphine and naloxone: Effects on conditioned fear as measured with the potentiated startle paradigm. Eur J Pharmacol 54:341-347,

1979b.

Davis, M. Pharmacological and anatomical analysis of fear conditioning

using the fear-potentiated startle paradigm. Behav Neurosci 100:814-824,

1986.

Davis, M., and Astrachan, D.I. Conditioned fear and startle magnitude:

Effects of different footshock or backshock intensities used in training.

J Exp Psychol [Anim Behav] 4:95-103, 1978.

Davis, M.; Cassella, J.V.; and Kehne, J.H. Serotonin does not mediate

anxiolytic effects of buspirone in the fear-potentiated startle paradigm:

Comparison with 8-0H-DPAT and ipsapirone. Psychopharmacology

94:14-20, 1988.

155
Davis, M.; Gendelman, D.S.; Tischler, M.D.; and Gendelman, P.M. A primary acoustic startle circuit: Lesions and stimulation studies, J Neurosci

6:791-805, 1982.

Davis, M.; Redmond, D.E., Jr.; and Baraban, J.M. Noradrenergic agonists

and antagonists: Effects on conditioned fear as measured by the potentiated startle paradigm. Psychopharmacology 65:111-118, 1979.

Davis, M., Schlesinger, L.S.; and Sorenson, C.A. Temporal specificity of

fear conditioning: Effects of different CS-US intervals on the fearpotentiated startle effect. J Exp Psychol [Anim Behav] 15:295-310, 1989.

Deutch, A.Y.; Goldstein, M.; and Roth, R.H. Activation of the locus coeruleus induced by selective stimulation of the ventral tegmental area. Brain

Res 363:307-314, 1986.

Ellis, M.E., and Kesner, R.P. The noradrenergic system of the amygdala

and aversive information processing. Behav Neurosci 97:399-415, 1983.

Estes, W.K., and Skinner, B.F. Some quantitative properties of anxiety.

J Exp Psychol 29:390-400, 1941.

Fanselow, M.S. Naloxone and Pavlovian fear conditioning. Learn Motiv

12:398-419, 1981.

Fanselow, M.S. The postshock activity burst. Anim Learn Behav 10:448-

454, 1982.

Fanselow, M.S. Shock-induced analgesia on the formalin test: Effect of

shock severity, naloxone, hypophysectomy, and associative variables.

Behav Neurosci 98:79-95, 1984.

Fanselow, M.S., and Bolles, R.C. Naloxone and shock-elicited freezing in

the rat. J Comp Physiol Psychol 93:736-744, 1979.

File, S.E. The use of social interaction as a method for detecting anxiolytic

activity of chlordiazepoxide-like drugs. J Neurosci Methods 2:219-238,

1980.

Gallagher, M., and Kapp, B.S. Manipulation of opiate activity in the amygdala alters memory processes. Life Sci 23:1973-1978, 1978.

Gallagher, M., and Kapp, B.S. Effect of phentolamine administration into

the amygdala complex of rats on time-dependent memory processes.

Behav Neural Biol 31:90-95, 1981.

Geller, I., and Seifter, J. The effects of memprobamate, barbiturates, damphetamine and promazine on experimentally induced conflict in the rat.

Psychopharmacologia 1:482-492, 1960.

Gentile. C.G.; Jarrel, T.W.; Teich, A.; McCabe, P.M.; and Schneiderman, N.

The role of amygdaloid central nucleus in the retention of differential

pavlovian conditioning of bradycardia in rabbits. Behav Brain Res

20:263-273, 1986.

Gerfen, C.R., and Sawchenko, P.E. An antegrade neuroanatomical tracing

method that shows the detailed morphology of neurons, their axons and

terminals: Immunohistochemical localization of an axonally transported

plant lectin, phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res

290:219-238, 1984.

156
Gloor, P. Amygdala. In: Field, J., ed. Hardbook of Physiology: Sec. I.

Neurophysiology. Vol. II. Washington, DC: American Physiological

Society, 1960. pp. 1395-1420.

Gloor, P.; Oliver, A.; and Quesney, L.F. The role of the amygdala in the

expression of psychic phenomena in temporal lobe seizures. In: Ben-Ari,

Y., ed. The Amygdaloid Complex. New York: Elsevier/North-Holland,

1981.

Gold, P.E.; Hankins, I,.; Edwards, R.M.; Chester, J.; and McGaugh, J.L.

Memory inference and facilitation with posttrial amygdala stimulation:

Effect varies with footshock level. Brain Res 86:509-513, 1975.

Goldenberg, M.; Snyder, C.H.; and Aranow, H., Jr. New test for hypertension due to circulating epinephrine. JAMA 135:971-976, 1947.

Guyenct, P.G., and Byrum, C.E. Comparative effects of sciatic nerve

stimulation, blood pressure, and morphine on the activity of A5 and A6

pontine noradrenergic neurons. Brain Res 327:191-201, 1985.

Handforth, A. Implications of stimulus factors governing kindled seizure

threshold. Exp Neurol 86:33-39, 1984.

Handwerker, M.J.; Gold, P.E.; and McGaugh, J.L. Impairment of active

avoidance learning with posttraining amygdala stimulation. Brain Res

75:324-327, 1974.

Harper, R.M.; Frysinger, R.C.; Trelease, R.B.; and Marks, J.D. Statedependent alteration of respiratory cycle timing by stimulation of the

central nucleus of the amygdala. Brain Res 306:1-8, 1984.

Hawkins, R.D.; Abrams, T.W.; Carew, T.J.; and Kandel, E.R. A cellular

mechanism of classical conditioning in Aplysia: Activity-dependent

amplification of prcsynaptic facilitation. Science 219:400-405, 1983.

Henke P.G. The centromedial amygdala and gastric pathology in rats.

Physiol Behav 25:107-112, 1980a.

Henke, P.G. The amygdala and restraint ulcers in rats. J. Comp Physiol

Psychol 94:313-323, 1980b.

Henke, P.G. Unit-activity in the central amygdalar nucleus of rats in

response to immobilization-stress. Brain Res 10:833-837, 1983.

Henkel, C.K. Afferent sources of a lateral midbrain tegmental zone associated with the pinnac in the cat as mapped by retrograde transport of

horseradish peroxidase. J Comp Neurol 203:213-226, 1981.

Henkel, C.K., and Edwards, S.B. The superior colliculus control of pinna

movements in the cat: Possible anatomical connections. J. Comp Neurol

182:763-776, 1978.

Hitchcock, J.M., and Davis, M. Lesions of the amygdala, but not of the

cerebellum or red nucleus, block conditioned fear as measured with the

potentiated startle paradigm. Behav Neurosci 100:11-22, 1986.

Hitchcock, J.M., and Davis, M. Fear-potentiated startle using an auditory

conditioned stimulus: Effect of lesions of the amygdala. Physiol Behav

39:403-408, 1987.

Hitchcock, J.M., and Davis, M. The efferent pathway of the amygdala

involved in conditioned fear as measured with the fear-potentiated startle

pathway. Behav Neurosci, in press.

157
Hitchcock, J.M.; Sananes, C.B.; and Davis, M. Sensitization of the startle

reflex by footshock: Blockade by lesions of the central nucleus of the

amygdala or its efferent pathway to the brainstem. Behav Neurosci

103:509-518, 1989.

Holmberg, G., and Gershon, S. Autonomic and psychic effects of yohimbine hydrochloride. Psychopharmacologia (Berl) 2:93-104, 1961.

Hopkins, D.A., and Holstege, G. Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp Brain Res 32:529-

547, 1978.

Inagaki, S.; Kawai, Y.; Matsuzak, T.; Shiosaka, S.; and Tohyama, M. Precise terminal fields of the descending somatostatinergic neuron system

from the amygdala complex of the rat. J für Hirnfurschung 24:345-356,

1983.

Innes, D.L., and Tansy, M.F. Gastric mucosal ulceration associated with

electrochemical stimulation of the limbic system. Brain Res Bull 5:33-36,

1980.

Ison, J.R., and Hammond, G. Modification of the startle reflex in the rat

by changes in the auditory and visual environment. J Comp Physiol

Psychol 75:435-452, 1971.

Ison, J.R.; McAdam, D.W.; and Hammond, G.R. Latency and amplitude

changes in the acoustic startle reflex of the rat produced by variation in

auditory prestimulation. Physiol Behav 10:1035-1039, 1973.

Iwata, J.; LcDoux, J.E.; Meeley, M.P.; Americ, S.; and Reis, D.J. Intrinsic

neurons in the amygdala field projected to by the medial geniculate body

mediate emotional responses conditioned to acoustic stimuli. Brain Res

383:195-214, 1986.

Jarrell, T.W.; McCabe, P.M.; Teich, A.; Gentile, C.G.; VanDercar, D.H.;

and Schneidrman, N. Lateral subthalamic area as mediator of classically

conditioned bradycardia in rabbits. Behav Neurosci 100:3-10, 1986.

Kaada, B.R. Stimulation and regional ablation of the amygdaloid complex

with reference to functional representations. In: Elcftheriou. B.E., ed.

The Neurobiology of the Amygdala. New York: Plenum Press, 1972.

pp. 205-281.

Kapp, B.S.; Frysinger, R.C.; Gallagher, M.; and Haselton, J.R. Amygdala

central nucleus lesions: Effects on heart rate conditioning in the rabbit.

Physiol Behav 23:1109-1117, 1979.

Kapp, B.S.; Gallagher, M.; Underwood, M.D.; McNall, C.L.; and

Whitehorn, D. Cardiovascular responses elicited by electrical stimulation

of the amygdala central nucleus in the rabbit. Brain Res 234:251-262,

1982.

Kapp, B.S., and Pascoe, J.P. Correlation aspects of memory: Vertebrate

model systems. In: Kesner, R.P., and Martinez, J.L., eds. Learning and

Memory: A Biological View. New York: Academic Press, 1984.

Kapp, B.S.; Pascoe, J.P.; and Bixler, M.A. The amygdala: A neuroanatomical systems approach to its contribution to aversive conditioning. In:

Butlers, N., and Squire, L.S., eds. The Neuropsychology of Memory.

New York: The Guilford Press, 1984.

158
Kehne, J.H.; Cassella, J.V.; and Davis, M. Anxiolytic effects of buspirone

and gepirone in the fear-potentiated startle paradigm. Psychopharmacology 94:8-13, 1988.

Kehne, J.H.; Gallager, D.W.; and Davis, M. Strychnine: Brainstem and

spinal mediation of excitatory effects on acoustic startle. Eur J

Pharmacol 76:177-186, 1981.

Kellicut, M.H., and Schwartzbaum, J.S. Formation of a conditioned emotional response (CER) following lesions of the amygdaloid complex in

rats. Psychol Rev 12:351-358, 1963.

Kesner, R.P. Brain stimulation: Effects on memory. Behav Neural Biol

36:315-367, 1982.

Krettek, J.E., and Price, J.L. Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat.

J Comp Neurol 178:225-254, 1978.

Leaton, R.N., and Borszcz, G.S. Potentiated startle: Its relation to freezing

and shock intensity in rats. J Exp Psychol [Anim Behav] 11:421-428,

1985.

LeDoux, J.E.; Ruggiero, D.A.; and Reis, D.J. Projections to the subcortical

forebrain from anatomically defined regions of the medial geniculate body

in the rat. J Comp Neurol 242:182-213, 1985.

Liang, K.C.; Bennett, C.; and McGaugh, J.L. Peripheral epinephrine modulates the effects of post-training amygdala stimulation on memory. Behav

Brain Res 15:93-100, 198.5.

Liang, K.C.; Juler, R.G.; and McGaugh, J.L. Modulating effects of posttrainng epinephrine on memory: Involvement of the amygdala noradrenergic systems. Brain Res 368:125-133, 1986.

Liebman, J.M.; Mayer, D.J.; and Liebeskind, J.C. Mesencephalic central

gray lesions and fear-motivated behavior in rats. Brain Res 23:353-370,

1970.

McAllister, W.R., and McAllister, D.E. Behavioral measurement of conditioned fear. In: Brush, F.R., ed. Aversive Conditioning Learning. New

York: Academic Press, 1971. pp. 105-179.

Miller, N.E., and Barry, H., III. Motivational effects of drugs: Methods

which illustrate some general problems in psychopharmacology. Psychopharmacologia 1:169-199, 1960.

Mishkin, M., and Aggleton, J. Multiple functional contributions of the

amygdala in the monkey. In: Ben-Ari, Y., ed. The Amygdaloid

Complex. New York: Elsevier/North-Holland, 1981.

Mizuno, N.; Takahashi, O.; Satoda, T.; and Matsushima, R. Amygdalospinal

projections in the macaque monkey. Neurosci Left 53:327-330, 1985.

Moore, R.Y., and Card, J.P. Noradrenaline-containing neuron systems. In:

Bjorklund, A., and Hokfelt, T., eds. Handbook of Chemical Neuroanatomy. Amsterdam: Elsevier Science Publishers, 1984.

Morgenson, G.J., and Calaresu, F.R. Cardiovascular responses to electrical

stimulation of the amygdala in the rat. Exp Neurol 39:166-180, 1973.

Ohta, M. Amygdaloid and cortical facilitation or inhibition of trigeminal

motoneurons in the rat. Brain Res 291:39-48, 1984.

159
Ottersen. O.P., and Ben-Ari, Y. Afferent connections to the amygdaloid

complex of the rat and cat. I. Projections from the thalamus. J Comp

Neurol 187:401-424, 1979.

Pascoe, J.P., and Kapp, B.S. Electrophysiological characteristics of amygdaloid central nucleus neurons during Pavlovian fear conditioning in the

rabbit. Behav Brain Res 16:117-133, 1985.

Phillipson, O.T. Afferent projections to the ventral tegmental area of Tsai

and intrafascicular nucleus: A horseradish peroxidase study in the tat.

J Comp Neurol 187:117-143, 1979.

Pollock, B., Bock, P.R., Fuchs, A.M.; and Lohaus, R. Visually evoked potentials in cortical and subcortical brain structures of conscious rabbits

with chronically implanted electrodes. Arzneim-Forsch Drug Res 26:327-

334, 1976.

Post, S., and Mai, J.K. Contribution to the amygdaloid projection field in

the rat: A quantitative autoradiographic study. J Hirnforsch 21:199-225,

1980.

Price, J.L., and Amaral, D.G. An autoradiogrdphic study of the projections

of the central nucleus of the monkey amygdala. J Neurosci 1:1242-1259,

1981.

Redmond, D.E., Jr. Alteration in the function of the nucleus locus coeruleus: A possible model for studies on anxiety. In: Hanin. I., ed.

Animal Models in Psychiatry and Neurology. Oxford: Pergamon Press,

1977. pp. 293-304.

Rosen, J.B., and Davis, M. Enhancement of acoustic startle by electrical

stimulation of the amygdala. Behav Neurosci 102:195-202, 1988.

Sandrew, B.B.; Edwards. D.L.; Poletti, C.E.; and Foote, W.E. Amygdalospinal projections in the cat. Brain Res 373:235-239, 1986.

Sanghera, M.K.; Rolls, E.T.; and Roper-Hall, A. Visual responses of

neurons in the dorsolateral amygdala of the alert monkey. Exp Neurol

63:610-626, 1975).

Sarter, M., and Markowitsch, H.J. Involvement of the amygdala in learning

and memory: A critical review, with emphasis on anatomical relations.

Behav Neurosci 99:342-380, 1985.

Schwaber, J.S.; Kapp, B.S.; Higgins, G.A.; and Rapp, P.R. Amygdaloid

and basal forebrain direct connections with the nucleus of the solitary

tract and the dorsal motor nucleus. J Neurosci 2:1424-1438, 1982.

Schwarcz., M.; Hokfelt, T.; Fuxe, K.; Jonsson, G.; Goldstein, M.; and

Terenius, L. Ibotcnic acid-induced neuronal degeneration: A morphological and neurochemical study. Exp Brain Res 37:109-216, 1979.

Sen, R.N., and Anand, B.K. Effect of electrical stimulation of the limbic

system of brain (‘visceral brain’) on gastric secretory activity and

ulceration. Indian J Med Res 45:515-521, 1957.

Shibata, K.; Kataoka, Y.; Yamashita, K.; and Ueki, S. An important role of

the central amygdaloid nucleus and mammillary body in the mediation of

conflict behavior in rats. Bruin Res 372:159-162, 1986.
Shopsin, B.; Friedman, E.; and Gershon, S. Parachlorophenylalanine

reversal of tranylcypromine effects in depressed patients. Arch Gen

Psychiatry 33:811-819, 1976.

Siegel, A. Stimulus generalization of a classically conditioned response

along a temporal dimension. J Comp Physiol Psychol 64:461-466, 1967.

Soffer, A. Regitine and benodaine in the diagnosis of pheochromocytoma.

Med Clin North Am 38:375-384, 1954.

Sorenson, C.A., and Wilkinson, L.O. Behavioral evidence that nicotine

administration has anxiolytic actions in rats. Abst Soc Neurosci 9:137,

1983.

Soubrie, P. Reconciling the role of central serotonin neurons in human and

animal behavior. Behav Brain Sci 92:319-364, 1986.

Spevack, A.A.; Campbell, C.T.; and Drake, L. Effect of amygdalectomy on

habituation and CER in rats. Physiol Behav 15:199-207, 1975.

Takeuchi, Y.; Matsushima, S.; Matsushima, S.; and Hopkins, D.A. Direct

amygdaloid projections to the dorsal motor nucleus of the vagus nerve:

A tight and electron microscopic study in the rat. Brain Res 280:143-

147, 1983.

Takeuchi, Y.; McLean, J.H.; and Hopkins, D.A. Reciprocal connections

between the amygdala and parabrachial nuclei: Ultrastructural demonstration by degeneration and axonal transport of HRP in the cat. Brain

Res 239:583-588, 1982.

Taylor, D.P.; Riblet, L.A.; Stanton, H.C.; Eison, A.S.; Eison, M.S.; and

Temple, D.L., Jr. Dopamine and antianxiety activity. Pharmacol

Biochem Behav 17:25-35, 1982.

Thierry, A.M.; Tassin, J.P.; Blanc, G.; and Glowinski, J. Selective activation of the mesocortical DA system by stress. Nature 263:242-244,

1976.

Tischler, M.D., and Davis, M. A visual pathway that mediates fearconditioned enhancement of acoustic startle. Brain Res 276:55-71, 1983.

Turner, B.H., and Zimmer, J. The architecture and some of the interconnections of the rat’s amygdala and lateral periallocortex. J Comp

Neurol 227:540-557, 1984.

Ursin, H.; Jellestad, F.; and Cabrera, I.G. The amygdala, exploration and

fear. In: Ben-Ari, Y., ed. The Amygdaloid Complex. Amsterdam:

Elsevier/North-Holland, 1981.

Veening, J.G.; Swanson, L.W.; and Sawchenko, P.E. The organization of

projections from the central nucleus of the amygdala to brain stem sites

involved in central autonomic regulation: A combined retrograde

transport-immunohistochemical study. Brain Res 303:337-357, 1984.

Walters, E.T., and Byrne, J.H. Long-term enhancement produced by

activity-dependent modulation of Aplysia sensory neurons. J Neurosci

5:662-672, 1985.

Zaczek, R., and Coyle, J.T. Excitatory amino acid analogues: Neurotoxicity and seizures. Neuropharmacology 21:15-20, 1982.

161
ACKNOWLEDGMENTS

This research was supported by the National Institute of Neurological and

Communicative Disorders and Stroke grant NS-18033, Research Scientist

Development Award MH-00004, National Institute of Mental Health grants

MH-41928 and MH-25642, a grant from the Air Force Office of Scientific

Research and the State of Connecticut. The following are acknowledged for

their assistance in much of this research: W. Keith Berg, for his work on

electrically elicited startle; Janice M. Hitchcock, for her work on lesions of

the amygdala; Jeffrey B. Rosen, for his work on electrical stimulation of the

amygdala; and Lee Schlesinger, for her work on drug and fear-potentiated

startle.

AUTHOR

Michael Davis, Ph.D.

Professor of Psychiatry and Psychology

Department of Psychiatry

Ribicoff Research Facilities of the

Connecticut Mental Health Center

Yale University School of Medicine

34 Park Street

New Haven, CT 06508

162
Stress, Performance, and Arousal:

Focus on CRF

George F. Koob, Belinda J. Cole, Neal R. Swerdlow,

Michel Le Moal, and Karen T. Britton

INTRODUCTION

Corticotropin releasing factor (CRF) is a 41-amino-acid peptide (Vale et al.

1981) that has potent activating effects on the pituitary adrenal axis, as

shown by its ability to release adrenocorticotropin hormone (ACTH) and

ß-endorphin from the anterior pituitary (Rivier et al. 1984). CRF is now

thought to be the primary hypothalamic releasing factor with the specific

function of activating the release of ACTH from the anterior pituitary.

The significance of the chemical identification of CRF can perhaps best be

elaborated in terms of classical “stress” theory. Two generally accepted

definitions of stress are that a “stress is the nonspecific (common) result of

any demand upon the body (usually, but not always noxious)” (Selye 1980,

p. vii) or “anything which causes an alteration of psychological homeostatic

processes” (Burchfield 1979). Using this definition, one of the most reliable

and sensitive indications of a state of stress is an increase in the production

of ACTH (Selye 1936).

Internal or external demands are conveyed in the form of stimuli to the anterior pituitary via neurohumoral means (presumably CRF), and the pituitary

responds with a secretion of ACTH. ACTH, in turn, stimulates the adrenal

cortex to secrete glucocorticoids that have widespread effects on metabolism, such as gluconeogenesis, hyperinsulinemia, lysis of lymphoid tissue, increased gastric secretion, and reduced inflammatory and antibody responses.

These physiological changes, in response to increased hypothalamic-pituitary

action, are paralleled by alterations in behavior that have been associated

with increases in alertness and attention (De Wied 1977).

However, an alternative means by which behavioral or physiological responses to arousing stimuli and/or stressors might involve CRF is via a

direct neurotropic action of CRF in the brain itself. Thus, just as pathways

project to the hypothalamus from limbic areas to activate the pituitary

adrenal axis via CRF release in the hypothalamus, so may CRF release in

163
the central nervous system (CNS) mediate behavioral arousal and behavioral

responses to stress.

AROUSAL-STRESS CONSTRUCT CONTINUUM

The concept of stress and arousal as intervening variables or hypothetical

constructs that have important similarities has been elegantly elaborated in

terms of psychoendocrine measures (Hennessy and Levine 1979). These authors argued that “the pituitary adrenal system is part of the arousal system

of the animal and [activity in this system] may prove to be an extremely

reliable and sensitive measure of arousal as well as stress” (Hennessy and

Levine, p. 137). These authors further argued that, while the concept of

arousal may involve a wide range of independent and dependent variables,

stress primarily refers to the excess consequences of arousal that produce

tissue damage and toxic effects. The concept of stress, then, may be considered a parallel continuum subsumed by a basic arousal theory.

The thesis of this chapter is that the pituitary adrenal axis has a homologous representation in the brain in a basic brain arousal system that utilizes

the neurotransmitter CRF. Thus, just as CRF controls the release of ACTH

from the pituitary, and activity in the pituitary adrenal axis has proven to be

a reliable and sensitive measure of arousal as well as of stress, it is hypothesized that CRF in the CNS is part of a basic arousal system that, under

conditions of extreme activity or chronic activation, can produce the behavioral manifestations of stress as well as, ultimately, pathophysiology.

CRF AND BEHAVIORAL AROUSAL

CRF administered directly into the CNS produces behavioral and physiological activation. Intracerebroventricular (ICV) injections of CRF produce

elevations of plasma epinephrine. norepinephrine, and glucose (Brown et al.

1982a; Brown et al. 1982b). These effects are reproducible in hypophysectomized animals but are abolished by ganglionic blocking agents (Brown

and Fisher 1983). suggesting an involvement of the sympathetic autonomic

nervous system. CRF injected ICV also produces a profound dosedependent activation of the electroencephalogram (EEG) (Ehlers et al.

1983). Doses of 0.015 to 0.15 nmol produced a long-lasting activation of

EEG. At the cellular level, CRF produces increases in the firing rate of

norcpinephrine containing neurons within the locus coeruleus (Valentino

et al. 1983), a system thought to be of importance in the mechanisms by

which the brain is able to attend selectively to certain novel external events

(Foote et al. 1983).

The autonomic and electrophysiological activation produced by central administration of CRF is paralleled by a dose-dependent increase in locomotor

activity (Sutton et al. 1982; Koob et al. 1984; Sherman and Kalin 1987).

This effect also appears to be independent of the pituitary-adrenal system,

164
since locomotor activation is still observed in hypophysectomized and dexamethasone-treated rats (figure 1) (Eaves et al. 1985; Britton et al. 1986).

Given that this activation is not seen with systemic administration of CRF,

these observations suggested that this peptide exerts some of its effects by a

direct action on the CNS.

FIGURE 1. Effects of CRF injected intracerebroventricularly on locomotor

activity in photocell cages in hypophysectomized rats

treated chronically with rat growth hormone and in shamoperated animals

*Significantly different from saline injection. p<.05, main effects ANOVA.

