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Modulation of hyperactivity and reward by excitatory , , and receptors in

Layer, Richard Thomas, Ph.D.

The Ohio State University, 1992

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 MODULATION OF HYPERACTIVITY AND REWARD

BY EXCITATORY AMINO ACID, SEROTONIN, AND OPIATE RECEPTORS

IN NUCLEUS ACCUMBENS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Richard T. Layer, B.A., M.Sc.

The Ohio State University

1992

Dissertation Committee: Approved by

Dennis R. Feller, Ph.D

Norman J. Uretsky, Ph.D Acjyisor Lane J. Wallace, Ph.D. College of Pharmacy The Secret Sits

We dance round in a ring and suppose, But the Secret sits in the middle and knows.

-Robert Frost To Lynda, my partner, who stoically endured both my rantings and ravings, and the chaotic appearance of our home while this dissertation was in preparation, and who loved me throughout. I couldn’t have done it without you. ACKNOWLEDGEMENTS

I would like to thank my fellow students, past and present, comrades all. Together we shared the slings and arrows of outrageous fortune.

I also would like to thank Bob Warwick, friend and advisor, from whom I learned the basics of and banjo picking.

I would like to express my appreciation to my committee, Dennis R. Feller, and especially Norman J. Uretsky. Your humor, guidance and advice were invaluable.

I would like to especially thank my mentor, Lane J. Wallace, for his advice, insight, generosity, and friendship. You gave me the right combination of freedom and direction, and I am grateful. From you I have learned much, including, among other things, the beauty of a good fade away jump shot.

Finally, and most importantly, I would like to thank my parents, Rick and Carol, for their love and support. More than any others, you made my dream possible. VITA

July 7,1964 ...... Born - Abington, Pennsylvania

1986 ...... B.A. in Biology, Gettysburg College Gettysburg, PA

1986-1989 ...... Graduate Teaching Assistant, Philadelphia College of Pharmacy & Science, Philadelphia, PA

1990 ...... M.Sc. in Pharmacology, Philadelphia College of Pharmacy & Science, Philadelphia, PA

1990-1992 ...... Graduate Teaching Associate, College of Pharmacy, The Ohio State University, Columbus, OH

PUBLICATIONS

Layer, R.T., and R.O. Warwick. Contrasting effects of enalaprilat and phosphoramidon on bombesin-induced hypothermia in the rat. (1988) Society for Neurocience Abstract 14: 60.1.

Hill, R., R. Layer, L. Wallace, T. Farooqui, N. Uretsky, and D. Miller. Synthesis and preliminary biological evaluation of substituted o-tyrosines as potential quisqualate analogs. (1989) American Association of Pharmaceutical Scientists Meeting.

Layer, R.T., N.J. Uretsky, and L.J. Wallace. Competition between motor activating psychostimulants and injected into the nucleus accumbens. (1990) The FASEB Journal 4: 4130.

iv Layer, R.T., N.J. Uretsky, and L.J. Wallace. Effects of intra-accumbens of the glutamate antagonist DNQX on place preference and locomotor activity induced by . (1991) Society for Neurocience Abstract 17: 567.14.

Layer, R.T., N.J. Uretsky, and L.J. Wallace. Effects of Morphine in the Nucleus Accumbens on Stimulant-Induced Locomotion. (1991) Pharmacology, Biochemistry, and Behavior 40: 21-26.

Layer, R.T., N.J. Uretsky, and L.J. Wallace. Effect of in the Nucleus Accumbens on d-Amphetamine-lnduced Locomotion. (1991) Life Sciences 50: 813-820.

FIELDS OF STUDY

Major field: Pharmacy Studies in: Pharmacology

v TABLE OF CONTENTS

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... Hi

VITA ...... iv

LIST OF TA BLES...... x

LIST OF FIG U R E S...... xi

CHAPTER I INTRODUCTION...... 1

THEORY AND TERMINOLOGY...... 2

METHODS OF ASSESSING REW A RD...... 6

OPERANT REINFORCEMENT TECHNIQUES ...... 7 SELF-ADMINISTRATION...... 7 FACILITATION OF ELECTRICAL STIMULATION ...... 9 DRUG DISCRIMINATION ...... 9

NON-OPERANT TECHNIQUES...... 10 CONDITIONED PLACE PREFERENCE ...... 11 CONDITIONED REINFORCEMENT...... 16

THE LOCOMOTOR ASSAY AND ITS RELATIONSHIP TO REW A RD ...... 16

NEURAL SUBSTRATE OF DRUG REWARD AND LOCOMOTOR PHARMACOLOGY...... 18

MOTIVATION AND DRIVE ...... 19

vi ANATOMY OF THE REWARD SUBSTRATE 20

THE NUCLEUS ACCUMBENS...... 23

ROLE OF IN NUCLEUS ACCUMBENS ...... 30 ANATOMY OF DOPAMINERGIC PR O JEC TIO N S 30 BIOCHEMICAL PHARMACOLOGY OF DOPAMINE . . 31

ELECTROPHYSIOLOGY OF DOPAMINE IN NUCLEUS ACCUMBENS...... 32 ROLE OF NUCLEUS ACCUMBENS DOPAMINE IN BEHAVIOR...... 33

INTERACTION WITH OF ABUSE WITH THE REWARD SUBSTRATE...... 35

PSYCHOSTIMULANTS...... 35

OPIATES ...... 38

ETHANOL ...... 41

NICOTINE ...... 41

A9- ...... 42

OTHER SYSTEMS INVOLVED IN REWARD AND LOCOMOTION...... 43

PHARMACOLOGY OF THE EXCITATORY AMINO ACID SYSTEM ...... 43 ENDOGENOUS ...... 43 RECEPTORS AND PHARMACOLOGY...... 44 BEHAVIORAL FUNCTIONS OF THE SYSTEM 52

PHARMACOLOGY OF ...... 53 ENDOGENOUS LIGANDS...... 53 RECEPTORS AND PHARMACOLOGY...... 54 BEHAVIORAL FUNCTIONS OF THE SYSTEM 54

PHARMACOLOGY OF THE SEROTONIN SYSTEM ...... 56 ENDOGENOUS LIGANDS...... 56 RECEPTORS AND PHARMACOLOGY...... 56 BEHAVIORAL FUNCTIONS OF THE SYSTEM 58

vii STATEMENT OF THE PROBLEM 60

CHAPTER II THE ROLE OF EXCITATORY AMINO ACIDS IN DRUG REWARD ...... 63

THE NMDA MK-801 ELICITS CONDITIONED PLACE PREFERENCE IN R A TS ...... 64

INTRODUCTION...... 64

M ETH O D S ...... 65

RESULTS...... 67

DISCUSSION ...... 67

A ROLE FOR AMPA/KAINATE RECEPTORS IN THE NUCLEUS ACCUMBENS IN DRUG INDUCED REWARD ...... 72

INTRODUCTION...... 72

METHODS...... 75 ANIMALS...... 75 SURGERY ...... 76 DRUGS ...... 76 CONDITIONED PLACE PREFERENCE PROCEDURE 77 CATALEPSY T E ST ...... 78 HISTOLOGY...... 79 STATISTICS...... 79

RESULTS...... 80

DISCUSSION...... 94

EFFECTS OF DNQX ON PLACE PREFERENCE ACQUISITION...... 95 EFFECTS OF DNQX ON PLACE PREFERENCE EXPRESSION...... 96 EFFECTS OF DNQX ON AMPHETAMINE LOCOMOTOR ACTIVITY...... 97 EFFECTS OF DNQX ON MORPHINE MOTOR EFFECTS AND CATALEPSY...... 98

viii CHAPTER III EFFECTS OF MORPHINE IN THE NUCLEUS ACCUMBENS ON STIMULANT-INDUCED LOCOMOTION ...... 101

INTRODUCTION...... 101

METHOD ...... 105 MEASUREMENT OF LOCOMOTOR ACTIVITY 105 HISTOLOGY...... 106 DRUGS ...... 106 STATISTICS...... 107

RESULTS...... 107 EFFECTS OF MORPHINE AND DTLET ON NORMAL LOCOMOTOR ACTIVITY IN THE R A T ...... 107 EFFECTS OF MORPHINE ON AMPHETAMINE- AND AMPA-STIMULATED LOCOMOTOR ACTIVITY IN THE R A T ...... 108 EFFECTS OF MORPHINE ON OTHER AGENTS THAT STIMULATE LOCOMOTOR ACTIVITY IN THE R A T ...... 109

DISCUSSION...... 121

CHAPTER IV EFFECT OF SEROTONERGIC AGONISTS IN THE NUCLEUS ACCUMBENS ON d-AMPHETAMINE-STIMULATED LOCOMOTION...... 127

METHODS...... 129 MEASUREMENT OF LOCOMOTOR ACTIVITY...... 129 HISTOLOGY...... 130 DRUGS ...... 131 STATISTICS...... 131

RESULTS...... 132

DISCUSSION...... 137

CHAPTER V SUMMARY AND CONCLUSIONS...... 144

SUMMARY 144 A SIMPLE MODEL ...... 147

IMPLICATIONS FOR CLINICAL TREATMENT OF DRUG ADDICTION...... 152

APPENDIX...... 156

LIST OF R E FE R EN C ES...... 159

x LIST OF TABLES

TABLE PAGE

1. Pharmacological Classification of Excitatory Amino Acid R e c e p to rs ...... 45

2. Pharmacological Classification of Receptors .55

3. Pharmacological Classification of 5-HT Receptors ..58

xi LIST OF FIGURES

FIGURE PAGE

1. The conditioned place preference apparatus ...... 12

2. The conditioned place preference paradigm ...... 15

3. The proposed reward c irc u it ...... 22

4. The nucleus accumbens ...... 26

5. Place preference elicited by MK-801 and d-amphetamine paired with the less preferred compartment ...... 68

6. Locomotor activity elicited during conditioning in the same rats by MK-801, d-amphetamine, or saline ...... 69

7. The effect of DNQX on place preference elicited by only two pairings with d-amphetamine ...... 82

8. The effect of DNQX simultaneously administered into nucleus accumbens on the locomotor activity elicited by d-amphetamine 83

9. Effects of DNQX and AFQX on the aquisition of an amphetamine-induced conditioned place preference ...... 84

10. Effects of DNQX and AFQX on the locomotor activity induced by amphetamine during conditioned place preference training ...... 85

11. Effects of DNQX and AFQX on the expression of an amphetamine-induced conditioned place preference ...... 86

12. Effects of DNQX and AFQX on locomotor activity during the expression of an amphetamine-induced conditioned place p referen ce...... 87

xii 13. Effect of DNQX on the expression of morphine- induced place preference ...... 88