NOTE: Results represent the total activity counts over 3 hours (mean ± SEM), n=9 in each group.

SOURCE: Eaves et al. 1985, copyright 1985, Pergamon Press.

Although other peptides such as the endorphins and ACTH have been

shown to produce increases in spontaneous behavioral activity, the locomotor activation caused by CRF is not antagonized by the opiate antagonist

naloxone or by low doses of the dopaminc-receptor antagonist alpha

165
flupenthixol (Koob et al. 1984). Nor is this activation reversed by

6-hydroxydopamine lesions of the region of the nucleus accumbens, even

though such lesions reverse the locomotor-stimulating effects of indirect

sympathomimetics (Swerdlow and Koob 1985). ACTH injected centrally

failed to increase locomotor activity but instead produced an increase in

grooming behavior, as has been observed by others (Gispen et al. 1975).

To delineate further the site of action for this behaviorally activating effect

of CRF, experiments were conducted that examined lateral ventricular and

cistema magna injections of CRF combined with obstruction of the cerebral

aqueduct. Rats injected intracerebroventricularly with CRF at the level of

the lateral ventricle or cistema magna showed a dose-dependent increase in

locomotor activity. The increase in locomotor activity from injections of

CRF into the cistema magna was blocked by a cold cream plug in the cerebral aqueduct (Tazi et al. 1987a). An identical plug failed to block the

increase in locomotor activity produced by CRF injected into the lateral

ventricle. These results suggest that the activating effects of CRF depend

on forebrain CRF receptors and may not depend on hindbrain sites.

In a subsequent study, rats were surgically implanted with cannulae aimed

at either the lateral ventricle, frontal cortex (FC), nucleus accumbens,

amygdala centralis, substantia innominata/lateral preoptic area (SI/PO), or

pedunculopontine nucleus (PPN). Injection of 0.5 μg rat CRF into discrete

brain regions produced increases in locomotor activity that were inversely

related to the distance of the injection site from the SI/PO: activation was

least intense when produced by CRF injection into the FC and PPN and

most intense when produced by CRF injection into the SI/PO. Only

SI/PO injection sites yielded activation levels that exceeded those produced

by ICV CRF injection (Tazi et al. 1987a). These results suggest some

degree of neuroanatomical specificity for the neural substrales of CRFinduced behavioral activation.

CRF AND BEHAVIORAL RESPONSES TO STRESS

Early work with CRF showed that while CRF produced a behavioral activation in some situations, in other more stressful situations, the same doses of

CRF produced behavioral inhibition. For example, when mildly fooddeprived rats are exposed to an open-field situation where food is available,

they will eventually approach the food and eat. In contrast to untreated

animals that made increasingly frequent forays into the center of the field to

consume food (Britton and Britton 1981), rats previously injected intracerebroventricularly with CRF showed a marked decrease in exploratory and ingestive behavior and remained close to the comers of the field (Britton et

al. 1982). CRF also decreases food intake and muscimol-, norepinephrine-,

dynotphin-, and insulin-induced feeding, effects attributed to a stress-related

suppression of food intake (Levine et al. 1983; Morley and Levine 1983).

166
Similar anxiogeniclike results were observed with nondeprived rats in an

open field without food (Sutton et al. 1982). Rats tested in a novel open

field, following ICV injection of doses of CRF (0.0015 to 0.15 nmole),

showed responses that were consistent with increased “emotionality” or sensitivity to the stressful aspects of the situation. Here, rats showed decreases

in locomotion and rearing (Sutton et al. 1982). In this open-field test, a

typical saline-injected rat rapidly circled the outer squares of the open field

during the first 3 to 4 minutes of the 5-minute test. During the last 1 to 2

minutes of the test, these saline-injected animals then made some forays

into the center of the open field, usually also rearing on their hind legs.

Typically, a rat injected with 0.15 nmole of CRF and placed 60 minutes

later in the open field moved hesitantly to the outer squares and then either

circled the open field, remaining close to the floor, or remained in one of

the comers, grooming or hesitantly moving forwards and backwards (Sutton

et al. 1982). These behavioral changes are consistent with heightened

“emotionality” or increased sensitivity to the presumably stressful aspects of

the test.

CRF has also been tested in other behavioral paradigms that are sensitive to

antianxiety (anxiolytic) compounds such as the benzodiazepines. In an

operant conflict test, CRF produced a significant decrease in punished and

unpunished responding, an effect opposite to that observed with benzodiazepines; this “anxiogenic” effect was reversed by concomitant treatment with a

benzodiazepine (figure 2) (Thatcher-Britton et al. 1985). However, this

increased sensitivity to aversive events was not paralleled by an increased

sensitivity to pain. These effects appear to be independent of the pituitary

adrenal axis, in that dexamethasone treatment also failed to alter the suppression in operant behavior produced by CRF (Britton et al. 1986a).

In a recent study, CRF injected intracerebroventricularly in rats produced a

further suppression during the conditioned stimulus (CS) presentation in a

conditioned suppression task. The observed decrease in the suppression rate

is of significance, because it shows that the decrease in performance was

more dramatic during the CS period that signaled impending shock (Cole et

al., unpublished manuscript).

Another paradigm differentially sensitive to anxiogenic and anxiolytic compounds is the acoustic startle test. The acoustic startle reflex is an easily

quantified muscular contraction in response to an intense acoustic stimulus.

CRF (1 μg ICV) significantly potentiated acoustic startle amplitude

(Swerdlow et al. 1986). Pretreatment with the benzodiazepine chlordiazepoxide (CDP) in doses that did not by themselves lower startle baseline

attenuated this effect (figure 3). These results also supported the hypothesis

that CRF might act to potentiate behavioral responses normally expressed

during states of enhanced fear or anxiety.

167
FIGURE 2. The interaction of 0.5 μg corticotropin-releasing hormone

injected intracerebroventricularly and of 5.0 mg/kg CDP

injected intraperitoneally on responding during the random

interval and conflict components of an operant conflict test

*Significant difference from saline, main effect CDP.

‡Significant difference from saline, main effect CRF. p<.05, two-way ANOVA.

NOTE: Results are expressed as percent of baseline from the previous day.

SOURCE: Thatcher-Britton et al. 1985, copyright 1985, Springer Verlag.

168
FIGURE 3. Acoustic startle amplitude in rats following ICV infusion of

1 μg CRF and IP injection of chlordiazepoxide (0, 2.5, 5.0,

or 10.0 mg/kg

*p<.05, Newman-Keuls individual means comparison.

NOTE: Insert histogram indicates mean startle amplitude over 50 trials.

SOURCE: Swerdlow et al., 1986, copyright 1986, Springer-Verlag.

Other more recent work has extended and elaborated this stresslike effect of

CRF. CRF injected intracerebroventricularly into mice allowed to explore a

novel environment (multicompartment chamber) produced a decreased mean

time per contact with novel stimuli (Bet-ridge and Dunn 1986). Interestingly, this effect was reversed by naloxone, but it should be noted that previous studies have shown that naloxone can produce the opposite result on its

own, i.e., increased exploratory behavior (Arnsten and Segal 1979).

Although the effects of exogenous CRF in potentiating behavioral responses

to stress are interesting, the hypothesis that behavioral responses to stress

depend on activation of endogenous CRF in the brain would be bolstered by

169
evidence of an antistress effect of an antagonist to CRF. A peptide antagonist to CRF, helical CRF 9-41 (Rivier et al. 19843, has recently been

shown to have behavioral actions in some tests. This antagonist is 10 times

less potent than CRF in binding to CNS CRF receptors (De Souza 1987).

Alpha helical CRF has been shown to partially reverse stress-induced decreases in feeding (Krahn et al. 1986), and it attenuated stress-induced

fighting (Tazi et al. 1987b). This antagonist also appears to have activity in

various open-field tests. Alpha helical CRF injected into mice reversed

stress-induced changes in a novel environment (multicompartment chamber)

(Berridge and Dunn 1987). Alpha helical CRF also decreased the latency

for rats to emerge into a novel open field from a dark familiar enclosure

(Takahashi et al. 1988).

Howevcr, these results with the CRF antagonist in aversive situations are

reasonably subtle particularly in light of a significant amount of negative

results in some classic animal models sensitive to antianxiety drugs. Alpha

helical CRF ICV by itself does not produce a reliable release of punished

responding (Britton et al. 1986b). Nor does a helical CRF ICV block the

suppression of responding observed in a conditioned emotional response

(CER) (Cole et al., unpublished manuscript).

CRF AND LEARNING

There is a significant amount of empirical and theoretical evidence that

suggests that performance of learned behavior can vary with the level of

activation or “arousal” of the animal. Correlations have been obtained

between independent measures of arousal such as exploratory behavior,

EEG, galvanic skin response, and the speed of acquisition of a response

(Berlyne 1960). Indeed, it has been hypothesized that both animals and

humans strive to maintain intermediate amounts of arousal potential, and it

follows that optimal learning will correspond to these optimal levels of

arousal. Other treatments of this theoretical issue have centered on arousal

as the intervening variable in the incentive-motivation required for all

learning (Killeen et al. 1978).

Recent results in our laboratory with both CRF and the CRF antagonist

have revealed more robust effects in learning situations where arousal and/or

an aversive state are required for animals to learn an association at an

optimal rate. First, rats pretreated with CRF and trained on a two-way

active avoidance task showed an improvement in performance at low doses

of CRF (10 and 100 ng ICV) but less of an improvement at a higher dose

of 1,000 ng (figure 4). At this higher dose, the rats appeared incapable of

responding in a coherent way and, in fact, reacted to the CS as if it were

the shock itself by jumping back and forth across the two compartments.

In the CER paradigm, a baseline of food-reinforced operant responding is

established and then a CS is presented prior to the delivery of a brief,

170
FIGURE 4. Effects CRF injected intracerebroventricularly on acquisition

of two-way active avoidance in rats

*Significantly different from saline, p<.05 ANOVA.

NOTE: CRF was injected 1 hour prior to testing. Rats received 20 trials per day. Each trial

consisted of 5 seconds of a compound CS (tone and light) followed by 0.3 mA of AC shock

if the rat did not jump to the other side within 5 seconds. The intertrial interval was 20

seconds. Saline group is the same in each panel, n=8 per group.

171
inescapable foot shock (unconditioned stimulus (UCS)) while the animal is

responding on the baseline schedule. This results in a reduction of foodreinforced responding during the presence of the CS. During acquisition,

this reduction in performance is acquired rapidly. However, rats treated

intracerebroventricularly with the CRF antagonist a helical CRF, at doses of

1 to 25 μg, had significantly impaired acquisition of the CER. High doses

of the antagonist injected intravenously sufficient to attenuate plasma corticosterone levels did not impair acquisition, although a low-intravenous dose

did impair acquisition. Also, dexamethasone treatment, in doses sufficient

to block the pituitary adrenal axis, failed to alter the acquisition of the

CER. These results, taken together, suggest that the ability of a helical

CRF to attenuate the development of conditioned fear is independent of its

action on the hypothalamic pituitary adrenal axis. The results also suggest

a possible role for peripheral CRF in the development of conditioned fear.

CRF AROUSAL AND PERFORMANCE

To summarize, CNS manipulation of the putative neurotransmitter CRF produces changes in activation, behavioral responses to stress, and learning.

CRF administered directly into the CNS also produces physiological activation and can potentiate behavioral responses to stress. This activation can

result in a facilitation of performance in learning situations, especially those

involving aversive motivation. However, CRF administered prior to the

daily acquisition of an appetitively motivated visual-discrimination task also

significantly improved acquisition (Koob et al. 1987).

The effects of the CRF antagonist a helical CRF have been more subtle.

This peptide antagonist appears to be particularly active in situations where

plasticity of associations is involved. For example, a helical CRF blocks

the development of stress-induced fighting and the development of the CER

but has little effect when the behavioral response is well established, e.g.,

lack of effect of a helical CRF on established conflict or CER performance.

These results suggest that CRF itself may be part of a fundamental activation system that is engaged in situations of high arousal and stress. This

brain system may be required for animals to form associations between

aversive events and previously neutral stimuli. The fact that the CRF antagonist does not have basic sedative properties and does not produce a

benzodiazepinelike profile of disinhibition suggests that this system may be

active only under situations requiring behavioral adaptation. Clearly, CRF

effects on activation, anxiety, and learning could all be interpreted as

independent effects (mediated by separate neural substrates), or they all

could be considered different behavioral manifestations of a common

process-arousal. Whether activation of the CRF system proves crucial to

learning in general or simply to the learning associated with stress reduction

remains to be determined.

172
REFERENCES

Amsten, A.F.T., and Segal, D.S. Naloxone alters locomotion and interaction with environmental stimuli. Life Sci 25:1035-1042, 1979.

Berlyne, D.E. Conflict Arousal and Curiosity. New York: McGraw-Hill

Publishing Co., 1960.

Betridge, C.W., and Dunn, A.J. Corticotropin-releasing factor elicits

naloxone sensitive stress-like alterations in exploratory behavior in mice.

Regul Pept 16:83-93, 1986.

Betridge, C.W., and Dunn, A.J. A corticotropin-releasing factor antagonist

reverses the stress-induced changes of exploratory behavior in mice.

Horm Behav 21:393-401, 1987.

Britton, D.R., and Britton, K.T. A sensitive open field measure of analytic

drug activity. Pharmacol Biohem Behav 15:577-582, 1981.

Britton, D.R.; Koob, G.F.; Rivier, J.; and Vale, W. Intraventricular

corticotropin-releasing factor enhances behavioral effects of novelty. Life

Sci 31:363-367, 1982.

Britton, K.T.; Lee, G.; Dana, R.; Risch, S.C.; and Koob, G.F. Activating

and “anxiogenic” effects of CRF are not inhibited by blockade of the

pituitary-adrenal system with dexamethasone. Life Sci 39:1281-1286,

1986a.

Britton, K.T.; Lee, G.; Vale, W.; Rivier, J.; and Koob, G.F. Corticotropin

releasing factor antagonist blocks activating and “anxiogenic” actions of

CRF in the rat. Brain Res 369:303-306, 1986b.

Brown, M.R., and Fisher, L.A. Central nervous system effects of corticotropin releasing factor in the dog. Brain Res 280:75-79, 1983.

Brown, M.R.; Fisher, L.A.; Rivier, J.; Spiess, J.; Rivier, C.; and Vale, W.

Corticotropin-releasing factor: Effects on the sympathetic nervous system

and oxygen consumption. Life Sci 30:207-210, 1982a.

Brown, M.R.; Fisher, L.A.; Spiess, J.; Rivier, J.; Rivier, C.; and Vale, W.

Comparison of the biologic actions of corticottopin releasing factor and

sauvagine. Regul Pept 4:107-114, 1982b.

Burchfield, S. The stress responses: A new perspective. Psychosom Med

41:661-672, 1979.

Cole, B.J.; Britton, K.T.; Rivier, C.; Rivier, J.; Vale, W.; and Koob, G.F.

Corticotropin releasing factor and conditioned fear. Unpublished

manuscript.

De Souza, E.B. Corticotropin releasing factor receptors in the rat central

nervous system: Characterization and regional distribution. J Neurosci

788-100, 1987.

De Wied, D. Behavioral effects of neutopeptides related to ACTH, MSH

and ß-LPH. In: Krieger, D., and Ganong, W., eds. ACTH and Related

Peptides: Structure, Regulation and Action. New York: Ann NY

Acad Sci:263-274, 1977.

Eaves, M.; Britton, K.T.; Rivier, J.; Vale, W.; and Koob, G.F. Effects of

corticotropin releasing factor on locomotor activity in hypophysectomized

rats. Peptides 6:923-926, 1985.

173
Ehlers, C.L.; Henriksen, S.J.; Wang, M.; Rivier, J.; Vale, W.; and

Bloom, F.E. Corticotropin releasing factor produces increases in brain

excitability arid convulsive seizures in the rat. Brain Res 278:332-336,

1983.

Foote, S.L.; Bloom, F.E.; and Aston-Jones, G. Nucleus locus ceruleus:

New evidence of anatomical and physiological specificity. Physiol Rev

63:844-914, 1983.

Gispen, W.H.; Weigant, V.M.; Greven, H.H.; and De Wied, D. The induction of excessive grooming in the rat by intraventricular application of

peptides derived from ACTH: Structure activity. Life Sci 17:645-655,

1975.

Hennessy, J.W., and Levine, S. Stress, arousal, and the pituitary-adrenal

system: A psychoendocrine hypothesis. In: Sprague, J.M., and Epstein,

A.N., eds. Progress in Psychobiology and Physiological Psychology. 8th

ed. New York: Academic Press, 1979. pp. 133-178.

Killeen, P.R.; Hanson, S.J.; and Osboume, S.R. Arousal: Its genesis and

manifestation as response rate. Psychol Rev 85:571-581, 1978.

Koob, G.F.; Swerdlow, N.; Seelingson, M.; Eaves, M.; Sutton, R.;

Rivier, J.; and Vale, W. CRF-induced locomotor activation is antagonized by alpha flupenthixol but not naloxone. Neuroendocrinology

39:459-464, 1984.

Koob, G.F.; Tazi, A.; Le Moal, M.; and Britton, K.T. Corticotropin

releasing factor, stress and arousal. In: Tache, Y., ed. Neuropeptides

and Stress. New York: Springer-Verlag, 1987.

Krahn, D.D.; Gosnell, B.A.; Grace, M.; and Levine, A.S. CRF antagonist

partially reverses CRF- and stress-induced effects on feeding. Brain

Res Bull 17:285-289, 1986.

Levine, A.S.; Rogers, B.; Kneip, J.; Grace, M.; and Morley, J.E. Effect of

centrally administered corticotropin releasing factor (CRF) on multiple

feeding paradigms. Neuropharmacology 22:337-339, 1983.

Morley, J.E., and Levine, A.S. Corticotropin-releasing factor, grooming and

ingestive behavior. Life Sci 81:1459-1464, 1983.

Rivier, J.; Rivier, C.; and Vale, W. Synthetic competitive antagonists of

corticotropin-releasing factor: Effect on ACTH secretion in the rat.

Science 224:889-891, 1984.

Selye, H. A syndrome produced by diverse noxious agents. Nature

32:138, 1936.

Selye, H., ed. Selye’s Guide to Stress Research. New York: van Nostrand

Reinhold Co., 1980.

Sherman, J.E., and Kahn, N.H. The effects of ICV-CRH on noveltyinduced behavior. Biochem Biophys Res Common 26:699-703, 1987.

Sutton, R.E.; Koob, G.F.; Le Moal, M.; Rivier, J.; and Vale, W. Corticotropin releasing factor (CRF) produces behavioral activation in rats.

Nature 297:331-333, 1982.

Swerdlow, N.R.; Geyer, M.A.; Vale, W.W.; and Koob, G.F. Corticotropin

releasing factor potentiates acoustic startle in rats: Blockade by

chlordiazepoxide. Psychopharmacology 88:142-152. 1986.

174
Swerdlow, N.R., and Koob, G.F. Separate neural substrates of the

locomotor-activating properties of amphetamine, caffeine and corticotropin

releasing factor (CRF) in the rat. Pharmacol Biohem Behav 23:303-307,

1985.

Takahashi, L.K.; Kalin, N.H.; Vandenburgt, J.A.; and Sherman, J.E.

Corticotropin-releasing factor modulates defensive withdrawal and exploratory behavior in rats. Behav Neurosci 103:648-654, 1989.

Taxi, A.; Dantzer, R.; Le Moal, M.; Rivier, J.; Vale, W.; and Koob, G.F.

Corticotropin-releasing factor antagonist blocks stress-induced fighting in

rat. Regul Pept 18:37-62, 1987a.

Taxi, A.; Swerdlow, N.R.; Le Moal, M.; Rivier, J.; Vale, W.; and

Koob, G.F. Behavioral activation of CRF: Evidence for the involvement

of the ventral forebrain. Life Sci 41:41-50, 1987b.

Thatcher-Britton, K.; Morgan, J.; Rivier, J.; Vale, W.; and Koob, G.F.

Chlordiazepoxide attenuates CRF-induced responses suppression in the

conflict test. Psychopharmacology 86:170-174, 1985.

Vale, W.; Spiess, J.; Rivier, C.; and Rivier, J. Characterization of a 41

residue ovine hypothalamic peptide that stimulates the secretion of corticotropin and beta-endorphin. Science 213:1394-1397, 1981.

Valentino, R.J.; Foote, S.L.; and Aston-Jones, G. Corticotropin-releasing

factor activates noradrenergic neurons of the locus coeruleus. Brain Res

270:363-367, 1983.

ACKNOWLEDGMENTS

This investigation was supported in part by U.S. Public Health Service grant

AM 26741 from the National Institutes of Health.

AUTHORS

George F. Koob, Ph.D.

Associate Member

Department of Neuropharmacology

Research Institute of Scripps Clinic

10666 North Torrey Pines Road

La Jolla, CA 92037

Belinda J. Cole, Ph.D.

Postdoctoral Fellow

Research Institute of Scripps Clinic

10666 North Torrey Pines Road

La Jolla, CA 92037

Neal R. Swerdlow, M.D., Ph.D.

Psychiatry Resident

Psychiatric Services

University of California, San Diego

3427 Fourth Avenue

San Diego, CA 92103

175
Michel Le Moal

Professor and Director

INSERM Unite 259

Psychobiologie des Comportements Adaptatifs

Domaine de Carreire Rue Camille Saint Saens

Bordeaux 33077

FRANCE

Karen T. Britton, M.D., Ph.D.

Associate Professor

Department of Psychiatry

University of California Medical Center

La Jolla, CA 92093

176
Attentional and Motivational Effects

of Psychoactive Drugs

Conan Kornetsky, Joseph E.G. Williams, and

Michael Bird

INTRODUCTION

An important question in the study of abuse substances is how and to what

extent these drugs affect learning and memory. Is an enhancement or

impairment of performance due to drug effects on motivation or attention?

This chapter will deal with some of the problems associated with the

multiple effects of abuse substances.

Learning and memory usually do not take place in the absence of reward or

reinforcement, and, for the regulation of a stimulus in the central nervous

system (CNS), the attention of the subject must be engaged. A possible exception is classical or type I conditioning. This type of learning is obligatory, in that reinforcement does not seem to play a direct role. Even in

classical conditioning, however,’ the conditioned stimulus, as well as the unconditioned stimulus, must be of sufficient intensity to register and elicit a

response. If the subject is not attending to the stimulus, it fails as a conditioned stimulus. Thus, in considering the effects of drugs on learning and

memory, factors other than the direct effect of the drug on those neurons in

the brain that are specifically related to learning and memory must be

considered.

AROUSAL AND ATTENTION

Although there seems to be a paucity of specific information on the effects

of arousal level on learning and memory, there arc a number of studies that

have manipulated the arousal level of the subject by looking at the effects

of drugs on various cognitive, motor, and attention tasks. Arousal level

may be changed by manipulating the motivational level. A classic study of

this type, in which motivation was manipulated, thus changing inferred

arousal levels, was carried out by Hill et al. (1957). In this experiment,

they determined the effects of 250 mg of pentobarbital in human volunteers

on reaction time (RT) under three motivational conditions. The subjects

who volunteered to participate in the experiment were prisoner-patients at

the U.S. Public Health Service hospital. These subjects were recidivists,

177
heroin addicts who had multiple admissions at the hospital. At the time of

the experiment, they were not dependent on any drug, although it was most

likely that once released from the hospital they would once again become

dependent. Subjects were often rewarded for participation in the experiments with small amounts of morphine, which would be given a number of

days after the completion of the experiment. In this experiment. one group

of subjects (standard incentive) received the usual fixed amount of morphine

at the end of the experiment. Another group (low incentive) received the

same amount of reward, only it was given a number of weeks prior to participation in the experiment. A third group of subjects (high incentive)

received payment for participation at the end of the experiment, but, in this

case, the amount of payment was contingent upon their latency of response

in a simple RT test. Figure 1 summarizes the results of this experiment.

As shown, changing the motivational conditions altered the speed at which

the subjects responded.

FIGURE 1. Effects of pentobarbital on RT under three levels

of motivation

SOURCE: Adapted from Hill et al. 1957 in Kornetsky 1976. copyright 1976, John Wiley & Son, Inc.

178
Although this effect was not surprising, the effects of pentobarbital under

these three conditions were. Under the highest motivating conditions, the

RT after pentobarbital was not only faster than that under control conditions

for low and medium levels of motivation, but was actually faster than that

seen in the unmedicated group in the high-motivation condition. The operant conditioning “maven” would point out that the three levels of motivation

resulted in changes in the schedule, and that schedule differences have been

clearly shown to product differential drug effects.

A series of experiments demonstrating that changing the arousal level of the

subject can produce markedly different drug effects were carried out; in

these experiments, arousal level was changed by direct manipulation of

the mesencephalic reticular formation. In the first of these experiments

(Kometsky and Eliasson 1969), low-level electrical stimulation delivered by

means of bipolar electrodes impaired the performance of rats on a simple

go-no-go discrimination test that was an animal version of the continuous

performance test (CPT) (Rosvold et al. 1956). However, when a dose of

chlorpromazine that impaired performance on this test was combined with

the low-level electrical stimulation to the reticular formation, the result was

an animal whose performance was not different from that seen after saline

alone. The results of this experiment are shown in figure 2.

The role of the arousal level of the subject is further demonstrated in an

experiment on the effects of d-amphetamine on attention, simple and complcx RT, and learning in sleep-deprived subjects (Kometsky et al. 1959). In

an earlier experiment in nonsleep-deprived subjects, it was found that

d-amphetamine caused no significant improvement in the performance of the

subjects (Kometsky 1958) a finding that generally corroborated previous

observations (Hauty and Payne 1957). In the sleep deprivation experiment,

normal volunteers were tested on the CPT, RT, Digit Symbol Substitution

Test (DSST), and a learning task after 44 and 68 hours of sleep deprivation. The learning task consisted of having the subjects learn the proper

association between 10 lights and 10 buttons. These were arranged on a

console in front of them, with the lights at eye level and the buttons

arranged in a row at desk level. For each learning test, a different association of lights and buttons was presented to the subjects. Each subject was

sleep deprived twice, 1 week apart. In one case, the subjects were administered a placebo 60 minutes prior to both the 44-hour and 68-hour tests. In

the second experiment, the subjects were administered 10 mg of d-amphetamine 60 minutes before the 44-hour test and 15 mg 60 minutes before the

68-hour test. The experiment was a simple double-blind cross-over design.

After 44 hours of sleep deprivation, there was significant impairment in performance on the CPT and RT but not on the learning task. Only the RT

performance was significantly improved by d-amphetamine as compared to

placebo at 44 hours of sleep deprivation. At 68 hours of sleep deprivation,

performance after placebo on the two procedures and on learning was significantly impaired. This impairment on all procedures was significantly

179
FIGURE 2. The percentage of errors of omission for the three animals

after administration of chlorpromazine (1.0 mg/kg) or

during 60 Hz 0.2 ma of stimulation and the effect of this

dose and this amount of stimulation combined

NOTE Although errors are indicated for the combined treatment, none of the animals made errors of

omission when chlorpromazine and stimulation were given together.

SOURCE: Kometsky and Eliasson 1969, copyright 1969, AAAS.

improved by d-amphetamine. Figure 3 shows the percent of control

(nonsleep-deprived) errors in learning for the four conditions in this sleep

deprivation experiment.

The significance of these studies is that the effects of a drug on a variety

of performance tasks is very dependent on the arousal level of the subject.

A question that must be asked by every investigator who attempts to study

the effects of a drug on cognition is whether the observed effect is a direct

result of the action of the drug on those areas of the brain that have

180
specific functions in learning and memory or is due to alterations of motor

or perceptual-attentional systems. The sleep deprivation study described

above would suggest that the drug effects were not due to a direct effect on

cognitive functioning.

FIGURE 3. Effects of d-anlphetanzine on learning (percent errors in

acquisition) after 44 hours and 68 hours of sleep

deprivation

SOURCE: Adapted from Kornetsky et al. 1959.

THE BRAIN REWARD SYSTEM AND PERCEPTION OF BRAIN

STIMULATION

All drugs that act in the CNS at some dose will decrease the registration of

stimulation. Whether a drug can enhance the perception of a stimulus in a

normal, well-motivated subject is not clear. The sleep deprivation study

described in the previous section is an example of the enhancement of the

registration of a stimulus by d-amphetamine in an impaired subject. Some

drugs are believed, especially by the users, to enhance the awareness of the

physical environment and, specifically, to lower the threshold for the perception of colors, sounds, and smells. Lysergic acid diethylamide (LSD)

was believed by many users in the 1950s and 1960s to enhance awareness

181
of all sorts of stimuli. Despite these claims, there is no experimental evidence of such enhancement of perception (Abramson et al. 1955). Experimental studies of the effects of LSD on perception demonstrated that it

either had no effect or impaired perception in a number of modalities.

What subjects may have been reporting is an apparent enhancement of perception resulting from an impairment in perceptual constancies. Thus, instead of perceiving a room that they are in as a large cube, they see it as

trapezoidal in shape, the image that is actually impinging on the retina.

The walls of a room that are painted one color are seen as being of different colors. Subjects become very much aware of differences caused by different amounts of light on the walls. Normally, these differences are not

noticed unless there is specific attention directed toward these differences.

The laboratory work of Kometsky has focused on the study of the effects

of drugs on the reward system, more specifically, the effects of drugs on

the threshold for the perception of rewarding brain stimulation. This system

was first described in an experiment by Olds and Milner in 1954, in which

they demonstrated that animals will work to obtain electrical stimulation of

lateral hypothalamic areas of the brain. Almost all, if not all, drugs that

lower the threshold for such rewarding brain stimulation are abused or have

the potential for abuse; animals will self-administer them and, when such

drugs are given to human subjects, the subjects report feelings of euphoria

(Kometsky 1985). This has been interpreted as evidence that the drug has

hedonic effects. Drugs that raise the threshold are believed to be anhedonic

(Wise 1982). The question that is raised by studies of the effects of drugs

on brain-stimulation reward is whether the effects observed are due to

(1) an effect of the drug on motor systems; (2) in the case of threshold

studies, the ability of the animal to make the perceptual discrimination; or

(3) the result of a combination of these effects with an effect on the motivational system.

When rate of response is the dependent variable in intracranial selfstimulation studies, it is difficult to determine whether facilitation of responding is the result of psychomotor stimulation and whether decreased

responding is due to psychomotor slowing. Many investigators using rate

of response as the dependent variable have found that, after the first exposure to morphine, facilitation of rate of response only occurs 3 hours after

the morphine is administered (Lorens and Mitchell 1973; Hand and Franklin

1386). In our earliest studies using a rate-independent threshold procedure,

morphine caused an increased sensitivity to the stimulation that was observed within 30 minutes after the drug was administered for the first time

(Marcus and Kometsky 1974).

Although the threshold method used gives results that are independent of

rate of response, the method does not completely answer the question of

whether observed changes are due to an effect of the drug on the

attentional-discriminative aspect of cognitive functioning. In order to

182
answer that question, a technique, brain-stimulation detection, was employed

that is similar to the procedure used for determining the threshold for rewarding brain stimulation, but asks the animal a different question. In the

brain-stimulation reward paradigm, the threshold for a rewarding brain stimulation is determined. In the brain stimulation detection paradigm, the

threshold for detecting an intracranial stimulation that has no reinforcing

effects of its own is determined.

In the brain-stimulation reward procedure, Marcus and Kometsky (1974)

used bipolar electrodes stereotaxically implanted into the medial forebrain

bundle at the level of the lateral hypothalamus (MFB) or the ventral tegmental area (VTA) of F-344 rats. Animals were trained and tested in an

experimental chamber in which there was a cylindrical manipulandum in

one wall. A maximum of a quarter turn of the manipulandum operated a

microswitch that resulted in the animal receiving a reinforcing electrical

stimulation via the bipolar electrode.

Determination of the reinforcement threshold made use of a rate-free,

discrete-trial procedure. A schematic of the procedure is indicated in figure 4. A trial began with the delivery of a noncontingent 0.5-second pulse

train. A response within 7.5 seconds of the onset of the noncontingent

stimulus resulted in the immediate delivery of a contingent stimulus that

was identical to the noncontingent stimulus in all parameters. This response

terminated the trial. If the subject failed to respond, there was no scheduled consequence; the trial was terminated after 7.5 seconds. Intervals

between trials averaged 15 seconds (range: 7.5 to 22.5 seconds), and

responses during the inlet-trial interval resulted in a 15-second delay before

the start of the next trial. Stimulus intensities were varied, and thresholds

were determined according to a modification of the psychophysical method

of limits. For further details of the procedure, see Esposito and Kometsky

(1977).

In the brain-stimulation detection procedure, the paradigm used was a modification of the technique first used by Doty et al. (1956). The experimental

chamber, including the manipulanda, was identical for brain-stimulation reward and detection. Also, the surgical preparation of the animals was identical, with some variation if the area of the brain in which the cueing

electrode was implanted was different from the electrode used for

reinforcing the behavior.

When only a single stimulating electrode was used (Kometsky and Esposito

1981), the procedure for determining the brain-stimulation detection threshold was only slightly different from that used and indicated in figure 4 for

brain-stimulation reward. In the detection procedure, the noncontingent

stimulus was varied in intensity but at current levels that were not reinforcing, while the second or response-contingent stimulus was held constant at a

highly reinforcing level. Thus, the noncontingent stimulus functioned purely

183
as a cue or discriminative stimulus, indicating the presence of a rewarding

level of stimulation, The intensity of the initial stimulus was varied according to a modification of the psychophysical method of constant stimuli.

FIGURE 4. A schematic representation of the two possible outcomes

during a single trial of the brain-stimulation reward

procedure

NOTE: In the first example (I), the subject is presented with a nonreinforcing stimulus (S1), after

which it does not respond during the 7.5-second available response time. The animal’s failure

to respond has no scheduled consequences, and a new trial begins after a 15-second intertrial

interval. In the second example (II), a reinforcing stimulus (S1) is presented, and the subject

responds by turning the wheel manipulandum within the 7.5-second available response interval

and receives a second stimulus (S2) of the same intensity as the S1.

By employing two electrodes (Wheeling and Kometsky 1983), detection

thresholds could be determined from one intracranial locus, while behavior

was maintained by rewarding brain stimulation to a second site. The advantage of the two-electrode model is that it allows for the determination of

the threshold for detection from sites where stimulation is never reinforcing

at any intensity. Other than the fact that the cueing electrode is not the

same electrode that delivers the reinforcing stimulation that drives the

behavior, the two-electrode procedure is identical to that employed when

only one electrode is used. Using this technique, marked differences in

drug effects on absolute threshold for detection and reward were found, but

detection was from different brain sites (Wheeling and Kometsky 1984).

Comparison of the Effects of Cocaine and Pimozide on the Thresholds

for Brain-Stimulation Reward and Brain-Stimulation Detection

In these experiments, the single electrode procedure was used. In the

first of these experiments, Kometsky and Esposito (1981) compared the

effects of cocaine, a drug that increases an animal’s sensitivity to rewarding

brain stimulation, on the threshold for rewarding and detecting electrical

stimulation to the MFB. An important question posed by this experiment

was whether the threshold-lowering effects of cocaine for rewarding brain

184
stimulation was specific for the brain site being stimulated or specific for

the type of stimulation, which, in this case, was rewarding. The former

would be borne out, if the threshold for detection also was lowered with the

potency approximately the same.

The threshold for rewarding brain stimulation after various doses of cocaine

was determined in four animals. The results replicated a previous experiment (Esposito et al. 1978) that demonstrated that cocaine lowered the

threshold. After a cocaine dose-response curve was generated for each animal, the animals were retrained on the brain-stimulation detection procedure

using the same electrode as was used for determining the threshold for rewarding stimulation. The range of thresholds for the four animals after

saline was 65 to 125 μA for reward and 7 to 22 μA for detection.

Threshold change scores (postdrug threshold minus predrug threshold) for

each experimental day were converted to a standard score (z-score) based

on the mean and standard deviation of all the change scores after saline

treatment for each animal respectively. This was done for both reward and

detection. The mean effects of cocaine on both the reward and detection

thresholds expressed as z-scores are shown in figure 5, which indicates two

interesting phenomena. The first is the complete dissociation between the

effects of cocaine. While cocaine increases an animal’s sensitivity to rewarding brain stimulation, as indicated by the lowering of the threshold, it

decreases the sensitivity of the animal to the detection of stimulation to the

same intracranial site by means of the same electrode. Further, cocaine will

lower the threshold for rewarding brain stimulation at doses that have little

or no effect on the detection threshold. These results suggest that

attentional-perceptual functioning is not impaired by cocaine at doses that

are probably euphorigenic; however, as doses increase, there is impairment

of such functioning.

If drugs that increase dopamine availability at the synapse have euphorigenie properties, are self-administered by humans and animals, and lower

the threshold for rewarding brain stimulation, do dopamine blockers decrease euphoria or, as suggested by Wise (1982), are they anhedonic? In

every case where the effects of a neuroleptic have been tested on brainstimulation reward, the neuroleptic has either decreased response rate for

the stimulation (Wise 1982) or it has raised the threshold (Esposito et al.

1981), suggesting that it decreases the animal’s sensitivity to the stimulation.

The problem of the specificity of the effect is even greater when looking at

a behavior that is impaired by the drug, rather than a behavior that is facilitated. Some investigators have attempted to deal with this problem of performance capability in the study of neuroleptic effects on brain-stimulation

reward. For example, in attempts to dissociate the motivational from the

performance effects of pimozide, some investigators (Franklin 1978; Stellar

et al. 1983; Gallistel and Karras 1984) have determined that the pulse

frequency (the higher the frequency, the greater the amount of stimulation)

185
required to initiate running was raised at doses that did not decrease the

speed of the running.

FIGURE 5. Mean effect of various doses of cocaine on the threshold for

brain-stimulation reward (reinforcement) and on the threshold for brain-stimulation detection

NOTE: Data are expressed as z-scores based on the respective mean and standard deviation of the

effects of saline (n=4).

SOURCE: Kornetsky and Bain 1982, copyright 1982, Elsevier.

Although these findings suggest a specific decrease in the reinforcing value

of the electrical stimulation, they did not control for the possibility that

pimozide was impairing attentional capacity, and this could account for the

increase in pulse frequency necessary to initiate speed of running. In order

186
to more directly address the problem of performance deficits accounting for

the decreased sensitivity of animals to rewarding brain stimulation, the

effects of pimozide on both the reward and detection threshold were determined in the same animals (Bird and Kometsky 1988). The experiment

was similar to the above described experiment with cocaine. Also, because

of the delay in the distribution of pimozide in the CNS, testing was not

begun until 4 hours after injection. In this experiment, psychophysical

power functions were determined using the least-squares regression analyses

of the probit of percent response versus the log of the total electrical charge

of the stimulus (pico Coulombs). The threshold in pico Coulombs was

interpolated from the line of best fit using the 50-percent response criteria

(probit, 5). These thresholds were transformed to z-scores based on the

mean and standard deviation of all vehicle treatment days for each animal,

respectively.

The results of one of the four animals used in the experiment are shown in

figure 6. The figure shows the power function lines of best fit for both

detection and reward. At doses of pimozide that significantly raise the

threshold for brain-stimulation reward, there are no significant effects on the

detection of stimulation. The mean dose-response curve for the four animals studied is shown in figure 7. Using this technique, it can be concluded that there is clearly no effect on the ability of the animal to detect

stimulation at doses that cause a loss of sensitivity to rewarding stimulation.

To the extent that the brain-stimulation detection procedure reflects an important part of cognitive functioning, these data indicate a relatively specific

effect of pimozide on the sensitivity of the animal to rewarding stimulation.

Effects of Chlorpromazine on the Threshold for Brain-Stimulation

Reward and Brain-Stimulation Detection

Chlorpromazine, also a neuroleptic but with less specificity than pimozide as

a dopamine antagonist, was tested on brain stimulation reward as well as

brain stimulation detection. As expected, chlorpromazine raised the threshold for both types of stimulation (Esposito et al. 1981; Williams and

Kometsky 1985). Figure 8 shows the mean dose-response curve for the

effects of doses of chlorpromazine from .03 to 2.0 mg/kg intraperitoneal

(IP), on the threshold for brain-stimulation reward with stimulating electrodes in the MFB-lateral hypothalamic area, detection from the MFB, and

also from the mesencephalic reticular formation (MRF). Although not indicated, higher doses of chlorpromazine raised the reward threshold even

higher; however, higher doses of chlorpromazine resulted in the animal’s

inability to do the detection task, as indicated by the vertical arrows. Of

interest is that, in contrast to the results obtained with pimozide, chlorpromazine caused marked impairment in detection from at least two sites in the

brain, at doses far below those that decreased sensitivity to rewarding

187
FIGURE 6. The effects of pimozide on the detection and reward power

functions and threshold for a single animal is shown

NOTE: The intensity of stimulation needed to reinforce responding to the S1 in the detection

procedure is indicated by the large arrows. Probit of percent response is plotted against the

stimulation intensity expressed in log 10 pico Coulombs units. Threshold for each treatment

is indicated by daggers.

brain stimulation. From these two experiments, it can be concluded that

there seems to be a relatively specific effect of the pimozide on brain stimulation, while the much less specific dopamine antagonist chlorpromazine

affects reward only at doses that are impairing the perceptual attentional

ability of the animals.

CONCLUSIONS

The above described experiments, although not directly measuring the

effects of drugs on learning and memory, clearly demonstrate that motivation and attention influence the effects of abuse substances on a subject’s

cognitive performance. A significant aspect of cognitive function is the

ability of the animal to make fine discriminations and attend to the possible

presentation of a stimulus. This detection of stimuli is easily impaired by

both abuse and nonabuse substances. This decrease in sensitivity to stimuli,

188
- -

FIGURE 7. Mean z-scores ±SEM in the detection and reward thresholds

as a function of punozide dose for four animals

NOTE: Error bars not shown indicate SEM less than the diameter of the symbols in this illustration.

Each z-score is based on the difference between the mean postvehicle and postpimozide

threshold. A z-score of 2 denotes the standardized 95-percent confidence interval of

postvehicle thresholds and indicates a significant deviation from vehicle-treated sessions.

however, may be selective, in that doses of cocaine that impaired detection

were able to increase the animal’s sensitivity to rewarding brain stimulation,

and doses of pimozide that decreased sensitivity to rewarding brain stimulation failed to impair the detection of nonrewarding brain stimulation to the

same site. Thus, it is important to realize that, in all experiments designed

to elucidate the effects of drugs on performance, at least at lower doses,

behaviors can be dissociated, and, although sensitivity of an animal can be

increased in one modality, it may be impaired in others.

189
FIGURE 8. Effects of chlorpromazine on threshold for rewarding stimulation to the MFB and detection threshold to the MFB and

to the MRF

NOTE: Data are expressed as the mean z-scores of two animals for each of the detection placements.

For the reward threshold level, the data are the mean of four animals. Z-scores are based on

the mean and standard deviation for all saline treatments for the respective animal and task.

REFERENCES

Abramson, B.A.; Jarvik, M.E.; Kaufman, M.R.; Kometsky, C.; Levine, A.;

and Wagner, M. Lysergic acid diethylamide (LSD-25) I. Physiological

and perceptual responses. J Psychol 39:3-60, 1955.

Bird, M., and Kometsky, C. The effects of pimozide on stimulus discrimination and rewarding electrical brain stimulation. Abs Soc Neurosci

11(2):1190, 1988.

Doty, R.W.; Rutledge, L.T., Jr.; and Larsen, R.M. Conditioned reflexes

established to electrical stimulation of the cat cerebral cortex. J Neurophysiol 19:401-415, 1956.

Esposito, R.U., and Kometsky, C. Morphine lowering of self-stimulation

thresholds: Lack of tolerance with long term administration. Science

195:189-191, 1977.

190
Esposito, R.U.; Motola, A.H.D.; and Kometsky, C. Cocaine: Acute effects

on reinforcement thresholds for self-stimulation behavior to the medial

forebrain bundle. Pharmacol Biochem Behav 8:437-439, 1978.

Esposito, R.U.; Perry, W.; and Kometsky, C. Chlorpromazine and brainstimulation reward: Potentiation of effects by naloxone. Pharmacol

Biochem Behav 15:903-905, 1981.

Franklin, K.B.J. Catecholamines and self-stimulation: Reward and performance effects dissociated. Pharmacol Biochem Behav 9:813-820, 1978.

Gallistel, C.R., and Karras, D. Pimozide and amphetamine have opposing

effects on the reward summation function. Pharmacol Biochem Behav

20:73-77, 1984.

Hand, T.H., and Franklin, K.B.J. Associative factors in the effects of

morphine on self-stimulation. Psychopharmacology 88:472-479, 1986.

Hauty G.T., and Payne, R.B. Effects of dextro-amphetamine upon judgment. J Pharmacol Exp Ther 120:33-37, 1957.

Hill, H.E.; Belleville, R.E.; and Wikler, A. Motivational determinants in

modification of behavior by morphine and pentobarbital. AMA Arch

Neurol Psychiatr 77:28-35, 1957.

Kometsky, C. Effects of meprobamate, phenobarbital and dextro-amphetamine on reaction time and learning in man. J Pharmacol Exp Ther

123:216-219, 1958.

Kometsky, C. Pharmacology: Drugs Affecting Behavior. New York:

Wiley Interscience, 1976. p. 143.

Kometsky, C. Brain-stimulation reward: A model for the neuronal bases

for drug induced euphoria. In: Brown, R.M., and Friedman, D.P., eds.

Neuroscience Methods in Drug Abuse Research. National Institute on

Drug Abuse Research Monograph 62. DHHS Pub. No. ADM(85)-1415.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1985.

Kometsky, C., and Bain, G. Effects of opiates on rewarding electrical

stimulation. In: Smith, J.E., and Lane, J.D., eds. Neurobiology of

Opiate Reward Mechanisms. Amsterdam: Elsevier, 1982. pp. 237-256.

Kometsky, C., and Eliasson, M. Reticular stimulation and chlorpromazine:

An animal model for schizophrenic overarousal. Science 165:1273-1274,

1969.

Kometsky, C., and Esposito, R.U. Reward and detection thresholds for

brain stimulation: Dissociative effects of cocaine. Brain Res 209:496-

500, 1981.

Kometsky, C.; Mirsky, A.F.; Kessler, E.K.; and Dorff, J.E. The effects of

dextro-amphetamine on behavioral deficits produced by sleep loss in

humans. J Pharmacol Exp Ther 127:46-50, 1959.

Lorens, S.A., and Mitchell, C.L. Influence of morphine on lateral hypothalamic self-stimulation in the rat. Psychopharmacologia 32:271-277, 1973.

Marcus, R., and Kometsky, C. Negative and positive intracranial reinforcement thresholds: Effects of morphine. Psychopharmacologia 38:1-13,

1974.

Olds, J., and Milner, P. Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol

Psychol 47:419-427, 1954.

191
Rosvold, H.E.; Mirsky, A.F.; Sarason, I.; Bransome, E.D., Jr.; and

Beck, L.H. A continuous performance test of brain damage. J Consult

Psychol 20:343-350, 1956.

Stellar, J.R.; Kelley, A.E.; and Corbett, D. Effects of peripheral and central

dopamine blockade on lateral hypothalamic self-stimulation: Evidence for

both reward and motor deticits. Pharmacol Biochem Behav 18:433-442,

1983.

Wheeling, H.S., and Kometsky, C. Detection thresholds for electrical

stimulation of forebrain and midbrain loci in the rat. Brain Res

272:13-19, 1983.

Wheeling, H.S., and Kometsky, C. Effects of antipsychotic drugs on brainstimulation detection in preliminary observations. Pharmacol Biochem

Behav 21:645-649, 1984.

Williams, J.E.G., and Kometsky, C. Chlorpromazine: Effects on the detection of non-rewarding brain stimulation. Abs Soc Neurosci 1(1):670,

1985.

Wise, R.A. Neuroleptics and operant behavior, the anhedonia hypothesis.

Behav Brain Sci 5:39-87. 1982.

ACKNOWLEDGMENTS

Much of the recent work in this manuscript is supported by U.S. Public

Health Service grant DA 02326 from the National Institute on Drug Abuse

and Research Scientist Award DA 00099.

AUTHORS

Conan Kometsky, Ph.D.

Professor

Departments of Psychiatry and Pharmacology

Boston University School of Medicine

80 East Concord Street, Room L-602

Boston, MA 02118

Joseph E.G. Williams, Ph.D.

Research Associate

Department of Psychiatry

University of Chicago

5841 South Maryland Avenue, Box 411

Chicago, IL 60637

Michael Bird

Boston University School of Medicine

80 East Concord Street, Room L-602

Boston, MA 02118

192
Learning as a Factor in Ethanol

Tolerance

A.D. Lê and H. Kalant

INTRODUCTION

Tolerance is usually defined as a reduction in the behavioral and/or physiological effects produced by a given dose of a drug as a consequence of

chronic exposure to the drug. Tolerance can result from either a diminution

in the sensitivity of the central nervous system (CNS) to the drug (i.e.,

functional tolerance), an increase in the rate of elimination of the drug

(dispositional or metabolic tolerance), or both (Kalant et al. 1971).

Functional or CNS tolerance has been a subject of interest for multiple

reasons, ranging from clinical pharmacological to forensic issues. From the

research viewpoint, however, tolerance has been studied for two main reasons. First, tolerance is a cardinal sign of drug addiction and is believed to

contribute to the strength of addiction. Therefore, a knowledge of its

mechanism(s) may facilitate the development of rational measures for treating addiction (Cappell and LeBlanc 1981). Secondly, tolerance is generally

viewed as a form of neuroadaptation (Kalant et al. 1971; LeBlanc and

Cappell 1977) and knowledge of its mechanism(s) may facilitate or contribute to our understanding of CNS adaptive mechanisms or neuronal plasticity

in general.

CNS tolerance has been a subject of investigation for more than 60 years.