14. Effect of DNQX on the aquisition of morphine- induced place preference ...... 89

15. Effects of DNQX on the locomotor activity induced by morphine during conditioned place preference train in g ...... 90

16. Effects of DNQX on locomotor activity during the expression of a morphine-induced conditioned place preference ...... 91

17. Effects of DNQX on the catalepsy induced by morphine during conditioned place preference training ...... 92

18. Unilateral administration of AMPA into the nucleus accumbens does not elicit a conditioned place preference ...... 93

19. Effects of morphine and DTLET on locomotor activity in the rat after bilateral injection into the nucleus accumbens ...... 111

20. Morphine inhibits d-amphetamine-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens ...... 112

21. Effect of morphine on AMPA-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens ...... 113

22. Effect of morphine on locomotor activity induced by high concentrations of AMPA or d-amphetamine in the rat after bilateral injection into the nucleus accum bens ...... 114

23. Effect of morphine on hypermotility elicited by bilateral injection of MK-801 into the nucleus accumbens or by systemic administration of caffeine or ...... 115

24. Effect of morphine on dopamine-induced locomotor activity in the rat after bilateral injection into the nucleus a ccu m b en s...... 116

xiii 25. Lack of effect of morphine on picrotoxin-induced locomotor activity in the rat after bilateral injection into the nucleus accum bens ...... 117

26. Possible sites of action of morphine within the nucleus accumbens; the "downstream" m odel ...... 118

27. Possible sites of action of morphine within the nucleus accumbens; the "multiple pathways" m odel ...... 119

28. DNQX (1.0 pg/0.5 pi) inhibits the locomotor activity elicited by picrotxin (0.5 pg/0.5 pi) when both are administered simultaneously into the nucleus accumbens ...... 120

29. Effects of 5-HT bilaterally injected into the nucleus accumbens on systemic d-amphetamine stimulated locomotor activity in the rat ...... 133

30. Effects of several 5-HT agonists on systemic d-amphetamine-stimulated locomotor activity in the rat after bilateral injection into the nucleus accumbens ...... 134

31. Effects of 8-OH-DPAT (5-HT-1a ) and CGS-12066B (5-HT-1b agonist) on systemic d-amphetamine-stimulated locomotor activity in the rat after bilateral injection into the nucleus a cc u m b e n s ...... 135

32. Lack of effect of serotonergic agonists in the nucleus accumbens on normal locomotor activity ...... 136

33. Possible sites of action of serotonin in the nucleus accumbens ...... 141

34. A model of neuronal interactions in the nucleus accumbens, involving phasic and tomic dopamine release ...... 151

xiv CHAPTER I

INTRODUCTION

Drug abuse is a major health problem affecting society. In 1988, the abuse of

and drugs by Americans was estimated to have cost over $144 billion,

including cost of treatment, prevention, lost productivity and violence (Carey,

1990). Furthermore, this figure does not address the cost in human suffering bom by both addicts and their loved ones. Despite the considerable progress that has been made in recent years towards understanding the phenomena of addiction (for review see Koob and Bloom, 1988), effective strategies for treating drug addicted persons remain elusive.

In the past, theories of addiction were based on the observation that upon cessation of drug intake, a painful withdrawal occurred. Thus, further use of the drug was seen as an attempt by the user to alleviate the and distress of withdrawal. In recent years, however, evidence has mounted which suggests that addicts maintain drug use due to the positively reinforcing effects of the drug.

Many researchers have therefore focused their efforts on the mechanisms of reward. While much progress has been made, the neural substrates of drug reward remain poorly understood. A better understanding of the interaction of

1 drugs with the neural substrates of reward will undoubtedly result In novel therapies for the treatment of drug addiction. The purpose of this dissertation, therefore, is to contribute to the growing database from which, hopefully, an

understanding of the neural substrates of drug reward will come.

THEORY AND TERMINOLOGY

Any discussion of theories of drug addiction requires the use of terminology which is often loosely defined. It is therefore important to provide definitions prior to any discussion. Jaffe (1980) defines the phrase drug abuse as the self­ administration of any drug in a manner that deviates from the approved medical or social patterns within a given culture. While this definition is not useful scientifically, it is useful in a sociological context. The phrase abuse potential refers to drugs whose habitual use could result in addiction, and abused drugs are those which are compulsively used by and which have addiction potential. The term drug addiction refers to "a behavioral pattern of drug use, characterized by overwhelming involvement with the use of a drug (compulsive use), the securing of its supply, and a high tendency to relapse after withdrawal" (Jaffe, 1980).

Traditional theories concerning drug addiction involve the development of drug dependence, involving both physical and psychological dependence. Physical dependence refers to "an adaptive state that manifests itself by intense physical disturbances when the administration of the drug is suspended" (Eddy etal, 1965) and is typical of opiate withdrawal. It is often, but not always, associated with tolerance, in which increasingly larger doses of a drug must be administered in order to obtain the effects observed with the original (Jaffe, 1985).

Psychological dependence, on the other hand, is "a condition in which a drug produces a feeling of satisfaction and a psychic drive that requires periodic or continuous administration of the drug to produce pleasure or avoid discomfort"

(Eddy et al, 1965). The Diagnostic and Statistical Manual of Mental Disorders

(American Psychiatric Association, 1987) defines drug dependence, however, as

"a cluster of cognitive, behavioral, and physiologic symptoms that indicate that the person has impaired control of psychoactive substance use and .continues use of the substance despite adverse consequences."

In operant conditioning, the term reinforcement refers to the occurrence of an event, such a s obtaining food or alleviating pain, following a desired response, such as pressing a lever. A positive reinforcer, as defined by Skinner (1938), is a , such as the presentation of a food pellet to a rat, which increases the probability of the response (lever press) which preceded its presentation. In contrast, a negative reinforcer is a stimulus, such as an electric shock, which, when removed following a response (i.e. a lever press), increases the probability of the response. Skinner (1938) labeled these phenomena as operant reinforcement.

Older theories of addiction emphasized the ability of abused drugs such as to produce physical or psychological dependence and associated noxious withdrawal symptoms and suggested that avoidance of this dysphoric state was 4

the primary motivation for drug administration (Bishop, 1920; Collier, 1968; Dole

and Nyswander, 1967; Lindesmith, 1947; Lindesmith, 1968; Wickler 1952). Thus,

in the case of compulsive drug use, withdrawal served as a negative reinforcer,

removed following drug administration. Furthermore, tolerance to the

elicited by certain drugs was proposed (Lindesmith, 1968; Wikler, 1973). These

observations led to the opponent process theory of motivation (Solomon, 1977).

This theory holds that a reinforcer arouses positive hedonic processes that are opposed by negative anhedonic processes (in a sort of neural tug of war). A drug user initially experiences a stimulation of the hedonic process. Repeated stimulation of the reward substrate which elicits the hedonic process, however,

results in its desensitization, and tolerance develops to the positive hedonic process. Negative hedonic processes (aversive withdrawal symptoms) thus become more pronounced. Dependence results when negative hedonic processes predominate in the normal, drug free state, and the drug user must repeatedly take the drug just to maintain a normal opposition between these opposing processes.

An important tenet of this theory (the negative reinforcement model) is that the negative reinforcement mechanism is the result of the desensitization of the positive reinforcement mechanism. Several lines of evidence, however, do not support the negative reinforcement model, in which physical dependence is necessary for addiction. First, behaviors can be reinforced by opiates and psychostimulants in the absence of physical dependence (Mucha etal, 1982; Koob etal, 1984; Bozarth and Wise, 1984). Second, the opioid 5

is self-administered and elicits place preference (measures of reward) in monkeys

(Mello etal, 1981; Lucas etal, 1986; Young e ta l 1984; Brown etal, 1991) and is

reported to have morphine-like euphoric effects in humans (Lewis, 1985) but

produces only a very mild withdrawal syndrome (Jasinski et al, 1978); while LI-

50,488, an experimental opioid agonist, is not rewarding but produces physical dependence (Tang and Collins, 1985). Third, brain sites which mediate reward

(nucleus accumbens and ventral tegmental area) and physical dependence

(periaquiductal grey region and dorsal thalamus) appear to be anatomically distinct

(Bozarth and Wise, 1983; Bozarth and Wise, 1984; but also see Baumeister e ta l

1989; Koob et al, 1989). Fourth, repeated exposure to some abused drugs appears to intensify their rewarding effect, not produce tolerance to it (Lett, 1989;

Vezina and Stewart, 1984).

While negative reinforcement may be involved to some extent (Koob et al,

1989; Koob and Bloom, 1988; Wise, 1990), in recent years evidence has accumulated which suggests that it is the positive reinforcing effect of drugs which is the critical determinant of their abuse (Wise, 1987; Wise, 1990; Negus and

Dykstra, 1989; Lett, 1989; Stewart etal, 1984). Thus, the drug-elicited euphoria serves as a positive reinforcer, and drug use is explained as a repeated attempt by the user to regain that euphoria.

In humans, euphoria can be defined as a feeling of extreme pleasure, happiness and well-being. All drugs which elicit the feeling of euphoria as reported by human users, most notably the opiates (morphine, ) and psychostimulants 6

(amphetamine, cocaine), are also addicting and have abuse potential. Animals will

also readily self-administer these drugs. However, as euphoria is a human,

subjective, inner experience, one must resist the inappropriate and anthropomorphic conclusion that an animal is experiencing euphoria. The term

reward, referring vaguely to an emotional state thought to be pleasurable, is preferable, and is best defined as an environmental stimuli which elicits approach

(White, 1989b; Bozarth, 1991). For example, detecting a food pellet activates the neural substrates of reward in a rat, which then approaches the pellet. Thus the pellet is considered rewarding. Drugs such as opiates and psychostimulants, when given to animals, bypass the sensory organs, but nonetheless activate the neural substrates of reward, and are therefore considered rewarding.

METHODS OF ASSESSING DRUG REWARD

Drug addiction is characterized by compulsive drug administration. In the positive reinforcement model, drug-induced reward is the motivating factor for repeated drug use. Therefore, the study of drug reward will ultimately provide valuable insight into the phenomena of drug addiction.

There are currently several methods used for assessing the rewarding properties of drugs in animals (for review see Bozarth, 1987; Evenden, 1990;

Woolverton and Nader, 1990). These methods have a high degree of correlation with data collected from humans. The most widely employed methods include direct drug self-administration, facilitation of electrical brain stimulation, drug discrimination, which all involve operant responding, and conditioned reinforcement and conditioned place preference, which do not.

Ooerant Reinforcement Techniques

Operant reinforcement, as originally defined by Skinner (1938), is typically studied using operant conditioning methods in which a reward is contingent upon an appropriate response by the subject. These methods are reviewed in the following section.