In earlier times, drug tolerance was an exclusive domain of pharmacologists

or neurobiologists. It was viewed as a general physiological adaptation to

the presence of the drug in the CNS (Himmelsbach 1943). Such adaptation

was thought to occur at the molecular level and to be specific for the type

of drug used or to the mechanism of action of the drug (Collier 1965;

Goldstein and Goldstein 1968; Cochin 1971). It was assumed that tolerance

developed equally to various drug effects following chronic drug treatment.

Beginning in the late sixties, the traditional view of tolerance was challenged by the work of Mitchell and colleagues on morphine tolerance

(Kayan et al. 1969; Kayan and Mitchell 1972) and that of Chen on ethanol

tolerance (Chen 1968). Essentially, these investigators demonstrated that

pharmacological treatment might not necessarily produce tolerance (Chen

193
1968), or that similar pharmacological history does not necessarily lead to

the same level of tolerance development (Kayan et al. 1969). Factors such

as repeated testing while under intoxication or resemblance between the procedure and circumstances associated with drug treatment and with testing

conditions (Gebhart and Mitchell 1972) might dictate the manifestation of

tolerance or the level of tolerance attained. These findings led to the view

that tolerance might be a learning process rather than a physiological adaptation. This, of course, makes the questionable assumption that learning is

not based on physiological adaptation.

Research in the last several years has indicated that tolerance is a multifaceted phenomenon. At present, it is quite clear that tolerance can develop

as a consequence of drug exposure per se (Lieber et al. 1965; Goldstein and

Pal 1971). For example, tolerance to ethanol (Goldstein and Pal 1971) can

be induced by continuous vapor inhalation, and tolerance to morphine can

follow morphine pellet implantation (Bhargava 1978). In such cases, behavioral factors are absent or minimally involved. However, various learning

processes such as associative learning or Pavlovian conditioning and opportunity to practice while under intoxication can be critical factors in tolerance

development.

There are two different bodies of experimental data that support the role of

learning in drug tolerance. The direct involvement of learning in tolerance

was demonstrated by the fact that behavioral manipulations such as practice

while under intoxication (Chen 1968; LeBlanc et al. 1973; LeBlanc et al.

1976a) and Pavlovian conditioning (Siegel 1975) can affect tolerance development. The second body of data was derived from experimental procedures that demonstrated that neurobiological manipulations that affect

learning also affect tolerance development in a similar manner. Although

the behavioral and neurobiological factors in tolerance have been studied on

a variety of drugs or drug classes, this chapter will concentrate mainly on

how these factors affect ethanol tolerance. Where relevant, tolerance to

other drugs will be discussed.

BEHAVIORAL MANIPULATIONS

Intoxicated Practice

The Before-and-After design has been used extensively to demonstrate the

role of intoxicated practice or response contingency in ethanol tolerance.

Essentially, this design involves two groups of subjects receiving the same

total drug exposure, but differing with respect to the temporal relation

between the drug and the test behavior. On the treatment or training days,

subjects in the Before (or Behavioral) group are injected with ethanol and

then tested on the task concerned. The subjects in the After (or Physiological) group perform the test in the sober condition and are then injected with

194
ethanol afterward. On the test days, both groups receive the alcohol before

performing the test.

Employing this design, Chen (1968) demonstrated that tolerance to the disruptive effect of ethanol on maze performance did not develop unless ethanol was given prior to the test on training days. In other words, tolerance

did not develop unless the animal had the opportunity to practice the task

while under ethanol intoxication. For this reason, he suggested that tolerance was a consequence of learning to compensate for drug-induced impairment, rather than of simple drug exposure per se.

Subsequent work by LeBlanc and his coworkers using food-motivated or

shock-avoidance tests (LeBlanc et al. 1973; LeBlanc et al. 1976a) essentially

confirmed Chen’s finding. In these studies, however, LeBlanc and his coworkers found that the After group also developed tolerance to the same

extent as that of the Before group, if the ethanol treatment was extended

over a long enough duration. LeBlanc et al. 1973, therefore, suggested that

practice while intoxicated only enhanced the rate of tolerance development,

but may not be essential for tolerance development. The enhancement of

tolerance development by intoxicated practice was referred to as behavioral

augmentation of tolerance.

Intoxicated Practice and the Development of Ethanol Tolerance

The role of intoxicated practice or response contingency on ethanol tolerance has also been shown in a variety of ethanol effects such as analgesia

(Jorgensen and Hole 1984; Jorgensen et al. 1986), impairment of thermoregulation (Alkana et al. 1983; Lê et al. 1986b), sexual behavior (Pinel

et al. 1988), and anticonvulsant action (Pinel et al. 1983; Pinel et al. 198.5).

For example, Pinel and coworkers have demonstrated that tolerance to the

anticonvulsant effect of ethanol in a kindled seizure model develops only

when amygdaloid stimulation has been administered during ethanol intoxication (Pinel et al. 1983; Pinel et al. 1985).

There is, however, considerable debate on whether intoxicated practice is an

absolute prerequisite for, or simply accelerates, tolerance development.

Some of the difference in the interpretation might be accounted for, in part,

by the ethanol dose level employed. For example, in a recent study (Lê et

al. 1987a). it was found that the influence of intoxicated practice on tolerance to the motor impairment effect of ethanol was dependent on treatment

dosage employed. When animals were treated daily with a 3.5 g/kg dose of

ethanol for 30 days, the extent of tolerance was essentially similar between

the intoxicated practice and nonpractice groups. However, when a low

treatment dose was used (1.5 to 2.5 g/kg intrapetitoneal (IP) daily for 30

days), or when tolerance was not maximal, intoxicated practice resulted in a

significant enhancement of tolerance development. In a collaboration with

Pinel’s laboratory, it was also found that tolerance to the anticonvulsant

195
effect of ethanol in a kindled seizure model did not require amygdaloid

stimulation during ethanol intoxication if the treatment dosage employed was

high (Mana et al. 1988). Similarly, work by Jorgensen et al. (1986)

showed that tolerance to the analgesic effect of ethanol was contingent upon

the administration of the painful stimulus during ethanol intoxication when

the daily ethanol treatment dose was 2.5 g/kg, but not when the treatment

dose was 5 g/kg.

These studies thus demonstrated that intoxicated practice can influence tolerance development, but that the extent or nature of the influence of such

practice on ethanol tolerance is dependent on the ethanol treatment regimen

employed.

PAVLOVIAN CONDITIONING AND ETHANOL TOLERANCE

The role of Pavlovian conditioning in morphine tolerance was demonstrated

by Siegel during the mid-seventies in a series of elegant experiments (Siegel

1975; Siegel 1976). The central hypothesis of the Pavlovian model of tolerance is that an unconditioned compensatory response to the drug effect

becomes conditionally linked to the environmental cues accompanying drug

administration, and that tolerance reflects an antagonism of the acute drug

effect by this conditioned response (Siegel 1975; Siegel 1976).

Work from our laboratory (Lê et al. 1979b; Lê et al. 1987a) and from

others (Mansfield and Cunningham 1980; Crowell et al. 1981; Melchior and

Tabakoff 1981; Melchior and Tabakoff 1985) has shown that tolerance to

ethanol-induced hypothermia is also subject to Pavlovian control. Essentially, in these studies, tolerance to the hypothermic effect of ethanol was

observed only, or to a greater extent, when animals were tested in the

presence of environmental cues previously associated with ethanol treatment,

not in an environment previously associated with saline administration. In

the presence of the environmental cues associated with ethanol administration, a saline injection elicited an anticipatory hyperthermia in the ethanoltreated animals (Lê et al. 1979b; Mansfield and Cunningham 1980; Crowell

et al. 1981).

Conditioning has been shown to influence tolerance to other effects of ethanol such as narcosis (Melchior and Tabakoff 1981) and analgesia (Tiffany

et al. 1987). In addition, tolerance to other sedative-hypnotic drugs, such as

barbiturates and benzodiazepines (Cappell et al. 1981; Greeley and Cappell

1985; King et al. 1987) as well as cross-tolerance between ethanol and barbiturate (Cappell et al. 1981; El-Ghundi et al. 1989), have been shown to be

subject to Pavlovian control.

Similar to the effect of intoxicated practice on tolerance, the influence of

Pavlovian conditioning on ethanol tolerance varies with the size of the alcohol treatment dose used to produce tolerance. At low treatment dose (12 to

1%
14 trials of 2 g/kg of ethanol), tolerance was demonstrable only in the presence of environmental cues previously associated with ethanol administration. At high treatment dose (12 to 14 trials of 4 g/kg of ethanol),

however, tolerance was manifested when tested in the presence or absence

of environmental cues previously associated with ethanol administration (Lê

et al. 1987b).

NEUROBIOLOGICAL MANIPULATIONS

The role of learning in drug tolerance has also been supported by a different body of data derived from a different experimental strategy. Basically,

this strategy consists of examining similarities or parallels in the characteristics of drug tolerance and learning and in their susceptibility to neurobiological modification. For example, a task once learned, then forgotten,

can be reacquired more rapidly than on the original learning. In a similar

way, tolerance to ethanol has been shown to be reacquired more rapidly on

subsequent cycles of exposure (Kalant et al. 1978; de Souza Moreira et al.

1981). Similarly, a number of neurobiological manipulations, ranging from

cortical ablation to neurotransmitter modification, which have been shown to

affect learning, modify tolerance in the same pattern. A summary of these

studies is shown in table 1. It is clear from this table that the

manipulations employed did not influence tolerance to a specific drug but

affected the development of tolerance to a variety of drug classes.

It is beyond the scope of this chapter to examine in detail the findings from

all these studies. The role of serotonin and the pituitary peptide vasopressin, and their interaction in the regulation of ethanol tolerance, will be

examined as an example of the approach that attempts to draw parallels

between learning and tolerance.

INTERACTION BETWEEN SEROTONIN AND VASOPRESSIN IN

THE REGULATION OF ETHANOL TOLERANCE

Various studies have suggested that the brain serotonergic system may be

involved in learning and adaptive processes (Khanna et al. 1980). Depletion

of brain serotonin by administration of patachlorophenylalanine (pCPA),

which inhibits the synthesis of serotonin, or by intracranial administration of

5,7-dihydroxytryptamine retarded the development of tolerance to the motor

impairment and hypothermic effects of ethanol (Frankel et al. 1975; Frankel

et al. 1978a; Lê et al. 1980a). Conversely, administration of L-tryptophan,

in daily doses known to increase brain 5-hydroxytryptamine (5-HT) content,

also increased the rate of tolerance development (Lê et al. 1979a). The

retardation of ethanol tolerance by depletion of serotonin appears to be due

to an acceleration of the loss of tolerance: once tolerance was fully

acquired, administration of pCPA accelerated its loss (Frankel et al. 1978b).

197
TABLE 1. Effects on drug tolerance of neurobiological manipulations that

have been shown to disrupt learning

Inhibition of Protein Synthesis:

Morphine tolerance:

Ethanol tolerance:

Barbiturate tolerance:

Electroconvulsive Shock:

Morphine tolerance:

Frontal Cortical Ablation:

Ethanol tolerance:

Amphetamine tolerance:

Depletion of Brain Acetylcholine:

Barbiturate tolerance:

Depletion of Brain Norepinephrine:

Morphine tolerance:

Ethanol tolerance:

Barbiturate tolerance:

Depletion of Brain Serotonin:

Morphine tolerance:

Ethanol tolerance:

Barbiturate tolerance:

Inhibits tolerance development

Ginsberg and Cox 1972;

Feinberg and Cochin 1977

LeBlanc et al. 1976b;

Speisky and Kalant 1987

Hitzemann and Loh 1973

Impaired tolerance development

Kesner et al. 1976;

Stolerman et al. 1976

Impaired tolerance development

LeBlanc et al. 1976b

Glick 1973

Impaired tolerance development

Brezenoff and Mycek 1976

Impaired tolerance

Friedler et al. 1972

Tabakoff and Ritzmann 1977;

Melchoir and Tabakoff 1981

Tabakoff et al. 1978

Retarded tolerance development

Cheney and Goldstein 1971;

Ho et al. 1972

Frankel et al. 1975;

Frankel et al. 1978a;

Lê et al. 1980a;

Lê et al. 1981

Frankel et al. 1975;

Lyness and Mycek 1978;

Lê et al. 1980b

198
TABLE 1. (Continued)

Vasopressin and Its Analogs: Prolong the retention of tolerance

Morphine tolerance:

Ethanol tolerance:

Krivoy et al. 1974;

de Wied and Gispen 1976

Hoffman et al. 1978;

Hoffman and Tabakoff 1984;

Rigter et al. 1980;

Lê et al. 1982;

Pittman et al. 1982;

Hung et al. 1985;

Speisky and Kalant 1985

The cell bodies of central serotonin neurons are clustered in three raphe

nuclei, which project their fibers to different parts of the CNS. By lesioning the raphe nuclei selectively, it is possible to determine whether or not

the development of ethanol tolerance requires the participation of all central

serotonergic neurons. Destruction of the mesolimbic serotonergic pathway

by means of electrolytic lesions of the median raphe nucleus, which mainly

innervates the septum and hippocampal areas, was found to be necessary

and sufficient to retard tolerance development. Lesioning of the dorsal

raphe nucleus, which projects to the anterior cortex, the hypothalamus, and

the striatum, or of the raphe magnus, which projects fibers to the spinal

cord, did not affect tolerance development (Lê et al. 1981).

The pituitary peptide hormone vasopressin, and its analogs such as desglycinamide lysine vasopressin (DGLVP) or desglycinamide arginine vasopressin (DGAVP), have been shown to be able to prolong the retention of

learned avoidance behavior in the rat (de Wied et al. 1972). Brattleboro

rats, which in their homozygous state are deficient in vasopressin, also show

impairment in their learning ability (Bohus et al. 1975).

Similarly, administration of vasopressin (Hoffman et al. 1978; Hoffman and

Tabakoff 1984) or its analog DGAVP (Lê et al. 1982) can retain tolerance

to the hypothermic, hypnotic, or motor impairment effects of ethanol despite

the termination of ethanol treatment. Brattleboro rats also fail to develop

tolerance to ethanol (Pittman et al. 1982). The effect of vasopressin on the

retention of ethanol tolerance is mediated centrally by its interaction with V1

receptors, and intracerebral administration of vasopressin or the specific V1

agonist can maintain ethanol tolerance (Hung et al. 1985; Tabakoff and

Hoffman 1988).

The effects of vasopressin or its analogs on learning might be mediated, in

part, by its interaction with brain serotonin (Ramaekers et al. 1977). It was

199
also found that the retention of ethanol tolerance by DGAVP required an

intact mesolimbic serotonergic pathway; administration of DGAVP failed to

retain ethanol tolerance in median raphe-lesioned rats. Lesions of the dorsal

raphe nucleus did not affect the maintenance of ethanol tolerance by

DGAVP (Lê et al. 1982).

As mentioned earlier, the mesolimbic 5-HT pathway innervates other structures such as the parietal cortex, as well as the hippocampus. The

mesolimbic 5-HT pathway splits into two branches before reaching the

hippocampus. The infracallosal branch, traveling via the fornix and fimbria,

innervates the larger ventral hippocampus, while the supracallosal branch

traveling via the cingulum bundles innervates the dorsal hippocampus

(Azmitia and Segal 1978). Selective depletion of hippocampal serotonin by

means of intracranial injection of 5,7-dihydroxytryptamine into these two

pathways blocked the retention of ethanol tolerance by DGAVP (Speisky

and Kalant 1985). Thus, the actions of DGAVP in the retention of tolerance to ethanol might be mediated through its interaction with hippocampal

serotonin.

The exact nature of the cellular processes for which 5-HT and DGAVP are

required to regulate tolerance are still unknown. However, the fact that

their interaction in the regulation of tolerance occurs mainly in the hippocampus, an important structure for learning and memory, further heightens

the analogy between learning and drug tolerance.

CONCLUSION

From all the foregoing data on the effects of behavioral and neurobiological

manipulations on tolerance, one must conclude that, depending on certain

experimental conditions, learning can play a critical role in tolerance

development. However, it is also clear that tolerance can develop under

treatment conditions in which learning has little opportunity to take place.

One of the important questions that needs to be answered is whether these

factors interact to regulate the development of a single process of tolerance

or whether learning constitutes a different type or mechanism of tolerance.

In a situation in which both learning and pharmacological factors are optimal, the same maximum degree of tolerance can be achieved (LeBlanc et

al. 1976a; Lê et al. 1987a; Lê et al. 1987b). For this reason, it has been

proposed that there is no need to invoke more than one mechanism of tolerance. A large body of data has suggested that the stimulus for tolerance

development is the functional disturbance induced by the drug rather than

the drug itself (Kalant et al. 1971; Kalant 1987). If this is the case, then

neurons or synapses in an altered state associated with functional activity,

due to either behavioral or physiological manipulations, are more sensitive

to the effects of ethanol and other drugs and, therefore, experience a greater

stimulus to adapt to these effects (Kalant 1985; Kalant 1987).

200
Recent studies, however, have suggested that learning might reflect a separate mechanism of tolerance. Tolerance acquired under learning conditions

can be retained for a much longer time than that acquired by purely pharmacological means. For example, tolerance to the hypothermic effect of

ethanol induced by purely pharmacological means dissipated by 3 to 6 days

after the termination of ethanol treatment (Hoffman et al. 1978; Lê et al.

1982). Conditioned tolerance to the hypothermic effect of ethanol, however,

persisted for months after the termination of ethanol treatment (Crowell

et al. 1981). Furthermore, learned tolerance also resulted in a crosstolerance to the effects induced by other drugs that pharmacological tolerance failed to confer. When tolerance to various effects of ethanol was

produced by treatment conditions that involved minimal learning opportunity, little or no cross-tolerance to pentobarbital or hydralazine was observed

(Gougos et al. 1986; Lê et al. 1987b). However, in a conditioning paradigm of tolerance, or when opportunity to practice while under ethanol

intoxication was provided, cross-tolerance to the hypothermic effects of

pentobarbital and hydralazine (Lê et al. 1987b; El-Ghundi et al. 1989) or to

the motor impairment effect of pentobarbital (Lê et al. 1986a) could be

readily demonstrated. Similarly, work by Jorgensen et al. (1986) showed

that cross-tolerance to morphine- or clonidine-induced analgesia following

chronic ethanol treatment was demonstrable in animals that had been

exposed repeatedly to thermal stimulation under ethanol intoxication, but

was not demonstrable in those that had received the same ethanol treatment

without the repeated thermal stimulation.

The data derived from studies with neurobiological manipulation provide

evidence for the involvement of learning in tolerance. However, the experimental paradigms employed to produce tolerance in these studies confounded learning and pharmacological factors. In these studies, tolerance to

ethanol was induced by high treatment doses and involved repeated testing

of the animals under the intoxicating effects of ethanol (Frankel et al. 1975;

Lê et al. 1980a; Lê et al. 1981). It is, therefore, difficult to determine

which factor these manipulations affected. It is clear that further work is

required to clarify the manner in which these neurobiological manipulations

affect tolerance development. The effects of these manipulations on tolerance should be studied under experimental conditions in which either learning or pharmacological factors are operating exclusively. The results from

these studies will be of help in clarifying the question of unitary or multiple

mechanisms of tolerance.

REFERENCES

Alkana, R.L.; Finn, D.A.; and Malcolm, R.D. The importance of experience in the development of tolerance to ethanol hypothermia. Life Sci

32:2685-2692, 1983.

201
Azmitia, E., and Segal, M. An autoradiographic analysis of the differential

ascending projections of the dorsal and median raphe nuclei in the rat.

J Comp Neurol 179:641-668, 1978.

Bhargava, H.N. Quantitation of morphine tolerance induced by pellet implantation in the rat. J Pharm Pharmacol 30:133-135, 1978.

Bohus, B.; van Wimersma Greidanus, Tj.B.; and de Wied, D. Behavioral

and endocrine responses of rats with hereditary hypothalamic diabetes

insipidus (Brattleboro strain). Physiol Behav 14:609-615, 1975.

Brezenoff, H.E., and Mycek, M.J. Prevention and reversal of tolerance to

barbiturates by intraventricular injection of Hemicholinium-3. Eur J

Phartnacol 37:125-132, 1976.

Cappell, H., and LeBlanc, A.E. Tolerance and physical dependence: Do

they play a role in alcohol and drug self administration? In: Isreal, Y.;

Glaser, F.B.; Kalant, H.; Popham, R.E.; Schmidt, W.; and Smart, R.G.,

eds. Research Advances in Alcohol and Drug Problems. Vol. 6. New

York: Plenum Press, 1981. pp. 159-196.

Cappell, H.; Roach, C.; and Poulos, C.X. Pavlovian control of crosstolerance between pentobarbital and ethanol. Psychopharmacology

(Berlin) 74:54-57, 1981.

Chen C.-S. A study of the alcohol-tolerance effect and an introduction of a

new behavioral technique. Psychopharmacologia (Berlin) 63:433-440,

1968.

Cheney, D.L., and Goldstein, A. The effect of p-chlorophenylalanine on

opiate-induced running, analgesia, tolerance and physical dependence in

mice. J Pharmacol Exp Ther 177:309-315, 1971.

Cochin, J. Role of possible immune mechanism in the development of tolerance. In: Clouet, D.H., ed. Narcotic Drugs. Biochemical Pharmacology. New York: Plenum Press, 1971. pp. 431-448.

Collier, H.O.J. A general theory of the genesis of drug dependence by induction of receptors. Nature 205:181-182, 1965.

Crowell, C.R.; Hinson, R.E.; and Siegel, S. The role of conditional drug

responses in tolerance to the hypothermic effects of ethanol. Psychopharmacology (Berlin) 73:51-54, 1981.

de Souza Moreira, L.F.; Capriglione, M.J.; and Masur, J. Development and

reacquisition of tolerance to ethanol administered pre- and post-trial to

rats. Psychopharmacology (Berlin) 73:165-167, 1981.

de Wied, D., and Gispen, W.H. Impaired development of morphine analgesia in rats with heredity diabetes insipidus. Psychopharmacologia

(Berlin) 46:127-29, 1976.

de Wied, D.; Greven, H.M.; Lande, S.; and Witter, A. Dissociation of the

behavioral and endocrine effects of lysine vasopressin by tryptic digestion.

Br J Pharmacol 45:118-122, 1972.

El-Ghundi, M.; Kalant, H.; Lê, A.D.; and Khanna, J.M. The contribution of

environmental cues to cross-tolerance between ethanol and pentobarbital.

Psychopharmacology (Berlin) 97:194-221, 1989.

202
Feinberg, M.P., and Cochin, J. Studies on tolerance. II. The effect of

timing on inhibition of tolerance to morphine by cycloheximide.

J Pharmacol Exp Ther 203:332-339, 1977.

Frankel, D.; Khanna, J.M.; Kalant, H.; and LeBlanc, A.E. Effect of

p-chlorophenylalanine on the acquisition of tolerance to the hypothermic

effect of ethanol. Psychopharmacology (Berlin) 57:289-302, 1978a.

Frankel, D.; Khanna, J.M.; Kalant, H.; and LeBlanc, A.E. Effect of

p-chlorophenylalanine on the loss and maintenance of tolerance to ethanol.

Psychopharmacology (Berlin) 56:139-143, 1978b.

Frankel, D.; Khanna, J.M.; LeBlanc, A.E.; and Kalant, H. Effect of

p-chlorophenylalanine on the acquisition of tolerance to ethanol and

pentobarbital. Psychopharmacologia (Berlin) 44:247-252, 1975.

Friedler, G.; Bhargava, H.N.; Quock, R.M.; and Way, E.L. Effect of

6-hydroxydopamine on morphine tolerance and physical dependence in

mice. J Pharmacol Exp Ther 183:49-55, 1972.

Gebhart G.F., and Mitchell, C.L. The relative contribution of the testing

cylinder and the heated plate on the hot plate procedure to the develop

ment of tolerance to morphine in rats. Eur J Pharmacol 18:56-62, 1972.

Ginsberg, M., and Cox, B.M. Proteins and nucleic acids. In: Mule, S.J.,

and Brill, H., eds. Chemical and Biological Aspects of Drug Dependence. Cleveland: CRC Press, 1972. pp. 465-475.

Glick, S.D. Impaired tolerance to the effects of oral amphetamine intake in

rats with frontal cortical ablation. Psychopharmacologia (Berlin) 28:363-

371, 1973.

Goldstein, A., and Goldstein, D.B. Enzyme expansion theory of drug tolerance and physical dependence. In: Wikler, A., ed. The Addictive State.

Baltimore: Williams and Wilkins, 1968. pp. 265-267.

Goldstein, D.B., and Pal, N. Alcohol dependence produced in mice by inhalation of ethanol: Grading the withdrawal reaction. Science 172:288-

290, 1971.

Gougos, A.; Khanna, J.M.; Lê, A.D.; and Kalant, H. Tolerance to ethanol

and cross-tolerance to pentobarbital and barbital. Pharmacol Biochem

Behav 24:801-807, 1986.

Greeley, J.D., and Cappell, H. Associative control of tolerance to the sedative and hypothermic effects of chlordiazepoxide. Psychopharmacology

(Berlin) 86:487-493, 1985.

Himmelsbach, C.K. Symposium: Can the euphoric, analgetic and physical

dependence effects of drugs be separated? IV. With reference to physical

dependence. Fed Proc 2:201-203, 1943.