Self-Administration

The self-administration paradigm typically involves implanting an intravenous catheter into an animal and subsequently allowing the animal to self-administer a drug. The administration of drug is typically contingent upon a behavioral response such as lever pressing and is easily understood utilizing the principles of operant psychology. The ability of the drug to reinforce the behavior that led to its administration is then assessed. Most of the drugs abused by humans (but not all, hallucinogens such as LSD are not, see Jaffe, 1985) are self-administered by animals, and self-administration studies can be used to assess the abuse potential of novel drugs (see Woolverton and Nader, 1990).

The rate or responding (how often a rat will press a lever for a drug injection) for rewarding drugs generally follows an "inverted U" dose-effect curve. Thus, animals do not respond for low doses of a drug, as it is not reinforcing. The rate 8 of responding increases up to a certain level, and then as the dose increases further, responding drops off again. This may reflect an attempt by the animal to titrate the amount of drug it gets, or it may reflect motor impairment.

The two most common techniques for assessing the ability of a drug to reinforce a behavior are cross substitution and the direct acquisition. The direct acquisition technique involves using the proposed drug initially, and determining if an animal will learn to self administer it. With the cross substitution method, on the other hand, animals are trained to self-administer a standard rewarding drug such as cocaine. A new drug is then switched with the standard drug (i.e. cocaine), and the ability of the new drug to maintain the behavior is assessed.

The self-administration technique can be used to determine which brain areas are important in reward mechanism. For example, animals will self-administer drugs directly into specific brain regions associated with reward. Intracranial self­ administration techniques are advantageous in that specific anatomical questions can be addressed.

In another paradigm, attempts to disrupt self administration are made using antagonists or lesions. For example, if destruction of dopaminergic cell bodies disrupts amphetamine self administration, then these cell bodies must be involved in maintaining self administration behavior. Typically, disruption of reward results in an initial increase in lever pressing (presumably the animal is trying harder to get what is no longer there) followed by a decrease in responding, known as extinction (presumably, the animal realizes that its efforts are futile). Facilitation of Electrical Brain Stimulation

Similar to the drug self administration paradigm, animals can be trained to work

for electrical brain stimulation. Typically, an electrode is implanted in the medial

forebrain bundle where it passes through the lateral hypothalamus. A behavioral

event (lever press) is then followed by the administration of small current (in the form of a train of electrical pulses) into the median forebrain bundle. This current activates the reward pathways and thus reinforces the behavior just like a food pellet might.

Some drugs enhance or facilitate the ability of electrical stimulation to elicit a response interpreted as reward. This can be demonstrated in two ways; either a given dose of a drug can increase the rate of responding for electrical brain stimulation (animal will press the lever more often), or a given dose of a drug will lower the current threshold required to maintain responding for electrical brain stimulation. There is a high correlation between drugs which have abuse potential in humans and drugs which enhance or facilitate electrical brain stimulation.

Drug Discrimination

The drug discrimination paradigm is used to study the subjective effects of drugs. In this paradigm, animals are trained to make one of two alternate responses when under the influence of a specific drug. For example, a hungry rat will learn to press a lever for a food pellet. Next, the rat is administered a drug, such as morphine, in a chamber with two levers and trained to press one particular lever (the right lever) for the pellet. When administered saline, the rat learns to

press the left lever for the pellet. After a number of daily trials, the rat learns to

discriminate the morphine from the saline. At this point, a novel drug is administered. If after its administration the rat presses the lever associated with the morphine experience, then the novel drug is said to have similar (or, in this case, morphine-like) discriminitive stimulus properties. If the rat presses the lever associated with the saline, thenthe drug is considered not to substitute for the test drug. Animals can distinguish many different classes of psychoactive drugs (e.g. ethanol, benzodiazepines, psychostimulants, opiates), and drugs which have similar subjective effects in human studies typically generalize in animal drug discrimination studies.

Non-Operant Techniques

A qualitatively different type of conditioning was described by Pavlov (1927).

When rewarding stimuli are paired with neutral stimuli (those stimuli which elicit neither approach nor avoidance), then the formerly neutral stimuli gain the capacity to elicit approach and therefore become rewarding. Skinner (1938) referred to this as respondent conditioning. The ability to predict a goal object, such as food or a sexual partner, represents an enormous evolutionary advantage, and neural systems have developed which make this possible. The intrinsic properties of the goal, which make it rewarding (capable of eliciting approach), are referred to as the unconditioned stimulus (US). Any arbitrary stimulus with which it is associated is termed the conditioned stimulus (CS). Thus, if there is a predictive relationship between the CS and the US (such as a dinner bell signaling the arrival of food), then the CS will come to elicit the central representation of the US (Robbins etal,

1989; Mackintosh, 1983).

In the methods described above, administration of the reinforcing stimulus (i.e. food or drug reward) is contingent upon the expression of an appropriate behavior.

The methods described below, in which operant responding may play some role, emphasize the assessment of the behavior of an animal after it has been presented with a conditioned stimulus associated with reward (see Bozarth, 1987).

Conditioned Place Preference

In the conditioned place preference paradigm, rats are trained by pairing distinct environmental stimuli or cues with the administration of a rewarding stimulus. This occurs over the course of several training periods in which an animal is administered a rewarding stimulus such as food or a dose of a rewarding drug and placed in one chamber of a place preference apparatus (see Figure 1) or given a neutral stimulus such as saline or vehicle and placed in another distinct chamber. An association between the environmental cues in the first chamber and the rewarding stimulus then develops. Theoretically, the environmental cues in the first cham ber are now capable of eliciting approach. The animal is tested later to see if it then spends more time in the chamber paired with the rewarding stimulus.

Animals will form a place preference with a number of rewarding stimuli, including 12

CS+

Figure 1. The conditioned place preference apparatus. The drug is paired with the least prefered side, usually white (CS+ for conditioned stimulus +). Saline is given in the prefered side, usually black (CS- for conditioned stimulus-). 13 food (Spyraki, 1982a), sucrose solutions (White and Carr, 1985), electrical brain stimulation (Ettenberg and Duvauchelle, 1988), and many drugs abused by humans (see Hoffman, 1989; Swerdlow etal, 1989).

A major advantage of the conditioned place preference paradigm is that animals are (in most cases) drug free during the actual test. This is in contrast to the procedures for measuring reward, which described above, involve operant responding by an animal while under the influence of a drug. The conditioned place preference paradigm has numerous other advantages; and was the method used in the studies described in this dissertation. First, the rewarding effects of drugs are assessed in the drug free animal. Thus complications arising from increases or decreases in motor activity are avoided. Second, the procedure is rapid, in one case place conditioning was reported after only one pairing with morphine (Bardo and Neisewander, 1986). Third, the apparatus is simple to construct and maintain. Fourth, no complicated surgeries are required

(intravenous catheters are notorious for being difficult to maintain). Fifth, the method is sensitive and easy to quantitate.

There are two basic types of place preference procedure: the balanced and unbalanced paradigms. In the balanced paradigm, animals initially spend an equal amount of time in both chambers. In the unbalanced paradigm, the animals initially prefer one side over the other, and the rewarding drugs are paired with the non-preferred side. Each method has advantages and disadvantages (see van der

Kooy, 1987; Fibiger, 1987; Bozarth, 1987). For example, in the unbalanced 14

paradigm measures of aversion cannot be done, as there may be a ceiling effect.

That is, it may be that one cannot get an animal to dislike the least preferred side

more than it does initially. Also, since in the unbalanced paradigm the drug must

be paired with the least preferred side (for reasons of ceiling effect, i.e. one may

not get an animal to like the preferred side more than it does initially), counter balanced controls (i.e. morphine in the preferred side) cannot be run. On the other hand, using an unbalanced paradigm is easier, and it allows one to maximize the potential shift in place preference following drug pairing with the least preferred side.

The unbalanced procedure used in some of the experiments for this dissertation project is described here and is typical of most conditioned place preference paradigms (Figure 2). The place preference apparatus used was a 3-compartment apparatus arranged so that a small central compartment separates 2 larger compartments. (Figure 1). Guillotine doors separate the compartments. The small compartment is grey, while the one larger compartment is painted white and the other black. The white compartment has a floor composed of steel rods, and the black compartment has a floor composed of wire mesh. On the day prior to training animals were allowed to freely explore the apparatus for 15 minutes with the guillotine doors open, thus establishing a baseline preference, (some investigators use more than one pre-training session). As this apparatus provides an unbalanced paradigm, the chamber they spend the least time in is considered the non-preferred side. On alternate days, rats were injected with either the drug Day 1 saline \ j

Day 2, 4, 6# drag

Day 3,5,7,9 saline sfeihiss

Day 10 saline

V'“

Figure 2. The conditioned place preference paradigm (adapted from Swerdlow etal, 1989). 16 and confined to the non-preferred side for 35 minutes or were injected with saline and confined to the preferred side for 35 minutes. Rats were tested for place preference on the day following training. Rats were placed in the small grey compartment with the doors closed. The doors were then opened, and the amount of time spent in each compartment during a 15 minute test period was recorded

(see Figure 2).

Conditioned Reinforcement

In the conditioned reinforcement paradigm, an animal is trained to respond for a rewarding drug injection. The drug administration is accompanied, however, by another neutral stimulus, such as a light or a tone. An association is then presumably formed by the animal between the rewarding drug injection and the secondary stimulus (e.g. the tone). The animal is then tested to determine whether it will respond for the tone only. If the drug was indeed rewarding, and an appropriate association was made, the animal should lever press for the tone alone. This technique, along with conditioned place preference, has the advantage that animals are tested in a drug free state, and other effects of the drug such as motor disturbances will not directly interfere (Davis and Smith, 1987).

THE LOCOMOTOR ASSAY AND ITS RELATIONSHIP TO REWARD

Psychostimulants such as cocaine and amphetamine are characterized both by their positively reinforcing effects and their ability to increase locomotor activity 17

(Evenden and Ryan, 1990). In addition, other classes of rewarding drugs (at

appropriate doses), including methylxanthines such as caffeine and opiates such as heroin (Swerdlow etal., 1986), and electrical stimulation of the medial forebrain bundle (Glickman and Schiff, 1967) are simulators of locomotor activity.

Furthermore, treatments which reduce the locomotor response to psychostimulants frequently also reduce their ability to elicit reward. These correlations have led to the hypothesis that increased locomotor activity is intimately involved in the reward process.

It has been proposed that all. positive reinforcers (Glickman and Schiff, 1967), including rewarding drugs (Wise and Bozarth, 1987), will activate approach responses. Locomotor activity may therefore represent an approach response in the absence of a specific object to be approached. Thus a relationship between the neural substrates of locomotor activity and reward seems to exist. While all stimuli that activate locomotor activity are not positively reinforcing (as avoidance is also a form of locomotor activity), it is possible that all positively rewarding stimuli (including abused drugs) will activate locomotor activity. The locomotor activity assay is therefore useful for analyzing the motor activating properties of abused drugs.

Locomotor activity is typically assessed by measuring either the breaking of infrared beams in a photocell cage or directly measuring distance traveled using a digitized image analysis. Combined with stereotaxic microinjection techniques, the locomotor activity assay is useful for analyzing the anatomical substrates 18

necessary for motor activation elicited by many classes of abused drugs (for

review see Swerdlow et al, 1986; Geyer, 1990).

NEURAL SUBSTRATE OF DRUG REWARD AND LOCOMOTOR

PHARMACOLOGY

The previous sections of this dissertation have reviewed some of the more common methods used in studying the behavioral effects of rewarding drugs, (in a loose and anthropomorphic sense, animal models of human, drug-elicited pleasure). To elicit reward, however, a drug must gain access to .some critical part of the , which mediates natural reward. In recent years, the central sites which mediate the rewarding effects of abused drugs have begun to be elucidated. The following sections, therefore, will review what is currently known about the neural substrates of reward. Recent research involving the neurobiology of drug reward has suggested that the three major classes of abused drugs, the psychostimulants, the opioids, and ethanol, trigger activity in a common reward system (Wise and Bozarth, 1982; Wise and Bozarth, 1984; Koob and

Bloom, 1988; Di Chiara etal, 1991; Bozarth, 1991). Early studies suggested the presence of what came to be known as a pleasure center (Olds, 1956); however, it has become apparent in recent years that multiple elements in a circuit are involved (Olds, 1972). This common reward circuit involves which interconnect the ventral tegmentum, nucleus accumbens, ventral pallidum, and frontal cortex (Koob and Bloom, 1988). Elements of this circuit also appear to 19 comprise the neural substrate of locomotor activity. The overlap of the neural substrates for both locomotor activity and reward has led to the tempting hypothesis that this common substrate may mediate goal-directed behavior

(Swerdlow et al, 1986). The following sections will review the anatomy and possible functions of these areas, with a special emphasis on the nucleus accumbens.

Motivation and Drive

Motivation has been defined as the set of processes that enable an organism to control the availability, probability or proximity of stimuli (Salamone, 1991).

These processes underlie the initiation and termination of goal-directed behavior.

Two schools of thought have been developed to explain the phenomenon of goal directed behavior. The first emphasizes what came to be known as drive reduction theory, and the second emphasizes incentive motivation theory.

Drive reduction theory suggests that the internal conditions that arouse and direct voluntary goal-directed motor behaviors are known as motivational states, and specific motivational states are known as drives, (Hull, 1943; Kupfermann,

1985). Animals tend to maintain homeostasis, and when homeostatic imbalances occur, animals act in such a way as to correct them. Thus there are specific drives for things such as food, water, and sex, and the biological incentive is to reduce that drive. For example, a hungry rat can be described as being in a drive state for food. Thus, drive states tend to "push" an animal to act, if hungry then 20

seek food.

In contrast, incentive motivational theory (see Bindra, 1968) emphasizes the

ability of stimuli associated with a goal object to energize motivated behavior.

Thus, the animal is "pulled" toward the goal object because of its rewarding value.

If a rat is hungry it simply adds to the incentive value of stimuli associated with

food. One factor which contributes to rewarding value and controls motivated

behavior is pleasure. As we are all well aware, eating a delicious meal when one

is hungry elicits the feeling of pleasure, it is a pleasurable experience. Although

it is impossible to know if an animal such as a rat feels pleasure; it seems likely that they do. For instance, rats will eat more of a palatable meal (including chocolate chip cookies and salami) than an equally nutritious but bland meal of rat chow (Sclafani, 1976). While this investigator can attest to the payability of chocolate chip cookies and salami, I must let the reader assume that rat chow is bland, as I have never sampled it.

Anatomy of the Reward Substrate

In 1954 it was demonstrated that electrical stimulation of specific areas associated with the hypothalamus could reinforce operant behavior (Olds and

Milner, 1954). Thus, electrical brain stimulation of this area seemed to be rewarding, similar to food. However, only a hungry rat will respond for food, while electrical brain stimulation of certain brain areas is effective without a drive state.

This lead to the hypothesis that electrical brain stimulation both evokes a drive 21 state, and activates reward or "pleasure centers", (Deutsch and Howarth, 1963).

Since the discovery by Olds and Milner in 1954, evidence has been mounting that a single "pleasure center" does not exist in the brain. Rather a system of interacting areas seems to underlie reward (Olds, 1972). It has recently been proposed that neurons which form a ventral tegmentum-nucleus accumbens- ventral pallidum-frontal cortex pathway form the substrate for natural reward (Koob and Bloom, 1988). A diagram of this circuit is shown in Figure 3.

The first element of the reward circuit is the medial forebrain bundle, electrical stimulation of which appears to be rewarding (Liebman, 1989). While presumably most of the many fibers which course through the medial forebrain bundle are stimulated by an electrode, electrophysiological and histological studies have revealed that the critical fibers for the rewarding effect of electrical stimulation represent mylenated axons, with a diameter of 0.5 and 2.0 pm and with cell bodies in the lateral hypothalamus or the nucleus of the diagonal band (for review see

Wise and Bozarth, 1984; Carlson, 1986). This suggests that it is not the small and unmyelinated fibers (i.e. dopaminergic or noradrenergic which also course through the medial forebrain bundle) which are activated by electrical stimulation. These fibers then at the ventral tegmental area of the ventral mesencephalon, where they activate dopaminergic (and possibly other) neurons which project to the nucleus accumbens.

The dopaminergic cells of the ventral tegmental area (A10) then send projections (back through the medial forebrain bundle, see Figure 3) to the nucleus 22

LIMBIC CTX

PFC Glu Glu DMT HIP Glu

DA VTA m ft NA VP PPN

NDB 5HT

RN

Figure 3. The proposed reward circuit. Abbreviations: (CTX) cortex; (PFC) ; (HIP) ; (VTA) ventral tegmental area; (NDB) nucleus of the diagonal band; (NA) nucleus accumbens; (RN) raphe nuclei; (VP) ventral pallidum; (PPN) pedunculopontine nucleus; (DMT) dorsomedial thalamus; (mfb) medial forebrain bundle; (DA) dopamine; (GLU) glutamate; (5HT) serotonin. accumbens, a region of the basal forebrain. The nucleus accumbens then sends

projections (believed to be GABAergic and enkephalinergic) both to the ventral

pallidum and back to the ventral tegmental area. The ventral pallidum, as used

in this text, includes the subcommissural substantia innominata and the more

rostral sublenticular ventral pallidum (Heimer and Alheid, 1991). The ventral

pallidum sends projections to the frontal cortex, a site associated with reward, and to both the dorsomedial thalamus and the pedunculopontine nucleus (considered the rat homolog of the mesencephalic locomotor region), sites associated with the control of locomotor activity. The dopaminergic cells of the ventral tegmental area also project to the prefrontal cortex, which then sends a projection to the nucleus accumbens (for a comprehensive review, see Wise, 1990; Heimer and Alheid,

1991; Groenewegen etal, 1991).

The Nucleus Accumbens

Figuring prominently in the above circuit (see Figure 3) is the nucleus accumbens, a structure of the basal forebrain. This nucleus, and especially the dopaminergic within it, appears to be the critical substrate for a number of abused drugs. Recent advances in the anatomy and histology of the nucleus accumbens have raised questions about its precise definition. The questions arise; what is the nucleus accumbens, and what does it do? In the following section and in the subsequent chapters, I will try to address these questions.

The nucleus accumbens, part of the ventral , is located strategically 24 within the proposed reward circuit such that it receives afferent projections (input) from limbic structures and sends efferent projections (output) to areas involved with motor behavior. This has led to the intriguing suggestion that the nucleus accumbens may serve as an interface between limbic and motor areas, translating relevant motivational-emotional information into motor behavior (Mogenson and

Yim, 1981).

The anatomy, histochemistry, and synaptic organization are similar in nucleus accumbens and striatum (Groenewegen, 1991; Voorn etal, 1986). In fact, while the nucleus accumbens is considered to lie ventral to the striatum, dorsal to the olfactory tubercle, and lateral from the medial septal area (see Figure 4), its borders are difficult to discern. The majority of the neurons of the striatal complex

(>90%) are medium I spiny neurons (Cajal, 1911; Kemp and Powell, 1971). These cells are primarily GABAergic, and may also contain or substance P

(Kita and Kitai, 1988; Gerfen and Young, 1988). They are the primary output cells of the striatal complex (Fruend etal, 1984; Chang and Kitai, 1985; Grofova, 1975;

Bolam etal, 1981a; Bolam etal 1981b; Smith and Bolam, 1990) and receive many convergent inputs (Groenewegen 1991). Other cell types include large, aspiny cholinergic , medium aspiny GABAergic and somatostatinergic interneurons, and possibly interneurons (Fonnum and Walaas, 1981;

Bolam e ta l 1984; Phelps e ta l 1985; Graybiel and Ragsdale, 1983; Parent, 1986;

Bolam etal, 1983; Difiglia and Aronin, 1982).Furthermore, although the anatomy of the accumbens is similar to that of the striatum, important differences are also 25

present. The nucleus accumbens receives afferent (incoming) projections from the

following: neocortex; regions of the allocortex including entorhinal, pyriform, and

prefrontal cortex, and hippocampus; amygdala; thalamus; septum; olfactory tubercle; mesencephalic dopaminergic neurons including the A8 group, A9 group

(substantia nigra pars compacta), and those originating in the ventral tegmental area (A10 group); and dorsal raphe nuclei (Domesick, 1981; Parent et al, 1981).

It is now apparent, however, that the accumbens is not a homogeneous nucleus. Recently, it has been shown via anatomical and histological means that the accumbens can be divided into two distinct compartments: a central core and a partially surrounding shell which lies on the ventral and medial border of the core

(Zaborsky et al, 1985; Heimer and Alheid, 1991) (see Figure 4). The efferent projections from the core of the accumbens resemble those of the more dorsal striatum in cytoarchitecture and anatomy. Whereas both the core and the shell send efferent projections predominantly to the ventral pallidum and the substantia nigra/ventral tegmental area, the shell sends additional projections to the hypothalamus and extended amygdala (Heimer and Alheid, 1991). It has been suggested that the nucleus accumbens may represent a zone of transition between the ventral elements of the striatum, and the lateral portions of the extended amygdala (Heimer and Alheid, 1991).