Hitzemann, R.J., and Loh, H.H. On the possible role of protein synthesis

in functional barbiturate tolerance. Eur J Pharmacol 40:163-173, 1973.

Ho, I.K.; Lu, S.E.; Stolman, S.; Loh, H.H.; and Way, E.L. Influence of

p-chlorophenylalanine on morphine tolerance and physical dependence and

regional brain serotonin turnover studies in morphine tolerant-dependence

mice. J Pharmacol Exp Ther 182:155-165, 1972.

Hoffman, P.L.; Ritzmann, R.F.; Walter, R.; and Tabakoff, B. Arginine

vasopressin maintains ethanol tolerance. Nature 276:614-616, 1978.

203
Hoffman, P.L., and Tabakoff, B. Neurohypophyseal peptides maintain tolerance to the incoordinating effects of ethanol. Pharmacol Biochem Behav

21:539-543, 1984.

Hung, C.R.; Tabakoff, B.; Melchior, C.L.; and Hoffman, P.L. Intraventricular arginine vasopressin maintains ethanol tolerance. Eur J Pharmacol

106:645-648, 1985.

Jorgensen, H.A.; Fasmer, O.B.; and Hole, K. Learned and

pharmacologically-induced tolerance to ethanol and cross-tolerance to

morphine and clonidine. Pharmacol Biochem Behav 24:1083-1088, 1986.

Jorgensen, H.A., and Hole, K. Learned tolerance to ethanol in the spinal

cord. Pharmacol Biochem Behav 20:789-792, 1984.

Kalant, H. The 1985 Upjohn Award Lecture. Tolerance, learning, and

neurochemical adaptation. Can J Physiol Pharmacol 63:1485-1494, 1985.

Kalant, H. Nathan B. Eddy Memorial Award Lecture: Tolerance and its

significance for drug and alcohol dependence. In: Harris, L.A., ed.

Problems of Drug Dependence 1986. Proceedings of the 48th Annual

Meeting. The Committee on Problems of Drug Dependence. National

Institute on Drug Abuse Research Monograph 76. DHHS Pub. No.

(ADM)87-1508. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1987. pp. 9-19.

Kalant, II.; LeBlanc, A.E.; and Gibbins, R.J. Tolerance to, and dependence

on, some non-opiate psychotropic drugs. Pharmacol Rev 23:135-191,

1971.

Kalant, H.; LeBlanc, A.E.; Gibbins, R.J.; and Wilson, A. Accelerated

development of tolerance during repeated cycles of ethanol exposure.

Psychopharmacology (Berlin) 60:59-65, 1978.

Kayan, S., and Mitchell, C.L. Studies on tolerance development to morphine: Effect of the dose-interval on the development of a single dose

tolerance. Arch Int Pharmacodyn Ther 199:407-417, 1972.

Kayan, S.; Woods, L.A.; and Mitchell, C.L. Experience as a factor in the

development of tolerance to the analgesic effect of morphine. Eur J

Pharmacol 6:333-339, 1969.

Kesner, R.P.; Priano, D.J.; and De Witt, J.R. Time-dependent disruption of

morphine tolerance by electroconvulsive shock and frontal cortical stimulation. Science 194:1079-1081, 1976.

Khanna, J.M.; Kalant, H.; Lê A.D.; and LeBlanc, A.E. Role of serotonin

(5-HT) in drug tolerance and general adaptation. Acta Psychiatr Scand

[Suppl 286] 62:161-170, 1980.

King, D.A.; Bouton, M.E.; and Musty, R.E. Associative control of tolerance to the sedatives effects of short acting benzodiazepine. Behav

Neurosci 101:104-114, 1987.

Krivoy, W.A.; Zimmerman, E.; and Lande, S. Facilitation of development

of resistance to morphine analgesia by desglycinamide-lysine vasopressin.

Proc Natl Acad Sci USA 71:1852-1856, 1974.

204
Lê, A.D.; El-Ghundi, M.; Khanna, J.M.; and Kalant H. Learning as a

factor in the development of tolerance (T) to ethanol (E) and crosstolerance (XT) to pentobarbital (PB) with respect to their motor impairment (MI) effects. Alcohol Alcohol 21(2), Abs. #147:A43, 1986a.

Lê, A.D.; Kalant, H.; and Khanna, J.M. Interaction between des-gly9-

[Arg8]-vasopressin and serotonin on ethanol tolerance. Eur J Pharmacol

80:337-345, 1982.

Lê, A.D.; Kalant, H.; and Khanna, J.M. Influence of ambient temperature

on the development and maintenance of tolerance to ethanol-induced

hypothermia. Pharmacol Biochem Behav 25:567-672, 1986b.

Lê, A.D.; Kalant, H.; and Khanna, J.M. Effects of treatment dose and

intoxicated practice on the development of tolerance to ethanol-induced

motor impairment. Alcohol Alcohol 1(Suppl 1):435-439, 1987a.

Lê, A.D.; Khanna, J.M.; and Kalant, H. Role of Pavlovian conditioning in

the development of tolerance and cross-tolerance to the hypothermic effect

of ethanol and hydralazine. Psychopharmacology (Berlin) 92:210-214,

1987b.

Lê, A.D.; Khanna, J.M.; Kalant, H.; and LeBlanc, A.E. Effect of

L-tryptophan on the acquisition of tolerance to ethanol-induced motor

impairment and hypothermia. Psychopharmacology (Berlin) 61:125-

129, 1979a.

Lê, A.D.; Khanna, J.M.; Kalant, H.; and LeBlanc, A.E. Effect of 5,7-

dihydroxytryptamine on the acquisition of tolerance to ethanol. Psychopharmacology (Berlin) 67:143-146, 1980a.

Lê, A.D.; Khanna, J.M.; Kalant, H.; and LeBlanc, A.E. The effect of

lesions in the dorsal, median and magnus raphe nuclei on the development of tolerance to ethanol. J Pharmacol Exp Ther 218:525-529,

1981.

Lê, A.D.; Khanna, J.M.; LeBlanc, A.E.; and Kalant, H. Effect of parachlorophenylalanine (p-CPA) on the acquisition of tolerance to barbital.

Can J Physiol Pharmacol 58:12-16, 1980b.

Lê, A.D.; Poulos, C.X.; and Cappell, H. Conditioned tolerance to the hypothermic effect of ethyl alcohol. Science 206:1109-1110, 1979b.

LeBlanc, A.E., and Cappell, H. Tolerance as adaptation: Interaction with

behavior and parallels to other adaptive processes. In: Blum, K., ed.

Alcohol and Opiates: Neurochemical and Behavioral Mechanisms. New

York: Academic Press, 1977. pp. 65-77.

LeBlanc, A.E.; Gibbins, R.J.; and Kalant, H., Behavioral augmentation of

tolerance to ethanol in the rat. Psychopharmacologia (Berlin) 30:117-122,

1973.

LeBlanc, A.E.; Kalant, H.; and Gibbins, R.J. Acquisition and loss of behaviorally augmented tolerance to ethanol in the rat. Psychopharmacologia (Berlin) 48:153-158, 1976a.

LeBlanc, A.E.; Matsunaga, M.; and Kalant, H. Effects of frontal polar cortical ablation and cycloheximide on ethanol tolerance in rats. Pharmacol

Biochem Behav 4:175-179. 1976b.

205
Lieber, C.S.; Jones, D.P.; DeCarli, L.M. Effect of prolonged ethanol intake:

Production of fatty liver despite adequate diet. J Clin Invest 44:1009-

1021, 1965.

Lyness, W.H., and Mycek, M.J. The role of cerebral serotonin in the development of tolerance to phenobarbital. Fed Proc 37:3394, 1978.

Mana, M.J.; Pinel, J.P.J.; and Lê, A.D. Tolerance to the anticonvulsant

effect (ACE) of ethanol: Absence of response contingency. Alcohol

Alcohol 23(3), Abs. #50:A43, 1988.

Mansfield, J.G., and Cunningham, C.L. Conditioning and extinction of tolerance to the hypothermic effect of ethanol in rats. J Comp Physiol

Psychol 94:962-969, 1980.

Melchior, C.L., and Tabakoff, B. Modification of environmental cued tolerance to ethanol in mice. J Pharmacol Exp Ther 219:175-180, 1981.

Melchior, C.L., and Tabakoff, B. Features of environmental-dependent tolerance to ethanol. Psychopharmacology (Berlin) 87:94-100, 1985.

Pinel, J.P.J.; Colbome, B.; Sigalet, J.P.; and Renfrey, G. Learned tolerance

to the anticonvulsant effects of alcohol in rats. Pharmacol Biochem

Behav 18(Suppl 1):507-510, 1983.

Pinel, J.P.J.; Mana, M.J.; and Renfrey, G. Contingent tolerance to the anticonvulsant effects of alcohol. Alcohol 2:495-499, 1985.

Pinel, J.P.J.; Pfaus, J.; and Christensen, B. Contingent tolerance to the disruptive effects of alcohol on the sexual behavior of male rats. Alcohol

Alcohol 23, Abs. #57:A47, 1988.

Pittman, Q.J.; Rogers, J.; and Bloom, F.E. Arginine vasopressin deficient

Brattleboro rats fail to develop tolerance to the hypothermic effects of

ethanol. Regul Pept 4:33-41, 1982.

Ramaekers, F.; Rigter, H.; and Leonard, B.F. Parallel changes in behaviour

and hippocampal serotonin metabolism in rats following treatment with

desglycinamide lysine vasopressin. Brain Res 120:485-491, 1977.

Rigter, H.; Rijk, H.; and Crabbe, J.C. Tolerance to ethanol and severity of

withdrawal in mice and enhanced by a vasopressin fragment. Eur J

Pharmacol 64:53-68, 1980.

Siegel, S. Evidence from rats that morphine tolerance is a learned

response. J Comp Physiof Psychol 89:498-506, 1975.

Siegel. S. Morphine analgesia tolerance: Its situation specificity supports a

Pavlovian conditioning model. Science 193:323-325, 1976.

Speisky, M.B., and Kalant, H. Site of interaction of serotonin and

desglycinamide-arginine vasopressin in maintenance of ethanol tolerance.

Brain Res 326:281-290, 1985.

Speisky, M.B., and Kalant, H. Effects of anisomycin, PLG and DGAVP on

rapid tolerance to ethanol in the rats. 10th International Congress of

Pharmacology. Abs. #P781, Sydney, Australia, 1987.

Stolerman, I.P.; Bunker, P.; Johnson, C.A.; Jarvik, M.E.; Krivoy, W.; and

Zimmerman, E. Attenuation of morphine tolerance development in mice.

Neuropharmacology 15:309-313, 1976.

206
Tabakoff, B., and Hoffman, P.L. Tolerance and the etiology of alcoholism:

Hypothesis and mechanism, Alcoholism: Clin Exp Res 12:184-186,

1988.

Tabakoff, B., and Ritzmann, R.F. The effects of 6-hydroxydopamine on

tolerance to and dependence on ethanol. J Pharmacol Exp Ther 203:319-

331, 1977.

Tabakoff, B.; Yanai, J.; and Ritzmann, R.F. Brain noradrenergic system as

a prerequisite for developing tolerance to barbiturates. Science 200:499-

502, 1978.

Tiffany, S.T.; McCal, K.J.; and Maude-Griffin, P.M. The contribution of

classical conditioning on tolerance to the antinociceptive effects of

ethanol. Psychopharmacology (Berlin) 92:524-528, 1987.

Tilson, H.A., and Rech, R.H. The effect of p-chlorophenylalanine on morphine analgesia, tolerance and physical dependence development in two

strains of rats. Psychopharmacologia (Berlin) 35:45-61, 1974.

AUTHORS

A.D. Lê, Ph.D.

Research Scientist

Biobehavioral Research Department

Addiction Research Foundation of Ontario

33 Russell Street

Toronto, ON M5S 2S1

CANADA

H. Kalant, M.D., Ph.D.

Professor of Pharmacology

Department of Pharmacology

University of Toronto

Medical Sciences Building

Toronto, ON M5S 1A8

CANADA

207
Conditioning as a Critical Determinant of Sensitization Induced by

Psychomotor Stimulants

Agu Pert, Robert Post, and Susan R.B. Weiss

INTRODUCTION

The fact that pharmacological agents can act as unconditioned stimuli has

been known for some time. Collins and Tatum (1925) were the first to

report that stimuli associated with drug administration in dogs could develop

the ability to elicit conditioned reactions. These investigators found that

situational cues associated with multiple injections of morphine eventually

acquired the power to induce salivation, an effect produced by the drug

itself. Pavlov (1927) and his colleagues observed similar effects in the dog

following morphine as well as apomorphine injections:

A dog was given a small dose of apomorphine subcutaneously and after one or two minutes a note of a

definite pitch was sounded during a considerable time.

While the note was still sounding the drug began to

take effect upon the dog: the animal grew restless,

began to moisten its lips with its tongue, secreted saliva

and showed some disposition to vomit. After the

experimenter had reinforced the tone with apomorphine

several times it was found that the sound of the tone

alone sufficed to produce all the active symptoms of the

drug, only in a less degree. (Pavlov 1927, p. 35)

These observations were followed shortly by a more detailed analysis of the

conditioned salivary reflex following morphine injections in the dog

(Kleitman and Crisler 1927). These investigators extended the previous

Endings by showing that conditioning would also occur to stimuli

(situational) that were presented as much as 2 hours prior to morphine

injections and that extinction would take place if the unconditioned stimulus

(UCS) (morphine) was no longer presented. In addition, it was also found

that the conditioned reflex was not as robust if sessions in which the

conditioned stimulus (CS) was not followed by morphine were interspersed

208
with conditioning sessions. These phenomena were entirely analogous to

those seen in classical alimentary reflex conditioning (Pavlov 1927).

In addition to the salivary reflex, it soon became apparent that other drug

reactions were also amenable to conditioning, especially those associated

with psychomotor stimulants. Tatum and Seevers (1929) reported that after

a month of cocaine injections in dogs, the animals became very excitable

whenever the attendant came near, especially if they saw the hypodermic

syringe. Downs and Eddy (1932) also found that dogs injected with

cocaine showed increased activity, excitement, and eagerness as the

experimenter entered the room. Both of these early studies appear to

suggest that situational stimuli associated with cocaine had acquired

incentive motivational properties. Surprisingly, little attention was paid to

these intriguing findings over the next three decades, even though they

suggested the development of rather striking environmental control over

addictive behaviors.

CONDITIONING OF PSYCHOMOTOR-STIMULANT-INDUCED

MOTORIC EFFORTS

Contemporary studies with psychomotor stimulants have focused on conditioning the motoric effects of these compounds. A variety of studies with

amphetamine and cocaine have assessed the ability of stimuli temporally

paired with these drugs to elicit locomotor effects or to potentiate the

motoric actions of the psychomotor stimulants. Irwin and Armstrong (1961)

reported the development of conditioned locomotor excitation in response to

an environment where rats had been injected with methamphetamine. Surprisingly, however, little extinction was observed following testing with

saline. Furthermore, Pickens and Dougherty (1971) have offered alternative

explanations to account for the increase in activity during tests with saline

in this study. Ross and Schnitzer (1963) also claimed to have demonstrated

conditioning of locomotor activity following exposure to amphetamine.

Three groups of rats were employed in their study. Two of the groups

were injected with amphetamine. Rats from one group were placed in a

locomotor activity monitor, while those from the other group were returned

to their home cage. The third group had a needle introduced

intraperitoneally (IP) and were placed in the test apparatus. One week later,

when all animals were placed in the apparatus, the group that had

experienced the apparatus under amphetamine had higher activity scores

than the needle-insertion controls. Unfortunately, however, no differences

were found between the conditioned and unconditioned amphetamine groups.

In this study, it appeared that exposure to the drug in either environment

was sufficient to enhance activity 1 week later under no-drug conditions.

Rushton et al. (1963) also reported enhanced exploratory activity in a

Y-maze following prior amphetamine plus barbiturate exposure. However,

they interpreted their results in terms of alterations in habituation processes.

209
They asserted that the drug mixture may have interfered with the

habituation of the animals to the apparatus. Thus, when these animals were

tested subsequently with saline, their locomotor responses would be

expected to be higher than those trained (habituated) under saline. Pickens

and Crowder (1967) attempted to control for lack of habituation as a

variable in conditioning paradigms. In their study, rats were first habituated

to the activity chamber for 6 hours. One group was then injected with

amphetamine and the other with saline. Both groups were placed in the

activity monitor for 30 minutes immediately following injections. Four to

five hours following placement in their home cages, the amphetamine rats

were injected with saline, while the saline rats were injected with

amphetamine. After six daily conditioning sessions, all rats were tested in

the activity monitors following saline injections. Rats that had been trained

under amphetamine had higher activity scores than those trained under

saline despite extensive habituation.

While conditioning does appear to occur in situations where drugs are used

as unconditioned stimuli, it is often uncertain which environmental cues

serve as the conditioned stimuli. Pickens and Dougherty (1971) have very

elegantly demonstrated the development of conditioned locomotor behavior

to a more defined CS. In this study, the CS was a 2-minute tone that was

followed by a 25-second infusion of 0.5 mg/kg methamphetamine that overlapped the CS. A progressive increase in locomotor activity in response to

the tone was seen following repeated CS-UCS pairings. Pseudoconditioning

controls did not show such conditioning. Schiff (1982), Bridger

et al. (1982), and Barr et al. (1983) have also described conditioning of

locomotor activity to auditory stimuli associated with injetions of

amphetamine or apomorphine. In addition, Hayashi et al. (1980) have

reported that a flickering light that had been associated with amphetamine

injections enhanced the locomotor stimulatory actions of amphetamine.

Conditioned locomotor behaviors in studies in which psychomotor stimulants

were used as the unconditioned stimuli follow many of the principles of

classical conditioning. Tilson and Rech (1973), for example, reported that

the magnitude of the conditioned locomotor effects were related directly to

the dose of amphetamine used during conditioning. The CS-UCS interval

also appears to be a critical variable that determines the strength of

conditioning to the motoric effects elicited by amphetamine (Pickens and

Crowder 1967). In addition, the presentation of the CS in the absence of

the UCS has been found to result in the extinction or weakening of conditioning. Barr et al. (1983) as well as Hinson and Poulos (1981) and

Hayashi et al. (1980) reported that situational cues associated with cocaine

and amphetamine injections lost their effectiveness to elicit conditioned

locomotor behavior when the drugs were no longer presented. Finally, the

strength of the conditioned response (CR) appears to decay with time (Barr

et al. 1983).

210
Gross locomotor activity is not the only motor behavior that is amenable to

conditioning. Carey (1986a; Carey 1986b), for example, has recently

demonstrated conditioned rotational behavior following the administration of

amphetamine or apomorphine to rats with unilateral 6-hydroxydopamine

lesions of the substantia nigra. Interestingly, rats were found to rotate

ipsilaterally to the lesion in the environment in which they experienced

amphetamine and contralaterally in the environment in which they had

experienced apomorphine. Thus, the appropriate conditioned stimuli are

able to mimic either directly or indirectly acting dopamine (DA) agonists.

Conditioned rotational behavior has also been reported by Casas

et al. (1989) following a single injection of apomorphine in lesioned rats.

This type of conditioning appears to be relatively resistant to decay, since

significant CRs have still been detected 24 (Carey 1986b) or 180 days

(Casas et al. 1988) following conditioning. Drew and Glick (1987) as well

as Elias et al. (1983) have also found conditioned rotational behavior in

unlesioned rats following amphetamine administration. Robinson (1984), on

the other hand, found little evidence for significant conditioning of rotational

behavior following amphetamine, although a clear trend in that direction is

evident in the data. A variety of cocaine-elicited motor behaviors, including

stereotypy, have also been conditioned (Schiff 1982; Barr et al. 1983;

Wagner et al. 1982).

Psychomotor stimulants such as amphetamine and cocaine are not the only

drugs that can be used to produce conditioned increases in locomotor

activity. Kamat et al. (1979) were the first to report that apparatus and

situational cues paired with low doses of morphine were able to elicit

increases in locomotor activity when the drug injections were discontinued.

Conditioned increases in locomotor activity in rats produced by morphine

have also been reported by Mucha et al. (1981) and Hinson and Siegel

(1983). Conditioned locomotor activity has been observed also following

direct injections of morphine into the ventral tegmental area of the rat

mesencephalon (Stewart 1983). Interestingly, in this study, pretreatment

with pimozide prevented the development of morphine-induced conditioning,

suggesting that dopaminergic mechanisms are critically involved. This is

not surprising considering the considerable amount of evidence suggesting

that the locomotor stimulatory actions of opiate agonists involve mesolimbic

DA (Broekkamp et al. 1979; Joyce and Iversen 1979; Kalivas et al. 1983;

Kelley et al. 1980).

CONDITIONED DECREASES IN LOCOMOTOR BEHAVIORS

In contrast to psychomotor stimulants, neuroleptics and other behavioral

depressants have been found in a few studies to be effective unconditioned

stimuli for producing conditioned suppression of activity or motor

performance. Irwin and Armstrong (1961) reported that situational cues

associated with acute and chronic injections of perphenazine or chlorpromazine developed the ability to suppress behavior. However, appropriate

211
control groups were not employed, and the conditioned response was unpredictable in a series of replications. Conditioned cataleptic effects have been

reported for bulbocapnine (Perez-Cruet and Gantt 1959), while apparatus

cues associated with haloperidol injections acquired the ability (at least at

low doses) to suppress motor behaviors (Carey and Kemey 1987). The

authors of the latter study, however, interpreted this effect in terms of

operant conditioning processes. Finally, stimuli associated with scopolamine

injections, which normally suppress behavior, have been found to disrupt

operant lever pressing in rats (Hermstein 1962). Although the findings

from the studies above are suggestive, it is not readily apparent whether the

results are due to conditioned decreases in locomotor activity or conditioned

suppression of ongoing behavior by stimuli that have been associated with

aversive events, i.e., pharmacological agents that produce aversive states.

NEUROPHARMACOLOGICAL SUBSTRATES OF CONDITIONED

LOCOMOTOR BEHAVIORS

Psychomotor stimulants such as cocaine and amphetamine are thought to

enhance locomotor output by increasing the availability of extracellular

mesolimbic dopamine (Kelly and Iversen 1975) either through uptake

blockade and/or enhanced release (Hadfield and Nuggent 1983; Moore 1977;

Reith et al. 1986). Opiate agonists are also thought to increase locomotor

output by enhancing the activity of the mesolimbic DA system (Broekkamp

et al. 1979; Di Chiara and Imperato 1988; Joyce and Iversen 1979; Kalivas

et al. 1983; Kelley et al. 1980). These are the mechanisms underlying the

unconditioned responses of these drugs. Relatively little is known, however,

regarding the neurochemical mechanisms that determine the conditioned

response. Do conditioned stimuli activate the same neurochemical systems

that are activated by the unconditioned stimuli (drugs), or are other systems

recruited as well? Only a few studies have attempted to assess the

neurochemical concomitants of conditioned locomotor behavior. Perez-Cruet

(1976), for example, found that DA metabolism could be increased by an

auditory stimulus previously associated with methadone, morphine, and

bulbocapnine, which by themselves had similar effects on this parameter of

DA function. Schiff (1982) has also found increases in mesolimbic and

striatal DA following presentation of stimuli associated with apomorphine

and amphetamine injections. The changes found in this study could reflect

either increases in DA turnover or increases in release without concomitant

alterations in synthesis induced by the conditioned stimuli. Alterations in

DA metabolism, however, were not found following presentation of stimuli

that had been paired with cocaine injections (Barr et al. 1983). It is still

uncertain whether the CR in locomotor activity studies is determined solely

through DA pathways or whether there is an involvement of additional

mechanisms that are recruited during the conditioning process. In light of

the following recent studies, as well as others, the latter seems to be the

more likely alternative.

212
PSYCHOMOTOR-STIMULANT-INDUCED SENSITIZATION

Conditioning also appears to play a rather important role in the expression

of certain behavioral effects associated with the repetitive administration of

psychomotor stimulants. It has been known for some time that certain

behavioral effects of drugs such as amphetamine and cocaine are augmented

with repetitive injections (behavioral sensitization). Tatum and Seevers

(1929) as well as Downs and Eddy (1932) found that, in dogs, there was a

progressive increase in the severity of reactions to cocaine with repeated

administration. Subsequent to these early reports, numerous other studies

also have reported sensitization to the behavioral effects of repetitive

cocaine and amphetamine injections. Repetitive administration of cocaine

has been found to produce progressive increases in the locomotor output of

rats (Post and Rose 1976; Post 1981; Kalivas et al. 1988) as well as mice

(Shuster et al. 1977; Riffee et al. 1988). Similar effects in rats have been

found following chronic administration of either amphetamine (Browne and

Segal 1977; Segal and Mandell 1974; Rebec and Segal 1979; Gold et al.

1988) or monoamine oxidase inhibitors (Irwin 1961). Stereotypic effects

have also been reported to increase in intensity with repetitive administration of either cocaine (Stripling and Ellinwood 1977; Post et al. 1981)

or amphetamine (Nelson and Ellison 1978; Browne and Segal 1977; Segal

and Mandell 1974; Magos 1969). Rotational behavior in either lesioned

(Robinson 1984; Robinson et al. 1982) or unlesioned rats (Glick et al.

1986; Drew and Glick 1989) also increases in intensity with repeated injections of amphetamine. With high doses of cocaine, sensitization is also

seen for the convulsant effects of this agent (Stripling and Ellinwood 1977;

Post et al. 1988; Tatum and Seevers 1929). Such behavioral alterations are

relatively long lasting and have been shown to be related to dose (Shuster

et al. 1977), gender (Post and Contel 1983), and, in part, to the intermittency of administration (Post 1980; Post 1981; Post and Ballenger 1981;

Post and Kopanda 1976).

CONDITIONING AS A DETERMINANT OF SENSITIZATION

At least two factors appear to contribute to the development of behavioral

sensitization. One set of factors is related to possible neuropharmacological

alterations induced by the repetitive administration of pharmacological

agents. These would include alterations in receptor sensitivity, enhanced

release, enhanced reuptake blockade, or decreases in autoreceptor sensitivity

(Robinson and Becker 1986). The other set of factors contributing to the

development of sensitization are those related to the conditioned drug effects

elicited by situational and environmental cues. The relative contribution of

each set of factors is determined by the experimental conditions and setting.

One of the best illustrations of the relative importance of conditioning as a

determinant of sensitization is found in a study by Post and his colleagues

(Post et al. 1981). In this report, two groups of rats were injected with

213
cocaine daily for 10 days. One group received cocaine in a locomotor test

apparatus and saline in the home cage, while the other group received saline

in the test apparatus and cocaine in the home cage. Both groups, therefore,

received equal exposure to the drug as well as the experimental chamber

and differed only in terms of where they were injected with cocaine. In

subsequent sessions, the animals that had experienced cocaine in the test

chamber had adivity scores that were considerably higher than those that

had experienced cocaine in their home cages, when tested under either

saline or cocaine. A number of other studies have also revealed the

importance of conditioning in the development and expression of sensitization induced by psychomotor stimulants. Tilson and Rech (1973), for

example, reported that rats that had experienced amphetamine in the test

chamber had activity levels that were considerably higher than those of

animals that had experienced amphetamine in the home cage when both

were tested subsequently with amphetamine in the test chamber. In fact,

the latter group was not different from rats treated with saline during the

initial phase of the study. Hinson and Poulos (1981) also found that

sensitization to the behavioral effects of cocaine was more pronounced

following drug administration in the presence of cues previously associated

with the drug. Hayashi et al. (1980) have reported that a flickering light

that had been presented concurrently with amphetamine injections enhanced

sensitization during the test day. Context-dependent sensitization to

amphetamine has also been reported by Drew and Glick (1989) and Elias

et al. (1983). This is not surprising, considering the fact that rotational

behaviors induced by psychomotor stimulants can be conditioned readily.

Although there is considerable evidence to indicate that conditioning plays

an important role in the sensitization process, a number of investigators

have failed to find support for the operation of this factor. Segal and

Mandell (1974), for example, found that rats that were continuously exposed

to the experimental chambers showed a progressive augmentation of locomotor output as well as stereotypy following chronic administration of

amphetamine. A saline challenge (presumably the CS) did not induce

locomotor behavior that was quantitatively different from that seen prior to

the initiation of drug injections. They concluded that conditioning is not a

critical factor in locomotor sensitization and also found it difficult to

understand how conditioning could contribute to the replacement of ambulatory responses by stereotypy with repeated administration of amphetamine.

Browne and Segal (1977) also found that animals treated with high doses of

amphetamine in three different environments still exhibited enhanced

stereotypy when tested in a standard experimental chamber. Past and Rose

(1976) also failed to find significant conditioning in chronically

cocaine-treated rats when saline injections and apparatus exposure were used

as the CS. Finally, Robinson (1984) has else failed to find that conditioning plays a critical role in the sensitization of amphetamine-induced

increases in rotational behavior. Rats that had been injected with high

doses of amphetamine in the test apparatus were not different from animals

214
that had received the amphetamine in the home cage when tested subsequently with the drug in the test chamber.

Several factors may account for the failure of some studies to have found

an important contribution of conditioning in sensitization. First, conditions

should be optimal for the operation of the wnditioning process. Segal and

Mandell (1974), for example, injected their rats in the cages in which the

animals lived. The strongest conditioning, however, would be expected to

develop to environmental cues that are discriminable. Conditioning would

not be expected to occur in a paradigm in which the animals were injected

in their home environment. This possibility has also been acknowledged by

Browne and Segal (1977) in a subsequent publication. Second, while conditioning undoubtedly does play a critical role in the sensitization process

under a variety of conditions, it is also apparent that high doses of psychomotor stimulants given over a longer period of time will contribute to

sensitization through mechanisms independent of conditioning, perhaps

involving alterations in neurochemical or neurophysiological processes

(Robinson and Becker 1986). The emergence of steteotypy following

repeated administration of high doses of amphetamine, for example, may be

determined solely by such mechanisms (Segal and Mandell 1974). Third,

the use of a habituation period prior to conditioning (Post and Rose 1976)

may also work against the acquisition of a conditioned locomotor response.

The presentation of a CS prior to conditioning has been shown to significantly retard the acquisition of a CR in a variety of paradigms (Anderson

et al. 1969; Carlton and Vogel 1967; Kremer 1971; Lubow and Moore

1959; Siegel 1969). Finally, it has commonly been assumed that the

injection of saline in the context of the test chamber, needle insertion, or

exposure to the apparatus are adequate conditioned stimuli to test for the

presence of conditioning (Browne and Segal 1977; Carey 1986a; Carey

1986b; Casas et al. 1988; Drew and Glick 1987; Hayashi et al. 1980; Post

and Rose 1976; Robinson 1984; Ross and Schnitzer 1963; Schiff 1982).

Generally speaking, however, the conditioned responses (locomotor activity)

under such conditions are rather modest in most cases or not seen at all.

This may be due to the fact that a saline injection is not always the

appropriate CS.

What is conditioned precisely when a drug is used as an UCS (figure l)?

The drug itself, of course, produces a variety of physiological and

behavioral actions, all of which can serve as the UCR. Locomotor activity

is one of a number of reactions (Stewart and Eikelboom 1987) that are

conditioned to situational stimuli. It is the only one, however, that is

measured in the majority of studies. It may be important to consider how

these conditioned reactions and behaviors interface and interact during the

process of conditioning. There are also problems in precisely defining the

stimulus complex that comes to serve as the CS. In the majority of studies,

the apparatus cues are generally considered to be the primary stimuli in

215
FIGURE 1. Classical conditioning paradigm in which drugs acting at

specific receptor sites in the brain produce UCS that result

in the production of various unconditioned physiological,

behavioral, and pharmacological states (UCR)

NOTE: External environmental (CS(E)) as well as interoceptive (CS(I)) stimuli produced by the drug

through peripheral sites take on the ability to elicit responses [CR) that are similar to those

produced by the drug (UCS) itself when the CS (E + I) and UCS occur in temporal

contiguity.

the total complex. Stimuli produced by the injection procedure, however, as

well as interoceptive cues accompanying the needle insertion, must also play

a role in the entire complex. More important, however, drugs such as

cocaine and amphetamine produce interoceptive cues through actions on

peripheral targets (i.e., cardiovascular system) that also enter into

determining the entire complex of cues to which conditioning occurs. All

of the above stimulus components should be capable of eliciting the conditioned response to some extent depending on their relative saliency. It is

also apparent that the most robust conditioned reactions should be seen

when the entire stimulus complex is reinstated during the test for conditioning. Injections with saline only reinstate a part of the total complex and

would not necessarily reveal the presence of conditioning in every situation.

The inability of a number of investigators to find conditioning following a

saline challenge may be due simply to the fact that the CS is not entirely

appropriate.

216
The authors recently introduced a rather simple and powerful one-session

conditioning paradigm with cocaine that illustrates the importance of

conditioning in sensitization more clearly by reinstating the interoceptive

drug-produced stimulus component of the CS during the test phase (Weiss

et al., in press). Three groups of rats were employed in this design. One

group was injected with a high dose of cocaine (40 mg/kg, IP) prior to

placement in the activity test chamber and with saline upon return to the

home cage (conditioning group). The second group was injected with saline

prior to placement in the test chamber and cocaine upon return to the home

cage (pseudoconditioning group). The third group received saline in both

environments (conditioning control group). It is important to note that the

first two groups received equal exposure to cocaine as well as to the two

different environments and differed only in terms of where they experienced

cocaine. The next day animals in all three groups were injected with a low

dose of cocaine (10 mg/kg, IP) and were placed in the test chamber. Rats

that were treated with cocaine in the test chamber on the first day showed a

marked increase in locomotor activity, compared to controls, following an

injection of 10 mg/kg of cocaine on the second day (figure 2). Animals

that received injections of cocaine in the home cage on day 1, however,

were no different than the saline controls during the second day. The

difference between the two groups that had been exposed to cocaine can be

attributed entirely to conditioning.

FIGURE 2. The role of conditioning in cocaine-inched sensitization

NOTE: Left panel illustrates the mean horizontal activity of rats (n=10/group) that received the

following treatment on day 1: cocaine (40 mg/kg, IP) in the activity chamber and saline in

the home cage (filled circles). Saline in the test chamber and cocaine in the home cage [filled

squares). and saline in both environments (open circles). Right panel illustrates horizontal

activity for these three groups following a cocaine challenge (10 mg/kg, IP) on day 2. Only

the rats that received cocaine in the test chamber on day 1 showed sensitization to cocaine on

day 2.

217
As noted above, the strength of the CR is dependent on the similarity of

the stimulus complex during conditioning to that presented during the test

for conditioning. Figure 3 illustrates the locomotor response of four groups

of rats to an injection of 10 mg/kg of cocaine after experiencing 40 mg/kg

of cocaine in different environments during the previous day. Rats that

were tested on day 2 in an environment identical to that present during

training had activity levels that were significantly higher than groups trained

and tested in different environments. Besides illustrating the presence of

discrimination in this paradigm, the findings also rule out the possibility that

failure to habituate to apparatus cues on day 1 under the influence of

cocaine could be a possible explanation for the differences found between

the two cocaine exposure groups in the previous study. The group that was

exposed to the apparatus under cocaine on day 1 was in fact more active

than any of the other groups on day 2.

COCAINE-INDUCED BEHAVIORAL SENSITIZATION:

EFFECT OF SIMILARITY OF PRETREATMENT

AND TEST ENVIRONMENT

FIGURE 3. The effect of similarity of pretreatment and test environment

on cocaine-induced behavioral sensitization

NOTE: Group means and standard error values for horizontal activity over a 30-minute period are

illustrated for four different pretreatments. All groups were injected with 40 mg/kg of

cocaine on day 1, but in different environments as described on the abscissa. For comparison, the mean value and standard error for a group of animals that received Saline on day 1

is also illustrated by the filled circle on the left of the figure.

218
While conditioning plays an important role in behavioral sensitization, it is

unlikely to be the only factor. Under certain circumstances, noncontextual

variables also appear to determine the increase in responsiveness to repeated

injections of cocaine. For example, when rats are pretreated for 3 days

with 40 mg/kg of cocaine instead of just 1 day and challenged on day 4

with 10 mg/kg of cocaine, then, even animals that experienced the high

dose of cocaine in their home cage have activity levels as well as

stereotypy scores significantly higher than the saline controls (figure 4).

Noncontextual factors may become determinants of sensitization when

relatively high doses of psychomotor stimulants are administered over a

longer period of time.

NEUROPHARMACOLOGICAL SUBSTRATES OF’ CONDITIONED

SENSITIZATION

Since mesolimbic DA appears to be involved in mediating the locomotor

stimulant actions of cocaine (Kelly and Iversen 1975), it follows that this

system may also be involved in mediating cocaine-induced sensitization as

well as its conditioned components. If this assumption is correct, then it

should be possible to attenuate the development of cocaine-induced

sensitization and the expression of its conditioned components with DA

blockers such as haloperidol. In a recent study (Weiss et al., in press), the

one-session conditioning paradigm was utilized to test these assumptions. It

was found that haloperidol administered concurrently with cocaine on day 1

blocked the cocaine-induced hyperactivity and also prevented the development of cocaine-induced sensitization on day 2 (figure 5). Rats treated with

cocaine (40 mg/kg) plus saline on day 1 had a significantly greater response

to a 10 mg/kg cocaine challenge on day 2 than rats that had been pretreated

with cocaine plus halopetidol (0.5 mg/kg). Diazepam administered concurrently with cocaine on day 1 also blocked cocaine-induced hyperactivity and

prevented the development of cocaine-induced conditioned sensitization on

day 2. Surprisingly, however, haloperidol administered concurrently with

10 mg/kg of cocaine on day 2 did not block the expression of conditioned

cocaine-induced sensitization (right side of figure 5). Thus, blockade of DA

receptors with haloperidol prevents the development of conditioned sensitization to cocaine, whereas it was incapable of preventing the expression of

the CR once established. Blockade of DA receptors with pimozide has also

been found to block the establishment of conditioned locomotor excitation

but not the expression of conditioned activity elicited by saline injections

following conditioning with either amphetamine (Beninger and Hahn 1983)

or cocaine (Beninger and Herz 1986).

Not all studies, however, support the position that the conditioned locomotor

reactions elicited by stimuli paired with psychomotor stimulants ate resistant

to DA blockade. Schiff (1982), for example, found that small doses of

haloperidol were able to block conditioned motor responses such as headbobbing, sniffing, and hyperactivity elicited by stimuli that had been paired

219
EMERGENCE OF

CONTEXT-INDEPENDENT SENSITIZATION

FOLLOWING REPEATED COCAINE

HORIZONTAL ACTIVITY

FIGURE 4. Development of context-independent sensitization

NOTE: Three groups of rats received the following treatments on days 1 to 3: Cocaine (40 mg/kg,

IP) in the activity chamber and saline in home cage (filled circles), saline in test chamber and

cocaine in home cage (filled squares). and saline in both environments (open circles). On day

4, all animals were injected with 10 mg/kg cocaine in the test apparatus Following 3 days

of treatment with high doses of cocaine, even animals that had been injected with cocaine in

their home cages showed sensitization to both stereotypy (bottom) as well as horizontal

locomotor activity (top). The difference in locomotor activity between the two cocainepretreated groups can be attributed to the development of more robust stereotypy in the group

that had received injections of cocaine in the test apparatus. Stereotypy interfered with the

expression of horizontal locomotor activity.

with amphetamine injections. However, the authors concluded that only the

blockade of head-bobbing and sniffing were pharmacologically specific,

since haloperidol also produced a significant attenuation of motor activity in

the pseudoconditioned controls. Drew and Glick (1987) have also reported

that 0.1 mg/kg of haloperidol was effective in blocking conditioned rotational behavior in unlesioned rats. Finally, Gold et al. (1988) have found

220
FIGURE 5. The effect of haloperidol on the development and expression

of conditioned cocaine-induced sensitization

NOTE: The figure illustrates horizontal activity on day 2 in response to a 10 mg/kg cocaine challenge

of activity in the control group. Left side: The control group for day 1 cocaine (40) group

(open column) was pretreated with Saline on day 1. The control group for the haloperidol

plus cocaine group (shaded column) was the group pretreated with haloperidol plus saline on

day 1. Haloperidol pretreatment on day I blocked sensitization to cocaine on day 2 when all

animals were challenged with 10 mg/kg of cocaine. Right side: Haloperidol (0.5 mg/kg) was

administered to animals only prior to cocaine rechallenge on day 2 (shaded column). The

control group for rats that received haloperidol prior to cocaine on day 2 following treatment

with cocaine (40 mg/kg) on day 1 (shaded column) received similar treatment on day 2 but

were pretreated with saline on day 1. As illustrated, these haloperidol-pretreated (day 2) rats

showed as robust sensitization, based on prior cocaine exposure, as did animals tested without

haloperidol (open column). Thus, haloperidol did not block the expression of cocaine

sensitization (right panel) but did block its development (left panel).

that 6-OHDA lesions of the nucleus accumbens, which decreased DA content by 90 percent, prevented the expression of a conditioned locomotor

response that had been conditioned with amphetamine as the UCS. The

authors concluded that an intact DA system within the region of the nucleus

accumbens is necessary for the expression of conditioned locomotor

behavior.

MECHANISMS UNDERLYING NEUROLEPTIC BLOCKADE OF

CONDITIONING

The mechanisms whereby DA blockers inhibit the development of psychomotor-stimulant-induced conditioning are not fully understood. Several

possibilities exist. Neuroleptics, for example, could disrupt the acquisition

of a conditioned response by interfering with associative processes, by

altering the nature of the conditioned stimuli, or by disrupting the unconditioned response (locomotor activity). The simplest explanation for the

ability of haloperidol or pimozide to prevent the development of conditioned

221
locomotor activity is that they block the expression of the unconditioned

response. Hirabayashi and Alam (1981) have in fact found that mice whose

activity is physically restricted do not develop sensitization. These findings,

however, could be confounded by the development of aversive conditioning

to the apparatus cues induced by restricted movement. The aversive motivational state elicited by the situational cues could interfere with the

expression of locomotor behavior. In addition, Swerdlow and Koob (1984)

have failed to replicate these findings. They reported that restrained rats

demonstrated significant conditioned locomotor activation in an amphetamine-associated environment. A simple inhibition of the unconditioned

reaction to psychomotor stimulants may not be sufficient to account for the

acquisition deficits seen. Blockade of the unconditioned response in other

conditioning paradigms has not been sufficient to prevent the appearance of

a conditioned reaction during subsequent testing. For example, pretreatment

of dogs with atropine, which blocks salivation, prior to presentation of

morphine or acid as the UCS does not prevent the development of conditioned salivation to stimuli associated with these two manipulations (Crisler

1930; Finch 1938). In addition, flexion reflexes are still elicited by

conditioned stimuli in animals that had ventral roots innervating the limb

crushed during training (Light and Gantt 1936; Beck and Doty 1957).

Finally, curare, a drug that blocks skeletal reactions, does not prevent the

conditioned expression of such reactions when the drug has worn off

(Solomon and Turner 1962). If conditioning of locomotor behaviors follows

the principles of classical conditioning, it is unlikely that merely preventing

the occurrence of the locomotor behavior during conditioning is sufficient to

disrupt the formation of the association. It is possible, however, that

centrally mediated effects of drugs such as neuroleptics and diazepam could

inhibit or block the initiation of motor programs that could result in a

deficit in conditioning.

Neuroleptics also have been found to retard the acquisition of other conditioned reactions. Harvey and Gormezano (1981) and Schindler et al. (1985)

have found that haloperidol as well as pimozide inhibit the rate of acquisition of the nictitating membrane response in rabbits. Such disruptive effects

were not determined by interference with associative processes but by the

ability of the drugs to attenuate the conditioned and unconditioned excitatory

properties of the stimuli used as the CS. Neuroleptics such as chlorpromazine have also been reported to block the unconditioned as well as conditioned excitatory effects of auditory stimuli as measured by changes in the

threshold for behavioral and electroencephalogram arousal (Key and Bradley

1960; Killam and Killam 1958). A decrease in the excitatory properties of

stimuli would decrease their ability to enter into conditioning. Strength of

conditioning has been shown to be directly related to the strength of the CS

(Barnes 1956; Kamin and Schaub 1963; Kamin and Brimer 1963). The

main effect of CS intensity may be to increase its discriminability from the

background.

222
Even more likely is the possibility that haloperidol and pimozide by blocking DA receptors alter the strength of the UCS. It has been well established that strength of conditioning is directly related to the intensity of the

UCS (Wagner et al. 1961; Ost and Lauer 1965; Annau and Kamin 1961;

Kamin and Brimer 1963). Increases in UCS probably strengthen the conditioning process by increasing overall levels of arousal or drive (Spence

1956).

NATURE OF THE CONDITIONED RESPONSE

What is the nature of the conditioned response in situations in which

psychomotor stimulants are used as unconditioned stimuli? In the simplest

formulation, the conditioned response mimics, at least in part, the

unconditioned response. Since psychomotor stimulants induce increases in

locomotor activity and stereotypy as part of their unconditioned effects, the

conditioned responses could simply mimic these reactions. It is possible,

however, that the motor behaviors elicited by conditioned stimuli associated

with psychomotor stimulants reflect more than just simple conditioned motor

reactions. One possibility is that such conditioned behavior actually

represents the operation of underlying incentive motivation mechanisms. It

has been known for some time that stimuli associated with primary reinforcers, such as food, for example, can increase locomotor output in rats

(Sheffield and Campbell 1954; Baumeister et al. 1964; Bolles 1963; Finger

et al. 1960). It has been suggested that such stimuli acquire the ability to

arouse incentive-motivational mechanisms that play a role in energizing

instrumental responding (Bindra 1968; Rescorla and Solomon 1967).

Loviband (1983), for example, has demonstrated that stimuli associated with

sucrose reinforcement in rabbits enhanced subsequent instrumental responding. Drugs such as cocaine and amphetamine can also serve as primary

reinforcers by acting directly on the reward systems in the brain. It is

apparent that stimuli associated with administration of psychomotor stimulants also acquire motivational properties. For example, animals injected

with amphetamine (Reicher and Holman 1977; Sherman et al. 1980),

cocaine (Mucha et al. 1982). or opiate agonists (Kumar 1972; Katz and

Gormezano 1979; Mucha et al. 1982; Rossi and Reid 1976) in a specific

environment will spend more time in that environment when given a chance

in subsequent test sessions without the drug (conditioned place preference).

Furthermore, specific stimuli that are paired with injections of opiates and

psychomotor stimulants in self-administration paradigms are capable of

maintaining instrumental responding when they are made contingent on such

behavior (Davis and Smith 1976; O’Brien 1976; Schuster and Woods 1968).

It is possible that stimuli associated with enhanced DA activity acquire the

ability to generate positive affective motivational states that are reflected by

increases in general locomotor activity.

Neuroleptics not only block the conditioning of locomotor reactions to

stimuli associated with psychomotor stimulants (above), but also inhibit the

223
ability of stimuli associated with primary rewards such as food and drugs to

acquire incentive-motivational properties (incentive learning). For example,

administration of pimozide during the pairing of an auditory stimulus with

food prevented the stimulus from acquiring the ability to maintain instrumental responding following removal of the drug (Beninger and Phillips

1980a). Pimozide has also been shown to prevent a conditioned stimulus

from acquiring incentive-motivational properties in an operant discrimination

paradigm (Beninger and Phillips 1980b). Incentive learning has also been

shown to be inhibited by haloperidol when amphetamine has been used as

an UCS (Davis and Smith 1975; Davis and Smith 1977). Thus, the ability

of haloperidol to prevent conditioned locomotor behavior in the authors’

studies above as well as in the studies reported by Beninger and his

colleagues (Beninger and Hahn 1983; Beninger and Herz 1986) could reflect

its ability to interfere with incentive learning. Haloperidol may have

blocked the acquisition of incentive-motivational qualities by stimuli

associated with either cocaine or amphetamine.

NEUROCHEMICAL SUBSTRATES OF THE CONDITIONED

RESPONSE

The inability of haloperidol to block the expression of conditioned locomotor activity in the authors’ studies as well as in those reported by

Beninger and Hahn (1983; Beninger and Herz 1986) would suggest that the

neural events underlying the conditioned response are somewhat different

than those produced by the drug itself. Since cocaine produces its effects

on locomotor activity by enhancing the availability of DA in the nucleus

accumbens (Kelly and Iversen 1975). it appears that conditioned stimuli

activate the motor system through mechanisms that are independent of

mesolimbic DA. Alternatively, it is possible that the acquisition process is

more sensitive to DA blockade than is the expression of established

behavior. In fact, it has been shown that well-established instrumental

behaviors are relatively resistant to disruption by neuroleptics (Beninger

1983).

As noted above, there are very few studies that have evaluated alterations of

DA function by stimuli associated with psychomotor stimulants. Unfortunately, the results from those that have are somewhat equivocal (Schiff

1982; Barr et al. 1983). Studies with opiates, on the other hand, do seem

to suggest that stimuli associated with morphine acquire pharmacological

properties that are highly similar to those produced by the drug itself.

Roffman et al. (1973), for example, reported that an auditory stimulus

paired with morphine injections acquired the ability to prevent withdrawal

symptoms in the rat. Blockade of morphine withdrawal by stimuli that had

been paired with morphine has also been reported by Tye and Iversen

(1975). In addition, hyperthermia induced by stimuli associated with

morphine has been found to be antagonized by naloxone (Lal et al. 1976).

All of these observations indicate that conditioned stimuli are capable of

224
eliciting neuropharmacological states similar to those produced by the drug

itself, Paradoxically, it would seem that these stimuli had acquired the

ability to release endogenous opiates in the CNS. On the other hand, it is

possible that conditioned stimuli, under certain circumstances, may acquire

the ability to produce effects that are similar to the UCS (drug) by acting

on pathways beyond the initial site of action of the drug.