Additionally, anatomical projections and neurochemical markers are heterogeneously distributed within the nucleus accumbens (and similarly in the striatum) in a mosaic-like pattern referred to as the patch and matrix pattern cc

CP

lac AbS iAbC

OT

Figure 4. The nucleus accumbens. This cross section (1.2 mm anterior to bregma) demonstrates the major anatomical features of the accumbens and surrounding structures. Note the regions of core and shell denoted by dashed lines, and patch/striosome compartments (filled with dots) as opposed to the surrounding matrix. Abbreviations: (AbS) shell of nucleus accumbens; (AbC) core of nucleus accumbens; (ac) anterior commisure; (cc) corpus callosum; (CP) caudate-putamen or dorsal striatum; (CTX) cortex; (IC) islands of Calleja; (OT) olfactory tubercle. (Adapted from Paxinos and Watson, 1982). (Herkenham, 1984; Gerfen, 1984; Gerfen, 1987; Groenewegen et al, 1987;

Jimenez-Castellanos and Graybiel, 1987). This heterogeneity is similar in several species including rat, cat, monkey, and human (Graybiel, 1984). The geographic relationship between these two compartments has been likened to a piece of swiss cheese, in which the holes represent the patch or striosome compartment and the cheese represents the matrix compartment (Goldman-Rakic, 1982). While 95% of striatal neurons are of the medium spiny type (Kemp and Powell, 1971), and the cells of patch and matrix have very similar morphology and repetitive firing characteristics (Kawaguchi etal, 1989), there are important differences. The patch compartment is characterized by dense p opiate receptor binding and high enkephalin and substance P immunoreactivity (Graybiel, 1990), and patches are more numerous in the ventral striatum and nucleus accumbens (White, 1989a).

The matrix is characterized by high acetylcholinesterase activity (Graybiel and

Ragsdale, 1978; Graybiel, 1990), tyrosine hydroxylase, cytochrome oxidase, and calbindinD28k-like immunoreactivity (Agwood and Emson, 1992). These studies suggest that matrix cells may be more metabolicaily active. Neurons containing somatostatin seem to connect the two compartments, and communication between the compartments may occur through these cells (Gerfen, 1984; Gerfen, 1987).

The anatomical connections of these two compartments are similarly distinct.

While dopaminergic cells from the ventral mesencephalon project to both compartments, distinct subdivisions of these cells differentially innervate both patch and matrix. In rats, cells from the ventral tegmental area (A10) project principally 28 to the matrix in the nucleus accumbens and ventral striatum. Cells from the dorsal part of the substantia nigra pars compacta (A9) project to the matrix of the dorsal striatum, whereas cells from the ventral part of the substantia nigra pars compacta project to patches in the dorsal and ventral striatum and nucleus accumbens.

Cells from a cluster in the ventral substantia nigra pars reticulata project to patches in the dorsal striatum (Gerfen, 1987; Jimenez-Castellanos and Graybiel, 1987; also see White, 1989a).

Interestingly, a recent study (Gerfen et al, 1987) demonstrated that dopaminergic innervation of the patch appears to develop earlier than that of the matrix. 6-OHDA was administered unilaterally into the striatum of one day old neonatal rats. After thirty days., the rats were sacrificed, and their were stained for tyrosine hydroxylase activity (a marker for dopaminergic axon terminals). It was discovered that dopaminergic innervation of the patch was absent, but that of the matrix was normal. These and supporting findings (see

Fishell and van der Kooy, 1989) may explain much puzzling data involving neonatally lesioned rats.

Non-dopaminergic inputs to the patch and matrix compartments of the striatum and nucleus accumbens are equally distinct. The matrix compartment receives projections from areas including the hippocampus, sensory and motor cortex, and the cingulate gyrus, while the patch compartment receives projections from the amygdala and prefrontal cortex. Thus, a functional difference between these compartments has been proposed. The patch compartment is associated with 29

structures mediating affect related and , while the matrix

compartment is associated with spatial memory and sensory-motor processing

(Graybiel, 1990). In addition, it was recently demonstrated that modulation of

dopamine release by NMDA in the patch compartment is functionally distinct from

that of the matrix (Krebs et al, 1991). NMDA-evoked release appears to be

greater in the matrix compartment, relative to patch (Krebs etal, 1991).

Efferent projections from patch and matrix compartments are equally distinctive.

Whereas outputs from the patch compartment project to the area of the

dopaminergic cell bodies of the ventral mesencephalon (A8, A9, A10) (Gerfen,

1984; (Jimenez-Castellanos and Graybiel, 1989), outputs from the matrix project

mainly to the pallidum and the substantia nigra pars reticulata (Jimenez-

Castellanos and Graybiel, 1989).

Metabolic responses to drugs also vary between the two compartments.

Recently, it has been shown that the psychostimulants amphetamine and cocaine

induce distinct patterns of activation of the c-fos gene in the two compartments,

patch and matrix, of both dorsal striatum and nucleus accumbens (Graybiel et al,

1990). Thus, while c-fos induced by amphetamine was more pronounced in striosomes, c-fos induced by cocaine occurred in both compartments. A pharmacological distinction between effects of the two stimulants was also observed as induction of c-fos by cocaine was inhibited by reserpine, whereas induction of c-fos by amphetamine was not. Induction of c-fos by both stimulants,

however, was blocked by a D1 dopaminergic antagonist, suggesting that the D1 30 receptor mediates this effect and further suggesting that the adenylate cyclase/A or phospholipase/C kinase second messenger systems are involved. In support, 01, but not D2, selective agonists have been shown to induce c-fos in the

6-OHDA dopamine depleted striatum of the rat (Robertson etal, 1989).

Role of Dooamine in Nucleus Accumbens

Dopaminergic synapses within in the nucleus accumbens are critically involved in the phenomenon of reward. The following sections will review the anatomy, biochemistry, ; and behavioral role of dopamine in the nucleus accumbens.

Anatomy of Dopaminergic Projections

The dopaminergic cell bodies of the ventral mesencephalon have ascending projections to several forebrain areas (for review see Bjorkland and Lindvall, 1984;

Fuxe, 1965). The mesolimbic system originates in the ventral tegmental area

(A10) and projects mainly to the nucleus accumbens and amygdala. The nigrostriatal system originates in the substantia nigra pars compacta (A9) and projects predominantly to the striatum. The mesocortical system also originates in the ventral tegmental area and projects to the prefrontal, cingulate and entorhinal cortices. Grouped together, these systems form the mesotelencephalic dopamine system. Projection patterns from this system are similar in rodents, primates and man. The large majority of cells in the nucleus accumbens and striatum are medium

spiny neurons, that is, neurons with large spiny dendrites (Cajal, 1911; Kemp and

Powell, 1971; Freund etal, 1984; Smith and Bolam, 1990). While some dopamine

axons synapse on the cell bodies and dendritic shafts of the medium spiny output

neurons, most synapse on the spines themselves (Fruend et al, 1984). Axons from the cortex (Bouyer et al, 1984), hippocampus (Totterdell and Smith, 1989;

Groenwegen, 1991), and possibly amygdala (see Yim and Mogensen, 1986), which may use glutamate as a neurotransmitter (Fonnum et al, 1981; Christie et

al, 1987; Fuller etal, 1987; also see Yim and Mogensen, 1989) synapse in close apposition to the dopamine terminals. Thus the dopamine synapses are in an ideal position to modulate information coming from the cells projecting from cortical

regions (see Smith and Bolam, 1990; Groenewegen, 1991).

Biochemical Pharmacology of Dopamine

It now appears that there are five dopamine receptors, designated D1, D2, D3,

D4 and D5 (see Strange, 1991; Snyder, 1990). Each of these receptor subtypes have now been cloned (Bunzow etal, 1988; Dearry etal, 1990; Zhou etal, 1990;

Sunahara etal, 1990; Strange, 1990; and Sokoloff etal, 1990). These dopamine receptors display an amino acid sequence consistent with the motif of seven transmembrane hydrophobic regions typical of receptors linked to a G . The

D2 and D3 are closer in homology than with the D1 receptor, with the major differences lying in the third intracellular loop (shorter in D1) and carboxy-terminal 32 tail (longer in D1). Additionally, two new dopamine receptors have been cloned,

D4 and D5 (Sunahara et al, 1991; Van Tol et al, 1991). Interestingly, the D4

receptor appears to have a high affinity for the , and the D5 receptor appears to have a higher affinity for dopamine than the D1 receptor.

D1 receptors have been shown to activate the adenylate cyclase

(Kebabian and Caine, 1979). The molecular structure, similar to the 02-adrenergic receptor which interacts with Gs, is consistent with this finding. D2 receptors are associated with dopamine nerve terminals and may function as autoreceptors and may regulate dopamine synthesis and release (Roth etal, 1978).

D1 and D2 receptors are found in most of the areas innervated by dopaminergic cells, including the nucleus accumbens (Wamsley et al, 1989).

Interestingly, the D3 receptor has a limited distribution and appears to be enriched in limbic areas including the nucleus accumbens, olfactory tubercle, hypothalamus and the islands of calleja (Strange, 1991).

Electrophysiology of Dopamine in Nucleus Accumbens

Electrophysiological studies have revealed a complex role for dopamine.

Activation of dopaminergic projections to the nucleus accumbens typically inhibits accumbens neurons (Yim and Mogenson, 1982) in anesthetized rats, probably by increasing the membrane potassium conductance. Studies using specific agonists have revealed that activation of D1 receptors by SKF-38393 and activation of D2 33 receptors by LY-141865 inhibit both spontaneous and glutamate-induced firing of accumbens neurons (White and Wang, 1986). On the other hand, studies using specific antagonists have revealed a different picture. Exogenous dopamine in the presence of a specific D2 antagonist, , hyperpolarized accumbens cells via activation of D1 receptors; however, exogenous dopamine in the presence of a D1 antagonist, SCH-23390, lead to depolarization of accumbal neurons via activation of D2 receptors (Uchimara and North, 1990). In separate studies in the striatum, it was found that dopamine inhibited the basal firing rate of striatal cells without greatly affecting the firing rate of the same cells responding during the performance of a specific task (Rolls et al, 1984; Chiodo and Berger, 1986). It is becoming apparent that the electrophysiological actions of dopamine, although predominantly inhibitory, are more complex than previously thought.

Role of Nucleus Accumbens Dopamine in Behavior

There are basically three ways to study the contribution of dopaminergic systems in behavior (Evenden and Ryan, 1990). First is to destroy dopaminergic cells with the selective neurotoxin 6-hydroxydopamine (6-OHDA). 6-OHDA is taken up specifically by and selectively destroys catecholaminergic neurons. By using an appropriate uptake blocker, one can protect either dopaminergic or noradrenergic cells. For example, desmethylimipramine prevents the uptake of 6-

OHDA into noradrenergic cells, thus sparing them. The second method involves directly administering dopaminergic antagonists into dopamine terminal fields, and 34

the third involves the direct administration of agonists into the terminal fields.