NEUROANATOMICAL SUBSTRATES UNDERLYING

CONDITIONED SENSITIZATION

While dopamine appears to be critically involved in the acquisition of

conditioned locomotor behaviors in which the UCS is a psychomotor stimulant, little is known regarding the neuroanatomical substrates. One likely

candidate is nucleus accumbens, which has been shown to mediate the

excitatory (Kelly and Iversen 1976) as well as reinforcing effects (Roberts

et al. 1977) of cocaine as well as other psychomotor stimulants. Additional

structures that may play a role in such conditioning include the amygdala,

hippocampus, and cerebellum. The amygdala had been implicated in

mediating the acquisition of a variety of conditioned behaviors. Amygdala

lesions, for example, have been shown to produce deficits in the learning of

active avoidance (Brady et al. 1954; Coover et al. 1973; Goldstein 1974;

Ursin 1965) as well as passive avoidance (Coover et al. 1973; Grossman

et al. 1975; Slotnick 1973; Ursin 1965) behaviors. Deficits in these tasks

probably reflect disruption of conditioned fear mediated by the amygdala.

Lesions of the amygdala have also been found to attenuate the acquisition

of conditioned emotional responding (Kellicutt and Schwartzbaum 1967;

Spevack et al. 1975) as well as heart rate conditioning in rabbits (Kapp

et al. 1979). More recently, amygdalectomy has been reported to block the

potentiation of a startle response by stimuli that had been associated with

shock (Hitchcock and Davis 1986; Hitchcock and Davis 1987).

There is considerable evidence to indicate that the cerebellum may also be

involved in conditioned motor reactions. Lesions of the dentate and

interpositus nuclei of the cerebellum or superior cerebellar peduncle have

been found to disrupt the acquisition and expression of the conditioned

nictitating membrane response in rabbits (Lincoln et al. 1982; McCormick

et al. 1981; McCormick et al. 1982) and lesions of the deep cerebellar

nuclei have been shown to abolish conditioned leg-flexion responses in the

rabbit (Donegan et al. 1983). Lesions of the hippocampus have also been

found to disrupt the acquisition of a variety of learned behaviors (Isaacson

1974).

In order to evaluate the participation of these neural structures in the

conditioned aspects of sensitization, the authors lesioned each region and

observed the effect of this manipulation on the acquisition of conditioned

increases in locomotor output elicited by cocaine. In the first study, three

groups of rats were used. One group was lesioned bilaterally in the amyg225
dala with radiofrequency procedures, while the other group was injected

bilaterally in the nucleus accumbens with 6-hydroxydopamine. The third

group received sham lesions. The radiofrequency lesions destroyed a

substantial portion of the amygdala, including most of the basolateral and

corticomedial nuclei. Thc 6-OHDA lesion of the nucleus accumbens

decreased DA content in this structure by approximately 65 percent. The

1-day conditioning paradigm described above was used to assess the effect

of the lesions on context-dependent, cocaine-induced sensitization. On day

1, half of the animals in each group were injected with 40 mg/kg of

cocaine, while the other half received saline. On day 2, both subgroups

received 10 mg/kg of cocaine. Neither lesion on day 1 appeared to modify

the response to the high dose of cocaine (figure 6). This was somewhat

surprising, since 6-OHDA lesions have previously been found to attenuate

the motoric effects of cocaine (Kelly and Iversen 1975). The DA depletion

in our study, however, was considerably smaller than that reported by these

investigators. Apparently a considerable degree of dopaminergic destruction

is needed to attenuate cocaine-induced increase in locomotor output.

Although neither lesion had an effect on the initial response to cocaine,

both effectively prevented the appearance of context-dependent sensitization.

There was a clear difference in locomotor activity between the saline- and

cocaine-pretreated rats only for animals that had received sham lesions.

Some of these findings were confirmed and extended in a subsequent study.

Rats were lesioned in the amygdala with either radiofrequency procedures or

6-OIIDA injections that reduced the DA content by approximately

60 percent. These lesions also had relatively little effect on the initial

response to 40 mg/kg of cocaine on day 1. When tested on day 2 with

10 mg/kg of cocaine, both types of lesions were found to have prevented

the development of sensitization (figure 7). Thus, it appears that DA in the

amygdala is a critical neurotransmitter involved in the process by which

environmental, interoceptive and situational stimuli acquire the ability to

elicit conditioned responses similar to those produced by cocaine. Neither

dorsal and ventral hippocampal lesions (figure 8) nor lesions of the deep

cerebellar nuclei (data not shown) had a significant effect on cocaineinduced sensitization in this paradigm, although the dorsal hippocampal

lesion did appear to attenuate it to some degree.

The two primary structures involved in mediating context-dependent sensitization appear to be the amygdala and nucleus accumbens. It is likely that

these two structures mediate different aspects underlying the development of

this phenomenon (figure 9). It is probably unlikely that lesions of the

nucleus accumbens attenuate the conditioning process by interfering with

associative mechanisms. Wilson (1983) has, in fact, shown that lesions of

the nucleus accumbens actually enhance conditioned leg flexion and vocalization in the cat elicited by electrical shock delivered to the leg. Lesions

of the nucleus accumbens have also been shown to enhance the acquisition

226
of avoidance responses (Lorens et al. 1970) that are dependent on aversive

conditioning.

INHIBITION OF COCAINE SENSITIZATION BY

AMYGDALA AND NUCLEUS ACCUMBENS LESIONS

FIGURE 6. The effect of amygdala (radiofrequency) and nucleus

accumbens (6-OHDA) lesions on the development of

conditioned cocaine-induced sensitization

NOTE: The left side of the figure illustrates the effects of saline or cocaine (40 mg/kg, IP) injections

on horizontal activity in the three different lesion groups. Neither lesion appeared to have a

marked effect on cocaine-induced increases in locomotor output. When all animals were

tested on day 2 with 10 mg/kg of cocaine, it was apparent that the lesions were effective in

preventing the development of cocaine-induced sensitization.

Mogenson (1987), as well as Stevens (1973), has proposed that the nucleus

accumbens may serve to gate motivational input from limbic structures such

as the amygdala and hippocampus to the motor system. Presumably the

function of such a gating mechanism is to regulate how biologically significant stimuli gain access to the motor system. There is evidence that

227
AMYGDALA LESIONS BLOCK THE DIFFERENTIAL

IMPACT OF PRIOR COCAINE

FIGURE 7. The effect of radiofrequency and 6-OHDA lesions of the

amygdala on the development of cocaine-induced

sensitization

NOTE: The top part of the figure illustrates total horizontal activity in 30 minutes following injection

of saline or cocaine (40 mg/kg) to the various lesion groups. Neither radiofrequency- or 6-

OHDA-induced destruction of the amygdala had a marked effect on the initial response to

cociane. However, when all animals were tested with 10 mg/kg of cocaine on day 2

(bottom), it was apparent that both lesions had prevented the development of sensitization.

Thus, dopamine in the amygdala appears to be critical in mediating this behavior.

activation of mesolimbic DA neurons has a modulatory influence on the

transmission of information from the amygdala and hippocampus to the ventral pallidum via the nucleus accumbens. The ventral pallidum, in turn, is

thought to be involved in the control of motor behaviors through its connections with the mesencephalic locomotor region (Mogenson 1987). Thus,

DA input to the nucleus accumbens may serve to translate the motivational

228
determinants of behavior that are mediated by the limbic system into

biologically relevant actions. In this respect, mesolimbic DA may serve not

to mediate the hedonic quality of rewards (Wise 1982) but their ability to

arouse and activate behaviors (incentive motivation).

FIGURE 8. Effects of ventral and dorsal hippocampal lesions on cocaineinduced sensitization

NOTE: Left side of the figure illustrates the response of lesioned animals to the initial injection of

cocaine (40 mg/kg). Neither lesion appeared to have an effect on cocaine-induced

hyperactivity on day 1. On day 2 sensitization to 10 mg/kg of cocaine was still evident in

both lesion groups.

The amygdala is a structure that receives polysensory input from the cortex

and may be a focal point in the brain in which stimuli from all sensory

modalities are assigned an emotional valance (Mishkin et al. 1984).

Monkeys with amygdala lesions, for example, have been shown to be slow

to learn the association between a visual stimulus and reward (Aggleton and

Mishkin 1985). It may be that this is the brain region in which stimuli

gain either positive or negative association values (expectations).

229
FIGURE 9. Schematic illustrating some of the interrelationships between

structures that play a role in mediating the conditioned

components of cocaine sensitization

NOTE: The nucleus accumbens may serve to gate motivational input from limbic structures to motor

system. Dopamine input to the nucleus accumbens from the ventral tegmentum may be

involved in translating the motivational determinants of behavioral mediated by the limbic

system into biologically relevant actions. In the amygdala, mesolimbic DA might modulate

the attachment of emotional significance to a given stimulus.

Interestingly, the amygdala also receives DA input from the same mesencephalic perikaryal region (A-10) that provides DA innervation to the

nucleus accumbens. Dopaminergic pathways arising from the ventral tegmentum might have somewhat different functions in these two terminal

regions, although the behavioral endpoints are ultimately related. In the

amygdala, DA might modulate the attachment of emotional significance to

a given stimulus. In this sense, amygdala DA input might serve as a gating

mechanism to determine which stimuli gain access to structures efferent to

the amygdala including the nucleus accumbens. Dopamine in the nucleus

accumbens, on the other hand, serves to determine which limbic inputs gain

access to the motor pathways.

SUMMARY AND CONCLUSIONS

It is apparent that stimuli associated with psychomotor stimulants as well as

opiates acquire the ability to elicit motor behaviors that probably reflect the

230
acquisition and operation of incentive motivational processes. Such conditioning also appears to be a critical determinant of behavioral sensitization

seen with repetitive administration of these agents. The conditioning of

motor excitation to stimuli associated with psychomotor stimulants follows

the principles of classical conditioning and is relatively long lasting.

Dopaminergic mechanisms appear to be involved in the acquisition of such

conditioned behaviors, since neuroleptics are effective blockers of the

process. Dopaminergic blockade probably disrupts conditioning through

several different mechanisms including attenuation of the conditioned and

unconditioned excitatory properties of the CS and blockade of the US. DA

blockade prevents stimuli associated with psychomotor stimulants from

acquiring and subsequently generating positive affective motivational states

that are reflected by increases in motoric output. While dopamine appears

to be necessary for the formation of conditioned motor excitation, it is not

critically involved in the expression of the conditioned effects. This seems

to suggest that DA may serve only to modulate the formation of motivationally significant associations but is not involved in the expression of

conditioned drug effects that may be mediated through DA-independent

pathways.

The amygdala and nucleus accumbens are two structures in the CNS

involved in the acquisition of conditioned motor excitation. Interestingly,

both of these brain regions are the recipients of mesolimbic DA input.

Dopamine probably plays different roles in each region during the conditioning process. In the amygdala, mesolimbic DA may serve to modulate

processes that attach emotional significance to environmental stimuli; futher,

DA may play a role in determining which stimuli gain access to structures

afferent to the amygdala, including the nucleus accumbens. Dopamine in

the nucleus accumbens, on the other hand, serves to determine which limbic

inputs gain access to the motor pathways. In this way, DA in the nucleus

accumbens may translate the motivational determinants of behavior that are

mediated by limbic structures into biologically relevant actions.

Understanding the mechanisms that determine the conditioning of drug

effects to associated stimuli also has possible relevance for elucidating

processes underlying addictive behaviors. For example, it has been

proposed (Stewart et al. 1984) that the acquisition of incentive motivational

properties by stimuli associated with drugs determines the craving in

addicts. Craving may simply be the induction of incentive motivational

states by environmental stimuli that have been classically conditioned to

elicit physiological states resembling those produced by the drug itself. In

this context, it has been shown that conditioned stimuli have the power to

initiate drug-seeking and drug-taking behaviors or to reinitiate them

following abstinence (de Wit and Stewart 1981). Further analyses of

conditioned behaviors associated with psychomotor stimulant administration

will hopefully reveal new pharmacological and behavioral strategies for

extinguishing addictive behaviors.

231
REFERENCES

Aggleton, J.P., and Mishkin, M. The amygdala: Sensory gateway to the

emotions. In: Plutchik, R., and Kellerman, H., eds. Emotion: Theory,

Research and Experience. Vol. 3. Academic Press, 1985.

Anderson, D.C.; O’Farrell, T., Formica, R.; and Caponigri, V. Preconditioning CS exposure: Variation in place of conditioning and or

presentation. Psycho Sci 15:54-55, 1969.

Annau, Z., and Kamin, L.J. The conditioned emotional response as a

function of intensity of the U.S. J Comp Psychol 54:428-432, 16961.

Balles, R.C. Effect of food deprivation upon the rat’s behavior in its home

cage. J Comp Physiol Psychol 56:456-460, 1963.

Barnes, G.W. Conditioned stimulus intensity and temporal factors in

spaced-trial classical conditioning. J Exp Psychol 51:192-198, 1956.

Barr, G.A., Sharplus, N.S.; Cooper, S.; Schiff, S.R.; Paredes, W.; and

Bridges, W.H. Classical conditioning, decay and extinction of cocaineinduced hyperactivity and stereotypy. Life Sci 33:1341-1351, 1983.

Baumeister, A.; Hawkins, W.F.; and Cromwell, R.L. Need states and

activity levels. Psychol Bull 61:438-453, 1964.

Beck, E.C., and Doty, R.W. Conditioned flexion reflexes acquired during

combined catalepsy and de-efferentation. J Comp Physiol Psychol

50:211-216, 1957.

Beninger, R. The role of dopamine in locomotor activity and learning.

Brain Res Rev 6:173-196, 1983.

Beninger, R.J., and Hahn, B.L. Pimozide blocks establishment but not

expression of amphetamine-produced environment-specific conditioning.

Science 220:1304-1306, 1983.

Beninger, R.J.; Hanson, D.R.; and Phillips, A.G. The acquisition of

responding with conditioned reinforcement: Effects of cocaine, 2

amphetamine and piprodrol. Br J Pharmacol 74:149-154, 1981.

Beninger, R.J., and Herz, R.S. Pimozide blocks establishment but not

expression of cocaine-produced environment-specific conditioning. Life

Sci 38:1425-1431, 1986.

Beninger, R.J., and Phillips, A.G. The effect of pimozide on the establishment of conditioned reinforcement. Psychopharmacology 68:147-153,

1980a.

Beninger, R.J., and Phillips, A.G. The effects of pimozide during pairing

on the transfer of classical conditioning to an operant discrimination.

Pharmacol Biochem Behav 14:101-105, 1980b.

Bindra, D. Neuropsychological interpretation of the effects of drive and

incentive-motivation on general activity and instrumental behavior.

Psychol Rev 75:1-22, 1968.

Bolles, R.C. Effect of food deprivation upon the rat’s behavior in its home

cage. J Comp Physiol Psychol 56:456-460, 1963.

232
Brady, J.V.; Schreiver, L.; Geller, I.; and Kling, A. Subcortical mechanisms

in emotional behavior. The effect of rhinencephalic injury upon the

acquisition and retention of a conditioned avoidance response in cats.

J Comp Physiol Psychol 47:170-186, 1954.

Bridger, W.H.; Schiff, S.R.; Cooper, S.S.; Paredes, W.; and Barr, G.

Classical conditioning of cocaine’s stimulatory effects. Psychopharmacol

Bull 18:210-214, 1982.

Broekkamp, C.L.E.; Phillips, A.G.; and Cools, A.S. Stimulant effects of

enkephalin injection into the dopaminergic A10 area. Nature

278:560-562, 1979.

Browne, R.G., and Segal, D.S. Metabolic and experiential factors in the

behavioral response to repeated amphetamine. Pharmacol Biochem Behav

6:545-556, 1977.

Carey, R.J. Conditioned rotational behavior in rats with unilateral

6-hydroxydopamine lesions of the substantia nigra. Brain Res

365:379-382, 1986a.

Carey, R.J. A conditioned anti-parkinsonian drug effect in the hemiparkinsonian rat. Psychopharmacology 89:269-272, 1986b.

Carey, R.J., and Kenney, S. Operant conditioning and haloperidol-induced

hypokinetic effects. Neuropharnracology 18:199-204, 1987.

Carlton, P.L., and Vogel, J.R. Habituation and conditioning. J Comp

Physiol 63:348-351, 1967.

Casas, M.; Guix, T.; Prat, G.; Ferre, S.; Cadafalch, J.; and Jane, F.

Conditioning of rotational behavior after the administration of a single

dose of apomorphine in rats with unilateral denervation of the

dopaminergic nigrostriatal pathway: Relevance to drug addiction.

Pharmacol Biochem Behav 31:605-609, 1988.

Collins, K.H., and Tatum, A.L. A conditioned salivary reflex established by

chronic morphine poisoning. Am J Physiol 74:14-15, 1925.

Coover, G.; Ursin, H.; and Levine, S. Corticosterone and avoidance in rats

with basolateral amygdala lesions. J Comp Physiol Psychol 85:111-122,

1973.

Crisler, G. Salivation is unnecessary for the establishment of the salivary

conditioned reflex induced by morphine. Am J Physiol 94:553-556, 1930.

Davis, W.M., and Smith, S.G. Effect of haloperidol in (+)-amphetamine

self-administration. J Pharm Pharmacol 27:540-541, 1975.

Davis, W.M., and Smith, S.G. Role of conditioned reinforcers in the

initiation, maintenance and extinction of drug-seeking behavior. Pavlov J

Biol Sci 11:222-236, 1976.

Davis, W.M., and Smith, S.G. Catecholaminergic mechanisms of reinforcement: Direct assessment by drug self-administration. Life Sci

20:483-492, 1977.

de Wit, H., and Stewart, J. Reinstatement of cocaine-reinforced responding

in the rat. Psychopharmacology 75:134-143, 1981.

233
Di Chiara, G., and Imperato, A. Opposite effects of Mu and Kappa opiate

agonists on dopamine release in the nucleus accumbens and in the dorsal

caudate of freely moving rats. J Pharmacol Exp Ther 244:1067-1080,

1988.

Donegan, N.H.; Lowery, R.W.; and Thompson, R.F. Effects of lesioning

ccrebellar nuclei on conditioned leg-flexion responses. Soc Neurosci Abs

9:331, 1983.

Downs, A.W., and Eddy, N. The effect of repeated doses of cocaine on

the rat. J Pharmacol Exp Ther 46:199-200, 1932.

Drew, K.L.. and Glick, S.D. Classical conditioning of amphetamine-induced

lateralized and nonlateralized activity in rats. Psychopharmacology

92:52-57, 1987.

Drew, K.L., and Glick, S.D. Environment-dependent sensitization to

amphetamine-induced circling behavior. Pharmacol Biochem Behav

31:705-708, 1989.

Elias, J.W.; Yandell, L.R.; Graff, R.; Albrecht, J.W.; and Smith, C.J. Short

term augmentation of amphetamine-induced rotation bias. Physiol Behav

31:639-642, 1983.

Finch, G. Salivary conditioning in atropinized dogs. Am J Physiol

124:136-141, 1938.

Finger, F.W.; Reid, L.S.; and Warner, M.H. Activity changes as a

function of reinforcement under low rates. J Comp Physiol Psychol

53:385-387, 1960.

Glick, S.D.; Shapiro, R.M.; Drew, K.L.; Hinds, P.A.; and Carlson, J.N.

Differences in spontaneous and amphetamine induced rotational behavior,

and in sensitization to amphetamine, among Sprague-Dawley derived rats

from different sources. Physiol Behav 38:67-70, 1986.

Gold, L.H.; Swerdlow, N.R.; and Koob, G.F. The role of mesolimbic

dopamine in conditioned locomotion produced by amphetamine. Behav

Neurosci 102:544-552, 1988.

Goldstein, M.L. The effect of amygdalectomy on long-term retention of an

undertrained classically conditioned fear response. Bull Psychonomic Soc

4:545-550, 1974.

Grossman, S.P.; Grossman, L.; and Walsh, L. Functional organization of

the rat amygdala with respect to avoidance behavior. J Comp Physiol

Psychol 88:829-850, 1975.

Hadfield, M.G., and Nuggent, E.A. Cocaine: Comparative effect on

dopamine uptake in extrapyramidal and limbic systems. Biochem

Pharmacol 32:744-746, 1983.

Harvey, J.A., and Gormezano, I. Effects of halopctidol and pimozide on

classical conditioning of the rabbit nictitating membrane response.

J Pharmacol Exp Ther 218:712-719, 1981.

Hayashi, T.; Ohashi, K.; and Tadokara, S. Conditioned drug effects to

d-amphetamine- and morphine-induced motor acceleration in mice:

Experimental approach for placebo effect. Jpn J Pharmacol 30:93-100,

1980.

Hernstein, R.J. Placebo effect in the rat. Science 138:677-678, 1962.

234
Hinson, R.E., and Poulos, C.X. Sensitization to the behavioral effects of

cocaine: Modification by Pavlovial conditioning. Pharmacol Biochem

Behav 15:559-562, 1981.

Hinson, R.E., and Siegel, S. Anticipating hyperexcitability and tolerance to

the narcotizing effect of morphine in the rat. Behav Neurosci 97:759-767,

1983.

Hirabayashi, M., and Alam, M.R. Enhancing effect of methamphetamine on

ambulatory activity produced by repeated administration in mice.

Pharmacol Biochem Behav 15:925-932, 1981.

Hitchcock, J., and Davis, M. Lesions of the amygdala, but not of the

cerebellum or red nucleus, block conditioned fear as measured with the

potentiated startle paradigm. Behav Neurosci 100:11-22, 1986.

Hitchcock, J., and Davis, M. Fear-potentiated startle using an auditory

conditioned stimulus: Effect of lesions of the amygdala. Physiol Behav

39:403-408, 1987.

Irwin, S. The actions of drugs on psychomotor activity. Rev Canadian

Biol 20:239-250, 1961.

Irwin, S., and Armstrong, P.M. Conditioned locomotor response with drug

as the unconditioned stimulus: Individual differences. In: Rothlin, E.,

ed. Neuropsychopharmacology: Proceedings of the Second Meeting of

the Colleqium Internationale Neuro-psychopharmacologicum. Amsterdam:

Elsevier, 1961. pp. 151-157.

Isaacson, R.L. The Limbic System. New York: Plenum Press, 1974.

Joyce, E.M., and Iversen, S.D. The effect of morphine applied locally to

mesencephalic dopamine cell bodies on spontaneous motor activity in the

rat. Neurosci Lett 14:207-212, 1979.

Joyce, E.M.; Koob, G.F.; Shucker, R.; Iversen, S.D.; and Bloom, F. The

behavioral effects of enkephalin analogues injected into the ventral

tegmental area of the globus pollidus. Brain Res 221:359-370, 1981.

Kalivas, P.W.; Duffy, P.; DuMars, L.A.; and Skinner, C. Behavioral and

neurochemical effects of acute and daily cocaine administration in rats.

J Pharmacol Exp Ther 245:485-492, 1988.

Kalivas, P.W.; Widerlav, E.; Stanley, D.; Breese, G.R.; and Prange, A.J.

Enkephalin action on the mesolimbic system: A dopamine-dependent and

a dopamine-independent increase in locomotor activity. J Pharmacol Exp

Ther 227:229-237, 1983.

Kamat, K.A.; Dutta, S.N.; and Pradhan, S.N. Conditioning of morphineinduced enhancement of motor activity. Res Commun Chem Pathol

Pharmacol 7:367-373, 1974.

Kamin, L.J., and Brimer, C.J. The effects of intensity of conditioned and

unconditioned stimuli on a conditioned emotional response. Can J

Psychol 17:194-198, 1963.

Kamin, L.J., and Schaub, R.E. Effects of conditioned stimulus intensity on

the conditioned emotional response. J Comp Physiol Psychol 56:502-507,

1963.

235
Kapp, B.S.; Frysinger, R.C.; Gallagher, M.; and Haselton, J.R. Amygdala

central nucleus lesions: Effect on heart rate conditioning in the rabbit.

Physiol Behav 23:1109-1117, 1979.

Katz, R.J., and Gormezano, G. A rapid and inexpensive technique for

assessing the reinforcing effects of opiate drugs. Pharmacol Biochem

Behav 11:231-233, 1979.

Kelley, A.E.; Stinus, L; and Iversen, S.D. Interaction between

D-Ala-Met-enkephalin, A 10 dopaminergic neurons, and spontaneous

behavior in the rat. Behav Brain Res 1:3-24, 1980.

Kelly, P.H., and Iversen, S.D. Selective 6-OHDA-induced destruction of

mesolimbic dopamine neurons: Abolition of psychostimulant-induced

locomotor activity in rats. Am J Pharmacol 40:45-56, 1975.

Kellicutt, M.H., and Schmartzbaum, J.S. Formation of a conditioned

emotional response (CER) following lesions of the amygdaloid complex in

rats. Psychol Rev 12:351-358, 1967.

Key, B.J., and Bradley, P.B. The effects of drugs on conditioning and

habituation to arousal stimuli in animals. Psychopharmacologia

1:450-465 1960.

Killam, K.F, and Killam, E.K. Drug action on pathways involving the

reticular formation. In: Jasper, H.H.; Practor, L.D.; Knighton, S.S.;

Nashay, W.C.; and Catello, R.T., eds. Reticular Formation of the Brain.

Boston: Little Brown and Co., 1958.

Kleitman, N., and Crisler, G. A quantitative study of a salivary conditioned

reflex. Am J Physiol 79:571-614, 1927.

Kremer, E.F. Truly random and traditional control procedures in CER

conditioning in the rat. J Comp Physiol Psychol 76:441-448, 1971.

Kumar, R. Morphine dependence in rats: Secondary reinforcement from

environmental stimuli. Psychopharmacologia 25:332-338, 1972.

Lal, H.; Miksic, S.; and Smith, N. Naloxone antagonism of conditioned

hypothermia: An evidence for release of endogenous opioid. Life Sci

18:971-976, 1976.

Light, J.S., and Gantt, W.H. Essential part of reflex arc for establishment

of conditioned reflex. Formation of conditioned reflex after exclusion of

motor peripheral end. J Comp Psychol 21:19-36, 1936.

Lincoln, J.S.; McCormick, D.A.; and Thompson, R.F. Ipsilateral cerebellum

lesions prevent learning of the classicially conditioned nictitating

membrane/eyelid response. Brain Res 242:190-193, 1982.

Lorens, S.A.; Sorensen, J.P.; and Harvey, J.A. Lesions in the nucli

accumbens septi of the rat: Behavioral and neurochemical effects.

J Comp Physiol Psychol 73:284-290, 1970.

Loviband, D.F. Facilitation of instrumental behavior by a Pavlovian

appetitive conditioned stimulus. J Exp Psychol [Anim Behav] 9:225-247,

1983.

Lubow, R.E., and Moore, A.V. Latent inhibition: The effect of nonreinforced preexposure to the conditioned stimulus. J Comp Physiol

Psychol 52:415-519, 1959.

236
Magos, L. Persistence of the effect of amphetamine on sterotyped activity

in rats. Eur J Pharmacol 6:200-201, 1969.

McCormick, D.A.; Guyer, P.E.; and Thompson, R.F. Superior cerebellar

penduncle lesions selectively abolish the ipsilateral classically conditioned

nictitating membrane/eyelid response of the rabbit. Brain Res

244:347-350, 1982.

McCormick, D.A.; Lavand, D.G.; Clark, G.A.; Kettner, R.E.; Rising, C.E.;

and Thompson, R.F. The engram found? Role of the cerebellum in

classical conditioning of nictitating membrane and eyelid responses. Bull

Psychonomic Soc 18:103-105, 1981.

Mishkin, M.; Malamut, B.; and Bachevalier, J. Memories and habits: Two

neural systems. In: Lynch, G.; McGaugh, J.L.; and Weinberger, N.M.,

eds. Neurobiology of Learning and Memory. New York: Guilford

Press, 1984. pp. 65-77

Mogenson, G.J. Limbic-motor integration. Prog Psychobiol Physiol

Psychol 12:117-170, 1987.

Moore, K.E. The actions of amphetamine on neurotransmitters: A brief

review. Biol Psychiatry 12:451-462, 1977.

Mucha, R.F.; Valkavski, C.; and Kalant, H. Conditioned increases in

locomotor activity produced with morphine as an unconditioned stimulus,

and the relation of conditioning to acute morphine effect and tolerance.

J Comp Physiol Psychol 955:352-362, 1981.

Mucha, R.F.; Van Der Kooy, D.; O’Shaughnerry, M.; and Buceniehs, D.

Drug reinforcement studied by place conditioning in rat. Bruin Res

243:91-105, 1982.

Nelson, L.R., and Ellison, G. Enhanced stereotypics after repeated

injections but not continuous amphetamine. Neuropharmacology

17:1081-1084, 1978.

O’Brien, C.P. Experimental analysis of conditioning factors in human

narcotic addiction. Pharmacol Rev 27:533-534, 1976.

Ost, J.W.P., and Lauer, D.W. Some investigations of classical salivary

conditioning in the dog. In: Prokasy, W.F., ed. Classical Conditioning:

A Symposium. New York: Appleton-Century-Crafts, 1965. pp. 192-207.

Pavlov, I.P. Conditioned Reflexes: An Investigation of the Physiological

Activity of the Cerebral Cortex. New York: Dover Publications, 1927.

Perez-Cruet, J. Conditioning of striatal dopamine metabolism with

methadone, morphine or bulbocapnine as an unconditioned stimulus.

Pavlov J Biol Sci 11:237-250, 1976.

Perez-Cruet, J., and Gantt, W.H. Conditioned catalepsy to bulbocapnine:

Cardiac, respiratory and motor components. Fed Proc:467, 1959.

Pickens, R.W., and Crowder, W.F. Effects of CS-US interval on

conditioning of drug response, with assessment of speed of conditioning.

Psychopharmacologic 11:88-94, 1967.

Pickens, R., and Dougherty, J.D. Conditioning of the activity effects of

drugs. In: Thompson, T., and Schuster, C., eds. Stimulus Properties of

Drugs. New York: Appleton-Century-Crafts, 1971. pp. 39-50.

237
Post, R.M. Intermittent versus continuous stimulation: Effect of time

interval on the development of sensitization or tolerance. Life Sci

26:1275-1282, 1980.

Post, R.M. Central stimulants: Clinical and experimental evidence on

tolerance and sensitization. In: Israel, Y.; Glaser, F.B.; Kalant, H.;

Popham, R.E.; Schmidt, W.; and Smart, R.G., eds. Research Advances in

Alcohol and Drug Problems. New York: Plenum Press, 1981.

Post, R.M., and Ballenger, J.C. Kindling models for the progressive

development of behavioral psychopathology: Sensitization to electrical,

pharmacological, and psychological stimuli. In: Van Prang, H.M.; Lale,

M.H.; Rafaelien, O.J.; Sachar, E.J., eds. Handbook of Biological

Psychiatry, Part IV. New York: Marcel Dekker, 1981.

Post, R.M., and Contel, N.R. Human and animal studies of cocaine:

Implications for development of behavioral pathology. In: Creed, I., ed.

Stimulants: Neurochemical Behavioral, and Clinical Perspective. New

York: Raven Press, 1983.

Post, R.M., and Kopanda, R.T. Cocaine, kindling, and psychosis. Am J

Psychiatry 133:627-634, 1976.

Post, R.M.; Lockfeld, A.; Squillace, K.M.; and Contel, N.R. Drugenvironment interaction: Control dependency of cocaine-induced

behavioral sensitization. Life Sci 28:755-760, 1981.

Post, R.M., and Rose, H. Increasing effects of repetitive cocaine

administration in the rat. Nature 260:731-732, 1976.

Post, R.M.; Weiss, S.R.B.; and Pert, A. Cocaine-induced behavioral

sensitization and kindling: Implications for the emergence of

psychopathology and seizures. Ann NY Acad Sci 537:292-308, 1988.

Rebec, G.V., and Segal, D.S. Enhanced responsiveness to intraventricular

infusion of amphetamine following its repeated systemic administration.

Psychopharmacology 62:101-102, 1979.

Reicher, M.A., and Holman, E.W. Location preference and flavor aversion

reinforced by amphetamine in rats. Anim Learn Behav 5:343-346, 1977.

Reith, M.E.A.; Meisler, B.E.; Sershen, H.; and Iajtha, A. Structural

requirements for cocaine congemers to interact with dopamine and

serotonin uptake sites in mouse brain and to induce stereotyped behavior.

Biochem Pharmacol 35:1123-1129, 1986.

Rescorla, R.A., and Solomon, R.L. Two-process learning theory: Relationships between Pavlovian conditioning and instrumental learning. Psychol

Rev 74:151-152, 1967.

Riffee, W.H.; Wanek, E.; and Wilcox, R.E. Apomorphine fails to inhibit

cocaine-induced behavioral hypersensitivity. Pharmacol Biochem Behav

29:239-242, 1988.

Roberts, D.C.S.; Corcoran, M.E.; and Fibiger, H.C. On the rate of

ascending catecholaminergic systems in intravenous self-administration of

cocaine. Pharmacol Biochem Behav 6:615-620, 1977.

238
Robinson, T.E. Behavioral sensitization: Characterization of enduring

changes in rotational behavior produced by intermittent injections of

amphetamine in male and female rats. Psychopharmacology 84:466-475,

1984.

Robinson, T.E., and Becker, J.B. Enduring changes in brain and behavior

produced by chronic amphetamine administration: A review and

evaluation of animal models of amphetamine psychosis. Brain Res Rev

11:157-198, 1986.

Robinson, T.E.; Becker, J.B.; and Presty, S.K. Long-term facilitation of

amphetamine-induced rotational behavior and striatal dopamine release

produced by a single exposure to amphetamine: Sex differences. Brain

Res 253:231-241, 1982.

Roffman, M.; Reddy, C.; and Lal, H. Control of morphine-withdrawal

hypothermia by conditioned stimuli. Psychopharmacologia 29:197-201,

1973.

Ross, S., and Schnitzer, S.B. Further support for a placebo effect in the

rat. Psychol Rep 13:461-462, 1963.

Rossi, N.A., and Reid, L.D. Affective states associated with morphine

injections. Physiol Psychol 4:269-274, 1976.

Rushton, R.; Steinberg, H.; and Tinson, C. Effects of a single experience

on subsequent reactions to drugs. Br J Pharmacol 20:99-105, 1963.

Schiff, S.R. Conditioned dopaminergic activity. Biol Psychiatry

17:135-154, 1982.

Schindler, C.W.; Garmizano, I.; and Harvey, J.A. Effects of drugs on

classical conditioning. Behavioral Pharmacology: The Current Status.

New York: Alan Liss, Inc., 1985. pp. 55-71.

Schuster, C.R., and Woods, J.H. The conditioned reinforcing effects of

stimuli associated with morphine reinforcement. Int J Addict 3:223-230,

1968.

Segal, D.S., and Mandell, A.J. Long-term administration of d-amphetamine:

Progressive augmentation of motor activity and stereotypy. Pharmacol

Biochem Behav 2:249-255, 1974.

Sheffield, ED., and Campbell, B.A. The role of experience in the

“spontaneous” activity of hungry rats. J Comp Physiol Psychol

47:97-100, 1954.

Sherman, J.E.; Roberts, T.; Raskam, S.E.; and Holman, E.W. Temporal

properties of the rewarding and aversive effects of amphetamine in rats.

Pharmacol Biochem Behav 13:597-599, 1980.

Shuster, L; Yu, G.; and Bates, A. Sensitization to cocaine stimulation in

mice. Psychopharmacology 52:185-190, 1977.

Siegel, S. Effect of CS habituation on eyelid conditioning. J Comp

Physiol Psychol 688:245-248, 1969.

Slotnick, B.M. Fear behavior and passive avoidance deficits in mice with

amygdala lesions. Physiol Behav 11:717-720, 1973.

Solomon, R.L., and Turner, L.H. Discriminative classical conditioning in

dogs paralyzed by curare can later control discriminative avoidance

responses in the normal state. Psychol Rev 69:202-219, 1962.

239
Spence, K.W. Behavior Theory and Conditioning. New Haven: Yale

University Press, 1956.

Spevack, A.A.; Campbell, C.T, and Drake, L. Effect of amygdalectomy on

habituation and CER in rats. Physiol Behav 15:199-207, 1975.

Stevens, J.R. Psychomotor epilepsy and schizophrenia: A common

anatomy? In: Brazier, M.A.B., ed. Phenomenon in Man. New York:

Academic Press, 1973.

Stewart, J. Conditioned and unconditioned drug effects in relapse to opiate

and stimulant drug self-administration. Prog Neuropsychopharmacol Biol

Psychiatry 7:591-597, 1983.

Stewart, J.; de Wit, H.; and Eikelboom, R. Role of unconditioned and

conditioned drug effects in the self-adminstration of opiates and

stimulants. Psychol Res 91:251-268, 1984.

Stewart, J., and Eikelboom, R. Conditioned drug effects. In: Iversen,

L.L.; Iversen, S.D.; and Snyder, S.H., eds. Handbook of

Psychopharmacology. Vol. 19. New York: Plenum Press, 1987.

pp. 1-57.

Stripling, J.S., and Ellinwood, E.H., Jr. Sensitization to cocaine following

chronic administration in the rat. In: Ellinwood, E.H., and Kilbey,

M.M., eds. Cocaine and Other Stimulants. New York: Plenum Press,

1977. pp. 327-351.

Swerdlow, N.R., and Koob, G.F. Restrained rats learn amphetamineconditioned locomotion, but not place preference. Psychopharmacology

84:163-166, 1984.

Tatum, A.L., and Seevers, M.H. Experimental cocaine addiction.

J Pharmacol Exp Ther 36:401-403, 1929.

Tilson, H.A., and Rech, R.H. Conditioned drug effects and absence of

tolerance to d-amphetamine induced motor activity. Pharmacol Biochem

Behav 1:149-153, 1973.

Tye, N.C., and Iversen, S.D. Some behavioral signs of morphine

withdrawal blocked by conditioned stimuli. Nature 255:416-418, 1975.

Ursin, H. Effect of amygdaloid lesions on avoidance behavior and visual

discrimination in cats. Exp Neurol 11:298-317, 1965.

Van Der Kooy, D.; Mucha, R.F.; O’Shaughnessy, M.; and Buceniehs, P.

Reinforcing effects of brain microinjections of morphine revealed by

conditioned place preference. Brain Res 243:107-117, 1982.

Wagner, H.B.; Schiff, S.R.; Cooper, S.S.; Pardes, W.; and Barr, G.A.

Classical conditioning of cocaine’s stimulatory effects. Psychopharmacol

Bull 18:210-214, 1982.

Weiss, S.R.B.; Post, R.M.; Pert, A.; Woodward, R.; and Murman, D. Role

of conditioning in cocaine-induced behavioral sensitization. Pharmacol

Biochem Behav, in press.

Wilson, W.J. Nucleus accumbens inhibits specific motor but not

nonspecific classically conditioned responses. Brain Res Bull 10:505-515,

1983.

240
AUTHORS

Agu Pert, Ph.D.

Robert Post, M.D.

Susan R.B. Weiss, Ph.D.

Biological Psychiatry Branch

National Institute of Mental Health

Bethesda, MD 20892

241
monograph series

While limited supplies last, single copies of the monographs may be

obtained free of charge from the National Clearinghouse for Alcohol and

Drug Information (NCADI). Please contact NCADI also for information

about availability of coming issues and other publications of the National

Institute on Drug Abuse relevant to drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service (NTIS) as

indicated. NTIS prices are for paper copy; add $3.00 handling charge for

each order. Microfiche copies are also available from NTIS. Prices from

either source are subject to change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville. MD 20852

GPO

Superintendent of Documents

U.S. Government Printing Office

Washington, DC 20402

NTIS

National Technical Information Service

U.S. Department of Commerce

Springfield, VA 22161

(703) 487-4650

For information on availability of National Institute on Drug Abuse

Research Monographs 1 through 24 (1975-1979) and others not listed

write to NIDA, Community and Professional Education Branch,

Room 10A-54, 5600 Fishers Lane, Rockville, MD 20857.

242
25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE

ABUSE. Norman A. Krasnegor, Ph.D., ed.

GPO out of stock NCADI out of stock

NTIS PB #80-112428/AS $31

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor,

Ph.D., ed. (Reprint from 1979 Surgeon General’s Report on Smoking and

Health.)

GPO out of stock NTIS PB #80-118755/AS $23

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY

PERSPECTIVES. Dan J. Lettieri, Ph.D.; Mollie Sayers; and Helen W. Pearson,

eds.

GPO Stock #017-024-00997-1 NCADI out of stock

Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

ed.

GPO out of stock NTIS PB #80-215171/AS $31

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L.

Foltz, Ph.D.; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz.

GPO out of stock NCADI out of stock

NTIS PB #81-133746/AS $31

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A

REVIEW OF MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS.

Lorenz K.Y. Ng, M.D., ed.

GPO out of stock NCADI out of stock

NTIS PB #81-240913/AS $31

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE.

Travis Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

GPO out of stock NCADI out of stock

NTIS PB #82-136961/AS $39

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri,

Ph.D., and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO out of stock NCADI out of stock

NTIS PB #82-148198/AS $23

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES.

Herbert Hendin, M.D.; Ann Pollinger, Ph.D.; Richard Ulman, Ph.D.; and

Arthur Carr, Ph.D., eds.

GPO out of stock NCADI out of stock

NTIS PB #82-133117/AS $23

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS.

Richard L. Hawks, Ph.D., ed.

GPO out of stock NTIS PB #83-136044/AS $23

243
44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE

SYSTEMS. Monique C. Braude, Ph.D., and Jacqueline P. Ludford, M.S., eds.

A RAUS Review Report.

GPO out of stock NCADI out of stock

NTIS PB #85-150563/AS $23

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983.

Roger M. Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report.

GPO out of stock NCADI out of stock

NTIS PB #84-184670/AS $17

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE

TREATMENT. John Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D.; and Jack E.

Henningfield, Ph.D., eds.

GPO out of stock NCADI out of stock

NTIS PB #84-184688/AS $23

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION

STRATEGIES. Thomas J. Glynn, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01180-1 $5.50 NCADI out of stock

NTIS PB #85-159663/AS $31

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF

SMOKING BEHAVIOR. John Grabowski, Ph.D., and Catherine Bell, M.S.,

eds.

GPO Stock #017-024-01181-9 $4.50 NCADI out of stock

NTIS PB #84-145184/AS $23

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF

ABUSE. John Grabowski, Ph.D., ed.

GPO Stock #017-024-01214-9 $4 NTIS PB #85-150381/AS $23

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES,

PROGRESS, AND PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-024-01218-1 $4.50 NTIS PB #85-150365/AS $23

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 $23

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION.

John Grabowski, Ph.D., and Sharon M. Hall, Ph.D., eds.

GPO Stock #017-024-01266-1 $3.50 NCADI out of stock

NTIS PB #89-123186/AS $23

244
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm.

Sharp, Ph.D., ed.

GPO out of stock NCADI out of stock

NTIS PB #89-103279/AS $39

5.5 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 out of stock NCADI out of stock

NTIS PB #89-123194/AS $45

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 NTIS PB #89-123160/AS $31

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 NTIS PB #88-248083/AS $23

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 NTIS PB #89-125017/AS $23

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 NTIS PB #89-125025/AS $23

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS.

C. Nora Chiang, Ph.D., and Charles C. Lee, Ph.D., eds.

GPO Stock #017-024-01257-2 $3.50 NTIS PB #89-124564/AS $23

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECTIVES. Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S., eds.

GPO Stock #017-024-01258-1 $5 NTIS PB N39-131866/AS $31

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M.

Brown, Ph.D., and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01260-2 $3.50 NCADI out of stock

NTIS PB #89-130660/AS $23

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG

CHILDREN AND ADOLESCENTS. Catherine S. Bell, M.S., and Robert J.

Battjes, D.S.W., eds.

GPO Stock #017-024-01263-7 $5.50 NTIS PB #89-103287/AS $31

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

GPO Stock #017-024-01281-5 $6.50 NTIS PB #89-131858/AS $31

245
65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A.

Ray, Ph.D., and Monique C. Braude, Ph.D., eds.

GPO Stock #017-024-01283-1 $3.50 NTIS PB #89-130637/AS $23

66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND

ALCOHOLISM. Monique C. Braude, Ph.D., and Helen M. Chao, Ph.D., eds.

GPO Stock #017-024-01291-2 $3.50 NCADI out of stock

NTIS PB #89-134423/AS $23

68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS

OF ABUSE. Monique C. Braude, Ph.D., and Harold M. Ginzburg, M.D., J.D.,

eds.

GPO Stock #017-024-01296-3 $6.50 NCADI out of stock

NTIS PB #89-134936/AS $31

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

Gene Bamett, Ph.D.; and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1297-1 $11 NTIS PB #89-158422/AS $39

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY, BIOSYNTHESIS, AND ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D.,

eds.

GPO Stock #017-024-1298-0 $12 NTIS PB #89-158430/AS $45

71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M.

Brown, Ph.D.; Doris H. Clouet, Ph.D.; and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01303-0 $6 NTIS PB #89-151955/AS $31

72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims, Ph.D.,

and Carl G. Leukefeld, D.S.W., eds.

GPO Stock #017-024-01302-1 $6 NTIS PB #89-151963/AS $31

73 URINE TESTING FOR DRUGS OF ABUSE. Richard L. Hawks, Ph.D.,

and C. Nora Chiang, Ph.D., eds.

GPO Stock #017-024-01313-7 $3.75 NTIS PB #89-151971/AS $23

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

Stephen I. Szara, M.D., D.Sc., ed.

GPO Stock #017-024-1314-5 $3.75 NTIS PB #89-151989/AS $23

75 PROGRESS IN OPIOID RESEARCH. PROCEEDINGS OF THE 1986

INTERNATIONAL NARCOTICS RESEARCH CONFERENCE. John W.

Holaday, Ph.D.; Ping-Yee Law, Ph.D.; and Albert Herz, M.D., eds.

GPO Stock #017-024-01315-3 $21 NCADI out of stock

Not available from NTIS

246
76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF

THE 48TH ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01316-1 $16 NCADI out of stock

NTIS PB #88-208111/AS $53

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT

RESEARCH. Elizabeth R. Rahdert, Ph.D., and John Grabowski, Ph.D., eds.

GPO Stock #017-024-01348-0 $4 NTIS PB #89-125488/AS $23

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO DRUGS.

David P. Friedman. Ph.D., and Doris H. Clouet, Ph.D., eds.

GPO Stock #017-024-01330-7 $6 NTIS PB #88-245683/AS $31

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE CANNABINOIDS.

Rao S. Rapaka, Ph.D., and Alexandra Makriyannis, Ph.D., eds.

GPO Stock #017-024-01331-5 $6 NTIS PB #89-109201/AS $31

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

NATIONAL AND INTERNATIONAL PERSPECTIVES. Robert J. Battjea,

D.S.W., and Roy W. Pickens, Ph.D., eds.

GPO Stock #017-024-01345-5 $5.50 NTIS PB #88-236138/AS $31

81 PROBLEMS OF DRUG DEPENDENCE, 1987. PROCEEDINGS OF

THE 49TH ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01354-4 $17 NTIS PB #89-109227/AS

Contact NTIS for price

82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01344-7 $4.25 NTIS PB #88-245691/AS $23

83 HEALTH HAZARDS OF NITRITE INHALANTS. Harry W. Haverkos,

M.D., and John A. Dougherty, Ph.D., eds.

GPO Stock #017-024-01351-0 $3.25 NTIS PB #89-125496/AS $23

84 LEARNING FACTORS IN SUBSTANCE ABUSE. Barbara A. Ray,

Ph.D., ed.

GPO Stock #017-024-01353-6 $6 NTIS PB #89-125504/AS $31

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE. Raquel A.

Crider, Ph.D., and Beatrice A. Rouse, Ph.D., eds.

GPO Stock #017-024-01360-9 $5.50 NTIS PB #89-123178/AS $31

86 COMPULSORY TREATMENT OF DRUG ABUSE: RESEARCH AND

CLINICAL PRACTICE. Carl G. Leukefeld, D.S.W., and Frank M. Tims,

Ph.D., eds.

GPO Stock #017-024-01352-8 $7.50 NTIS PB #89-151997/AS $31

247
87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and Bhola N.

Dhawan, M.D., eds.

GPO Stock #017-024-01366-8 $7 NTIS PB #89-158430/AS $45

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H.

Clouet, Ph.D.; Khursheed Asghar, Ph.D.; and Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01359-5 $11 NTIS PB #89-125512/AS $39

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE. Roy W. Pickens,

Ph.D., and Dace S. Svikis, B.A., eds.

GPO Stock #017-022-01054-2 $5 NTIS PB #89-125520/AS $23

90 PROBLEMS OF DRUG DEPENDENCE, 1988. PROCEEDINGS OF

THE 50TH ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01362-5 $17

91 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION

DATA. Steven W. Gost, Ph.D., and J. Michael Walsh, Ph.D., eds.

GPO Stock #017-024-01384-6 $10 NTIS PB #90-147257/AS $39

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

GPO Stock #017-024-01379-0 $12 NTIS PB #90-148933/AS $45

93 AIDS AND INTRAVENOUS DRUG USE: FUTURE DIRECTIONS FOR

COMMUNITY-BASED PREVENTION RESEARCH. C.G. Leukefeld, D.S.W.;

R.J. Battjes, D.S.W.; and Z. Amsel, Ph.D., eds.

GPO Stock #017-024-01388-9 $10 NTIS PB #90-48941/AS $39

94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND

RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D.; Errol De Souza,

Ph.D., eds.

GPO Stock #017-024-01386-2 $11 NTIS PB #90-148958/AS $39

IN PRESS:

95 PROBLEMS OF DRUG DEPENDENCE, 1989. PROCEEDINGS OF

THE 51ST ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY, AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D.,

edS.

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY.

Lynda Erinoff. ed.

98 THE COLLECTION AND INTERPRETATION OF DATA FROM

HIDDEN POPULATIONS. Elizabeth Y. Lambert, M.S., ed.

248

U.S. GOVERNMENT PRINTING OFFICE: 1990 -0- 270-469
DHHS Publication No. (ADM) 90-1677

Alchohol, Drug Abuse, and Mental Health Administration

Printed 1990