Studies using the techniques described above have revealed that dopamine in

the nucleus accumbens plays an important role in the control of cognitive

processes and emotional behavior. Lesions of the dopaminergic projection from

the ventral tegmental area to the nucleus accumbens with 6-OHDA have been

shown to reduce motivated behaviors such as exploratory behavior in a novel

environment without producing motor impairment (Kelley and Stinus, 1983; Kelley

etal, 1989) and to reduce food pellet hoarding (Kelley and Stinus, 1985). 6-OHDA

lesions of the dopaminergic projection to the nucleus accumbens have also been

shown to reduce displacement behavior (Robbins and Koob, 1980). In this

paradigm, hungry rats were presented with a food pellet every 60 seconds, (more

time than it takes for a rat to consume a food pellet). The displacement behavior,

drinking while waiting for the next pellet, was increased in sham treated controls

but not in 6-OHDA treated rats. The theory holds that the motivational excitement

accompanying the delivery of a food pellet outlasts its consumption, in which case

alternative (displacement) behaviors are evoked by available environmental stimuli

(in this case a water bottle). Thus the 6-OHDA seems to produce a deficit not in the consumption of a pellet, but in the level of motivational excitement accompanying the pellet (Robbins and Koob, 1980). 35 INTERACTION OF DRUGS OF ABUSE WITH THE REWARD SUBSTRATE

As stated above, to elicit reward initially and ultimately result in addiction

according to the positive reinforcement model, rewarding drugs must gain access

to elements of the proposed reward system. The following sections will review the

evidence that the major drugs abused by humans do indeed interact at some level

with the proposed system.

Psychostimulants

Psychostimulants are characterized by their ability to increase locomotor activity and their ability to elicit reward. The prototype psychostimulants are amphetamine and cocaine, and these will be discussed in the following section.

Psychostimulants are readily self administered intravenously by rats, monkeys, and

humans (Petit e ta l, 1984; Roberts and Koob, 1982; Wilson and Schuster, 1972;

Zito etal, 1985) and lower reinforcement thresholds for brain stimulation reward

(Koob and Bloom, 1988; Kornetsky and Esposito, 1981). Cocaine blocks the reuptake of dopamine into the presynaptic terminal (Heikkila, 1975; Kuhar et at,

1991). Amphetamine acts similarly but also stimulates dopamine release (Axelrod,

1970; Carlsson, 1970; Kuczenski, 1983). Furthermore, psychostimulants increase extracellular concentrations of dopamine in the nucleus accumbens as measured by microdialysis (Di Chiara and Imperato, 1988). That this increase in synaptic dopamine is involved in the rewarding effects of psychostimulants is evidenced by the finding that dopamine antagonists inhibit the reward elicited by 36

both amphetamine (Wilson and Schuster, 1972; Yokel and Wise, 1975; Yokel and

Wise, 1978) and cocaine (de Witt and Wise, 1977; Ettenberg et al, 1982) in

animals. In addition, the euphoric effects of amphetamine are blocked by

dopamine antagonists (Gunne etal, 1972) and dopamine depletion (Jonsson etal,

1971) in man.

Several lines of evidence indicate that these psychostimulants elicit locomotor

activity and reward from augmentation of dopaminergic activity within the nucleus

accumbens. Injection of psychostimulants into the nucleus accumbens (Pijnenburg

etal, 1976; Kaddis etal, 1991), but not the caudate (Jackson etal, 1975), elicits

increased locomotor activity in rats. Blockade of dopamine receptors (Lyon and

Robbins, 1975; Swerdlow et al, 1986) or 6-OHDA lesions of the nucleus

accum bens (Kelly etal, 1975) block psychostimulant induced locomotor activity.

A role for the nucleus accumbens in mediating amphetamine reward is well

established. Indeed, amphetamine is readily self-administered into the nucleus

accumbens (Hoebel etal, 1983). Administration of amphetamine into the nucleus

accumbens elicits a conditioned place preference (Carr and White, 1983; Carr and

White, 1986). 6-OHDA lesions of the nucleus accumbens attenuate amphetamine

self-administration (Lyness et al, 1979) and a-flupenthixol co-injected into the

nucleus accumbens with amphetamine on training days inhibits the conditioned

place preference elicited by amphetamine (Aulisi and Hoebel, 1983).

The role of dopamine in the nucleus accumbens in mediating cocaine reward is less well established. 6-OHDA lesions of the nucleus accumbens (Petit et al, 37

1984) or ventral tegmental area (Roberts and Koob, 1982; Zito et al, 1985) attenuate or block cocaine-induced reward as measured by self-administration.

Cell body lesions of the nucleus accumbens or ventral pallidum attenuate cocaine reward as well (Zito etal, 1985).

In contrast, lesions with 6-OHDA of the nucleus accumbens (Spyraki et al,

1982b) and systemic administration of dopamine antagonists (Spyraki et al, 1982b;

Mackey and van der Kooy, 1985) have been reported not to inhibit a conditioned place preference elicited by intraperitoneally administered cocaine. However, dopamine antagonists do block the place preference elicited by intravenous

(Spyraki et al, 1987) and intracerebroventricular (Morency and Beninger, 1986) cocaine. Furthermore, intraperitoneally administered procaine, which shares cocaine's local anesthetic effect but not central stimulant effect, elicits place preference (Spyraki etal, 1982b). Thus Spyraki et a/have concluded that cocaine elicits place preference by enhancing dopamine transmission in the nucleus accumbens, and by producing local anesthetic effects in the periphery (Spyraki et al, 1982b; Spyraki etal, 1987).

While cocaine is readily self administered into the prefrontal cortex (which receives innervation from the mesocortical dopamine system originating in the ventral tegmental area), it is not self administered into nucleus accumbens

(Goeders and Smith, 1983). On the other hand, a separate but elegant study suggests a role for the nucleus accumbens (Wood and Emmett-Oglesby, 1989).

Rats were trained to distinguish a rewarding, systemic dose of cocaine. When rats 38

were trained sufficiently, cannulae were implanted into either the nucleus

accumbens or the prefrontal cortex. Intra-accumbens cocaine generalized with

systemic cocaine, but intra-prefrontal cortex cocaine did not. One hypothesis which integrates these two sets of conflicting data suggests that the prefrontal cortex mediates the initiation of cocaine action, while the nucleus accumbens is

necessary for maintenance of cocaine action.

The studies reviewed above suggest that the psychostimulants, amphetamine and cocaine, interact at some level in the proposed system to elicit locomotor activity and reward. Additionally, the above studies implicate the nucleus accumbens (and possibly the prefrontal cortex) as substrates of locomotor activity and reward. The finding that cell body lesions of the ventral pallidum, the main projection area of the nucleus accumbens in the proposed pathway, attenuate cocaine reward (Zito ef al, 1985) suggests that lesions "downstream" from the site of action will similarly attenuate reward.

Opiates

Opiates, morphine and heroin are prototypes, have been used (and abused) by humans for centuries. The use of (the juice derived from the opium poppy

Papaver somniferum and source of morphine) was first recorded by Theophrastus in the third century B.C., and its use probably started much earlier. A great deal of evidence indicates that opiates may also interact with the proposed reward circuit. It further appears that opioid jx and 8 receptors are involved: both are found in the nucleus accumbens and ventral pallidum, and p receptors are found in the ventral tegmental area (Atweh, 1983; Mansour et al, 1987). At appropriate doses, opiates increase locomotor activity when injected systemically (Swerdlow etal, 1986) or into the ventral tegmental area (Joyce and Iversen, 1979). Opiates are readily intravenously self-administered (Bonese et al, 1974; Van Ree et al,

1978; Weeks and Collins, 1964; Thompson and Schuster, 1964; Petit etal, 1984), lower thresholds for brain stimulation reward (Marcus and Kornetsky, 1974; Bush etal, 1976; Koob etal, 1975; Reid, 1987), and elicit place preference (Bozarth and

Wise, 1981; Phillips and Le Piane, 1982; van der Kooy etal, 1982; Spyraki etal,

1983; Bechara and van der Kooy, 1989; Hoffman, 1989). The precise neural substrate within the reward circuit, however, is a matter of som e debate.

A substantial body of evidence indicates that opiates may act at the level of the ventral tegmental area to increase firing rate of dopaminergic cells that project to the nucleus accumbens. Thus, opiates administered into the ventral tegmental area elicit place preference (Phillips and LePiane, 1980; Phillips and LePiane,

1982; Bozarth and Wise, 1981; Bozarth, 1987). Opiate antagonists administered into the ventral tegmental area inhibit intravenous heroin administration (Britt and

Wise, 1983; Vaccarino et al, 1985). Furthermore, opiates increase the extracellular concentration of dopamine in the nucleus accumbens (Di Chiara and Imperato,

1988). Morphine administered into the ventral tegmental area elicits dopamine dependent locomotor activity (Joyce and Iversen, 1979). Finally, neuroleptics inhibit intravenous heroin self-administration and heroin induced place preference 40

(Bozarth and Wise, 1981; Petit et al, 1984; Zito et al, 1985).

On the other hand, opiates may also act in the nucleus accumbens to elicit

reward. Morphine (Olds, 1982) and methionine enkephalin (Goeders etal, 1984)

are self administered into the nucleus accumbens, and intra-accumbens morphine

elicits place preference (van der Kooy etal, 1982). Intra-accumbens a-flupenthixol

(a dopaminergic antagonist), at a dose that blocks amphetamine locomotor activity, did not block heroin locomotor activity (Vaccarino et al, 1986). Administration of

an opiate receptor antagonist into the nucleus accumbens attenuates heroin self­ administration (Vaccarino etal, 1985) and locomotor activity (Amalric and Koob,

1985). While blockade or 6 -OHDA lesions of the nucleus inhibit cocaine or amphetamine self administration, these treatments spare heroin self administration (Petit et al, 1984; Zito et al, 1985). Neuroleptics block amphetamine elicited place preference but not morphine elicited place preference

(Mackey and van der Kooy, 1985). Cell body lesions of the nucleus accumbens or ventral pallidum attenuate opiate reward as well (Zito et al, 1985).

It is likely that some of the seemingly contradictory data described above reflect procedural differences. While further studies will be required to resolve this issue, it is clear that opiates act at a site in the proposed circuit to elicit reward. The results reviewed above suggest that opiates may interact with the circuit at two levels; at the p receptor in the ventral tegmental area (activating dopaminergic projections to the nucleus accumbens vja disinhibition) and/or in the nucleus accumbens (possibly at p or 5 receptors) via a mechanism independent of 41 dopaminergic activity (Koob and Bloom, 1988).

Ethanol

Ethanol is widely abused by humans. Ethanol has also been shown to be self­ administered intravenously by rats (Smith and Davis, 1974; but see also Collins et al, 1984) and rhesus monkeys (Deneau et al, 1969).

Ethanol increases firing of the A10 dopaminergic neurons of the ventral tegmentum which project to the nucleus accumbens (Gessa etal, 1985), resulting in an increase in the extracellular concentration of dopamine in the nucleus accumbens (Di Chiara and Imperato, 1988). These results also suggest an interaction with the proposed pathway.

Nicotine

Nicotine is self-administered intravenously in several species including rat, dog, rhesus monkey, squirrel monkey and baboon (Lang et al, 1977; Risner and

Goldberg, 1981; Deneau and Inoki, 1967; Goldberg etal, 1981; Ator and Griffiths,

1983). Furthermore, nicotine elicits locomotor activity in rats (Museo and Wise,

1990), increases the firing rate of dopaminergic neurons of the ventral tegmental area (Merey et al, 1987), and increases dopamine levels in the nucleus accumbens (Imperato et al, 1986). Nicotine appears to produce these effects either through action at nicotinic receptors at cell bodies in the ventral tegmental area which cause an increase in firing or by action at terminal fields which 42

enhance the release of dopamine (Misfud etal, 1989; Rowell etal, 1987). Indeed,

nicotine binding sites have been located autoradiographically in the ventral tegmental area and the nucleus accumbens (Clarke and Pert, 1985).

A9-Tetrahvdrocannabinol

The active ingredient in marijuana, A9-tetrahydrocannabinol (THC) has been shown to be habit forming in humans and has been used for many centuries for its effects. THC facilitates brain stimulation reward in rats (Gardner etal, 1988), is self-administered in monkeys (Pickens et al, 1973) and rats (Takahashi and

Singer, 1979), and enhances dopamine efflux from nucleus accumbens (Chen et al, 1989) and prefrontal cortex (Chen etal, 1990).

Thus it appears that drugs of abuse with different primary mechanisms of action initiate drug dependence by interacting with elements of a common reward pathway (Koob and Bloom, 1988; Wise and Bozarth, 1984). This reward circuit may become modified through chronic use, giving rise to opposing processes as the homeostatic mechanisms of the brain counter the drug effects (Koob and

Bloom, 1988). The opposing processes may result in the aversive effects (i.e., dysphoria, malaise) of abstinence and may be a major motivating force in the maintenance of behaviors associated with drug addiction (Koob and Bloom, 1988). 4a OTHER NEUROTRANSMITTER SYSTEMS INVOLVED IN REWARD AND

LOCOMOTION

Although a great deal of evidence implicates a role for dopamine in mediating reward and locomotion, other neurotransmitter systems certainly must play a role a s well. Of particular interest with respect to the following chapters, neural system s utilizing excitatory amino acids, endogenous opioids, and serotonin as appear to interact with dopamine systems and thus play a role in reward and locomotor activity. The following sections will review the anatomy, pharmacology, and possible functional role (with regards to reward and locomotion) of these systems.

Pharmacology of The Excitatory Amino Acid System

Endogenous Ligands

Excitatory amino acids, L-glutamate and related acidic amino acids, are considered to be the predominant excitatory neurotransmitters in the mammalian brain (Fagg and Foster, 1986; Fonnum, 1984). Aspartate, L-cysteine sulfinate, and quinolinate (all excitatory amino acids) are found endogenously and may be neurotransmitters as well. A great deal of evidence suggests that excitatory amino acids function as neurotransmitters (for review see Meldrum, 1985; Boldry, 1990).

Not surprisingly, it has been demonstrated that fibers utilizing excitatory amino acids as neurotransmitters are found in the nucleus accumbens. By using a combination of [ 3H]D-aspartate and wheat germ agglutinin-horse-radish peroxidase 44

(WGA-HRP) and studying that subset of cells labelled by WGA-HRP also labelled

by the [ 3H]D-aspartate, Fuller et al (1987) demonstrated that the nucleus

accumbens receives prominent glutamatergic projections from the prefrontal and

olfactory cortex, the subiculum, various nuclei of the amygdala (most prominent the

lateral and basolateral nuclei), and the midline nucleus of the thalamus. In

addition, glutamatergic interneurons have been demonstrated in the nucleus

accum bens (Fonnum and W alaas, 1981). Finally, all three of the functionally

defined ionotropic receptors - AMPA, kainate, and NMDA - have been reported in

the nucleus accumbens (Cotman e ta l 1987).

Receptors and Pharmacology

Several distinct receptor subtypes have been established at which excitatory

amino acids act. Unfortunately, different methods of analyzing function

(electrophysiology, pharmacology) have resulted in different classification schemes

and some degree of confusion. At present, however, excitatory amino acid

receptors are characterized by the agonist which activates them, and they appear to occur in three or four distinct groups; 1) the ionotropic NMDA receptor, 2) the ionotropic AMPA and Kainate receptors, 3) the metabotropic receptor, and possibly

4) an AP4 sensitive autoreceptor (see Table 1).

The most extensively studied is the NMDA receptor (see Stone and Burton,

1987) at which NMDA is a selective agonist in electrophysiological studies.

Activation of this receptor leads to the opening of an channel that is permeable 45

Table 1. Pharmacological Classification of Excitatory Amino Acid Receptors

Receptor Agonist Antagonist

NMDA NMDA Site: NMDA Site: Ibotenate AP-5 L-Glutamate AP-7 CPP CGS-19755 NPC12626 LY-233053

Glycine Site: Site: Glycine Cycloleucine ACPC

Channel Site: MK-801

AMPA AMPA DNQX Quisqualate CNQX Willardiine NBQX L-Glutamate GAMS GDEE Joro Spider Toxin

Kainate Kainate DNQX Domoate CNQX NBQX GAMS GDEE

Metabotropic Quisqualate AP-3 Trans-ACPD

Abbreviations: (ACPC) 1 -aminocyclopropanecarboxylic acid; (ACPD) 1-amino-1,3- cyclopentane-dicarboxylic acid; (ADCP) cis- 1 -amino- 1 ,3-dicarboxycyclopentane; (AMPA) a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; (AP-3) 2-amino-3- phosphonopropionic acid; (AP-5) 2-amino-5-phosphonovaleriv acid; (AP-7) 2- amino-7-phosphonoheptanoic acid; (CNQX) 6-cyano-7-nitroquinoxaline-2,3-dione; (CPP) 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate; (DNQX) 6,7- dinitroquinoxaline-2,3-dione; (GAMS) D-glutamylamino-methyl sulfonate; (GDEE) diethylester; (MK-801) [(+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine maleate]; (NBQX) 2,3-dihydroxy-6-nitro-7- sulfamoyl benzo(f)quinoxaline; (NMDA) N-methyl-D-aspartate. to , potassium and (MacDermott etal, 1986). This conductance,

however, is voltage dependent and inhibited by (Ascher and Nowak,

1988). In the absence of Mg++ the channel is not voltage dependent (Nowak et al,

1984; Mayer et al, 1984). Thus, the channel is closed at or below resting

membrane potential. As the cell is depolarized, the Mg++ mediated channel block

is lost, and current (carried mostly by sodium but also by potassium and calcium

) flows through the channel. Recently, a receptor has been cloned with

pharmacology consistent with the NMDA receptor (Moriyoshi et al, 1991). This

NMDA receptor shares some homology with the AMPA/kainate receptors. It is of

similar size and has a large N-terminal region and four transmembrane domains.

A negatively charged region of glutamate residues, which precedes the second

transmembrane domain, may be involved in voltage-dependent Mg++ block

(Moriyoshi et al, 1991). Although this receptor is fully functional, it probably

represents a subunit in a homomeric molecular complex. Since depolarizations

elicited in Xenopus oocytes by RNA synthesized from the clone were much

smaller than depolarizations elicited from RNA isolated from rat brain, it is likely that other receptor subunits exist. Furthermore, additional NMDA receptors have been proposed (Monaghan et al, 1989; Stone and Burton, 1987), and another possible NMDA receptor subunit has been cloned with different sequence (Kumar

et al, 1991; see also Mayer, 1991).

Several sites on the NMDA receptor-ionophore complex exist at which conductance can be regulated. Glycine, acting at a strychnine insensitive binding 47

site which colocalizes with the NMDA site, appears to be required for activation of

NMDA receptors (Kleckner and Dingledine, 1988). In fact, two classes of NMDA

receptor based on their differential regulation by glycine have been proposed

(Monoghan et al, 1988). The polyamines and appear to

enhance NMDA receptor function, while the polyamine (which may be

a spermine antagonist) appears to inhibit receptor function (Sacaan and Johnson,

1990). The transition metal has also been shown to block the action of NMDA on cortical neurons (Peters etal, 1987).

The pharmacology of the NMDA receptor has been extensively reviewed elsewhere, (see Meldrum, 1985; Boldry, 1990; Watkins and Collingridge, 1989;

Willetts et al, 1990), and NMDA is the agonist of choice for this receptor.

Antagonists are of three types, competitive antagonists at the NMDA recognition site, non-competitive antagonists which appear to act as channel blockers, and glycine antagonists. Among competitive antagonists, AP5, AP7, CPP, CGS-19755,

NPC12626, and LY233053 have been used, while among noncompetitive channel blockers, MK-801 () is the drug of choice (see Willetts etal, 1990). As the channel blockers such as MK801 and PCP appear to actually bind in the ion channel, they are use dependent; thus, the channel must be open in order for them to act as antagonists. D-Cycloserine is an agonist, while cycloleucine and

ACPC appear to function as antagonists at the strychnine-insensitive glycine site.

The second excitatory amino acid receptor group is made up of the AMPA and kainate ionotropic receptors, often defined negatively as non-NMDA receptors. 48

Activation of these receptors appears to open a voltage independent ion channel permeable to sodium and potassium (See Fagg et al, 1986). Furthermore, these

receptors (AMPA/kainate) are hypothesized to be the primary mediators of rapid excitatory synaptic transmission.(Fagg etal, 1986). While agonists seem to have some degree of selectivity in distinguishing between AMPA and kainate activated responses, antagonists for the AMPA and kainate excitatory amino acid receptors -

including GAMS and the quinoxilinediones DNQX, CNQX, and NBQX - do not.

Anatomical data suggest that the AMPA and kainate receptors are distinct, as these receptors appear to have different distributions in mammalian brain as measured by autoradiography (Monaghan et al, 1984). Functional data indicate that in primary afferent C fibers kainate evokes responses whereas AMPA does not (Agrawal and Evans, 1986). Much electrophysiological data seems to suggest that the AMPA and kainate receptors are actually the same (Jahr and Stevens,

1987; Dingledine, 1991). The use of molecular cloning techniques may, however, have resolved the issue.

Recently, a number of non-NMDA excitatory amino acid receptor subunits have been cloned and sequenced (Barnard and Henley, 1990; Miller, 1991; Dingledine,

1991). They are termed GluR 1 through GluR 6 , in order of their discovery, and KA-

1. These subunits may be arranged in a manner similar to that of the nicotinic acetylcholine receptor. Recall that the nicotinic receptor is composed of five subunits, arranged to form a channel. Thus the non-NMDA excitatory amino acid receptors may have a similar structure. Different combinations of the recently 49

discovered subunits may account for their functional properties. When expressed

in various combinations in Xenopus oocytes, GluR1 through GluR4 appear to have

the characteristics of an AMPA receptor: more sensitive to AMPA than kainate,

rapid desensitization after AMPA, and competitive blockade by CNQX. GluR5,

GluR6 , and KA -1 appear to have the characteristics of kainate receptors: bind

kainate with high affinity, insensitive to or only marginally activated by AMPA.

Thus the molecular biology data suggests the existence of distinct receptor

subtypes.

A third type of excitatory amino acid receptor is the metabotropic receptor. This

receptor is sensitive to pertussis toxin and appears to be linked via a G protein,

Gi, to the phosphoinositide second messenger system (Anwyl, 1991). Thus, activation of phospholipase C and subsequent liberation of inositol 1,4,5- triphosphate leads to calcium mobilization from internal stores. andtrans-ACPD appear to be agonists at this receptor, while AP3 is an antagonist.

A fourth possible excitatory amino acid receptor is sensitive to AP-4. While AP-4 antagonizes electrically evoked excitatory responses at glutamatergic synapses

(measured as inhibition of synaptic transmission) in the cortex, it doesn't block responses elicited by exogenous glutamatergic agonists. Therefore, AP-4 appears to act presynaptically (Davies and Watkins, 1982; Forsythe and Clements, 1988).

Thus there is some speculation that this receptor may represent a glutamatergic autoreceptor.

The interaction between these various receptor subtypes leads to interesting 50

functional implications. In many brain regions, the excitatory post synaptic

potential (EPSP) is composed of two components: a fast, rapidly decaying

component associated with AMPA/kainate receptors; and a slower NMDA

mediated component. Furthermore, AMPA and NMDA receptors appear to be

colocalized in many brain regions (Monoghan et al, 1984; Rainbow et al, 1984).

This evidence suggests that these receptors may lie on the same neurons. By

working together, the excitatory amino acid receptors confer unique abilities on the

cells that express them.

First, an amplification system is achieved. As glutamate is released, it acts at

AMPA/kainate receptors initially thereby depolarizing the cell. At higher levels of

depolarization, magnesium block is relieved at the NMDA receptor, and further

depolarization is achieved. Thus, the NMDA receptor acts as an amplifier of large

EPSPs.

Second, a filtering or selective-activation system may be achieved. As stated above, the NMDA-elicited component of the EPSP is slow, and several factors may account for this. The NMDA receptor has a lower dissociation constant for glutamate, thus, glutamate associates with the receptor longer. The rate of desensitization may also be different (longer for the NMDA receptor). Thus, if depolarizations mediated by AMPA/kainate receptors are occurring frequently, a cumulative depolarization occurs sufficient to open the NMDA channel. In fact, responses in cells of the ventrobasal thalamus evoked by high frequency electrical stimulation of an afferent pathway were blocked by AP5, but responses evoked by 51

low frequency electrical stimulation of the afferent pathway were not blocked by

AP5 (Salt, 1986). Thus it seems that cells expressing the NMDA and non-NMDA

receptors are able to distinguish low and high frequency stimulation.

Third, expressing both receptors may enable cells to act as pacemakers

(Walldn and Grillner, 1987). Following an initial depolarization by sodium influx

(possibly by AMPA/kainate receptors), NMDA receptors will open, allowing the

influx of calcium. This depolarization is initially opposed via voltage gated

potassium channels; however, as calcium enters the cell, calcium-dependent

potassium channels will also open, which gradually repolarize the cell. At a

sufficient level of repolarization, NMDA receptors will close, accelerating

repolarization. Because calcium ions mediate repolarization, when NMDA

receptors close (due to voltage dependent Mg++ block) and intracellular calcium

decreases, a gradual depolarization occurs. If this reaches a level which relieves

Mg++ block, the cycle is ready to begin again (Wallen and Grillner, 1987).

Fourth, the entry of calcium through the NMDA receptor is probably responsible for the induction of long term potentiation (LTP), (for review see Cotman and

Monagham, 1988). LTP was first described in hippocampal cells, in which brief but high frequency electrical stimulation of hippocampal neurons was followed by a long-lasting enhancement of synaptic function (Bliss and Lomo, 1973). Following this stimulation, possibly occurring via non-NMDA ionophore receptors, sufficient depolarization is reached to relieve magnesium blockade and open the NMDA channel, allowing the influx of calcium. The increase in the intracellular 52 concentration of calcium causes the activation of kinase and calmodulin.

These events may cause induction of gene expression in the post synaptic , such as induction of c-fos, and subsequent transcription of which may elicit structural changes at active synapses or, upon secretion, elicit changes in presynaptic terminals. These changes ultimately result in a long term enhancement in the way the cell responds to a subsequent stimulation, or, in other words, a long term modification at the synaptic level. LTP has been implicated in learning and memory, , and satisfies the definition of a Hebbian synapse (Hebb, 1949). A Hebbian synapse is one in which the strength of the synapse will be increased when pre and post synaptic activity occur simultaneously. Neurons rich in NMDA receptors fit this description, as both postsynaptic depolarization (sufficient to relieve magnesium blockade) and presynaptic transmitter release (glutamate) must both occur.

Furthermore, the metabotropic receptor may be involved. There is evidence suggesting that it plays a role in both LTP and the down sensitization of AMPA receptors seen in the cerebellum during another form of neuronal memory, long term depression (see Anwyl, 1991).

Behavioral Functions of the System

There are numerous glutamatergic projections to the accumbens (Fuller et al,

1987), and three functionally defined ionotropic receptors (see below) have been reported in the accumbens (Cotman et al 1987). Behaviorally, direct injection of 53

excitatory amino acids into the nucleus accumbens results in increases in

locomotor activity (Arnt, 1981; Donzanti and Uretsky, 1983). The locomotor activity

elicited by excitatory amino .acids can be inhibited by intra-accumbens

administration of excitatory amino acid antagonists, including GDEE, GAMS, and

DNQX (Donzanti and Uretsky, 1984a; Donzanti and Uretsky 1984b; Shreve and

Uretsky, 1988; Boldry, 1990). Interestingly, increases in locomotion elicited by

psychostimulants can also be reduced by excitatory amino acid antagonists

including AP5, GDEE, and DNQX (Pulverenti et al, 1991; Willins et al, 1992;

Kaddis et al, 1991), and locomotor activity elicited by excitatory amino acids is

reduced by treatments which impair dopaminergic transmission (Boldry, 1990;

Donzanti and Uretsky, 1984a). These data suggest interactions at the level of the

nucleus accumbens between dopaminergic and excitatory amino acid systems

(see Chapter II).

Pharmacology of Opioids

Endogenous Ligands

Endogenous opioids (i.e., compounds that are made in the brain and act on the same receptors as morphine-like compounds) derive from one of three precursors which comprise the family. These precursors include (source of 8 -, and y-endorphin), (source of the ), and proenkephalin B (source of ). All the opioid peptides contain the sequence Tyr-Gly-Gly-Phe-X, with X being Met or Leu (for review see 54

Simon and Hiller, 1989).

Receptors and Pharmacology

Opiate binding sites were first discovered in 1973 (Pert and Snyder, 1973).

Since then a number of receptors have been characterized, including the p 1 , p 2 ,

8 , k , and e receptors (for review see Chang, 1984). Of these \i, 8 , and k receptors

are the best characterized, with morphine, enkephalin, and ketocyclazocine acting

as respective agonists. All of these receptors have more selective agonists and

antagonists (see Table 2), but all are antagonized by . These receptors

display a regional distribution within mammalian brain (for review see Atweh, 1983;

Mansour et al, 1987). The neurophysiological role of these receptors appears to

be activation of a G protein, which results in either an increase in potassium

current (thus hyperpolarizing the cell and decreasing firing rate); or a decrease in

calcium influx (thus acounting for a decrease in transmitter release).

Behavioral Functions of the System

Opiate receptors appear to play a major role as neuromodulators, affecting the

release or post-synaptic response to a wide variety of neurotransmitters including dopamine, epinephrine, serotonin, acetylcholine, and substance P. Opiate

receptors have been implicated in a host of physiological and behavioral functions.

Physiological roles include analgesia and pain perception, stress regulation, and

respiratory and temperature regulation. Opioid receptors also appear to play a role 55

Table 2. Pharmacological Classification of Opioid Receptors

Receptor Agonist Antagonist

Morphine Naloxone DAMGO CTAP Sufentanyl PL017

5 DTLET Naloxone DPDPE IC1174864

K Ketocyclazocine Naloxone U69593 nor- CI977 ICI197067 U50488.H

Abbreviations: (CTAP) D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; (DAGO) Tyr-D- Ala-Gly-MePhe-Glyol; (DPDPE) Tyr-D-Pen-Gly-Phe-D-Pen; (DTLET) [D-Thr2]- leucine enkephalin-Thr; (ICI74864) N.N-bisallyl-tyr-Aib-Aib-Phe-Leu-OH {Aib: aminoisobutyrate}; (U50488H) trans-(±)-3,4-dichloro-n-methyl-[2-(1 - pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulphonate 56 in behavioral functions including feeding, movement regulation including both catalepsy and locomotor activity, and emotional behavior involving regulation of stress and reward. Disorders of opioid systems may play a role in the etiology of and depression (for a general review, see Simon and Hiller, 1989;

Hughes et al, 1984).

Pharmacology of the Serotonin System

Endogenous Ligands

Serotonin (5-hydroxytryptamine, 5-HT) neurons originate in raphe nuclei of the mesencephalon. The firing of serotonin cells of the raphe nuclei is normally regular and appears to correlate with the state of arousal. Thus, raphe neuron activity is high during waking hours and low during REM sleep (Aghajanian and

Vandermaelen, 1986). On the other hand, lesions of the serotonergic system generally produce behavioral activation (Fibiger and Campbell, 1971). The nucleus accumbens receives serotonergic projections from the medial and dorsal raphe nuclei (Parent et al, 1981).

Receptors and Pharmacology

Serotonin exerts its actions by binding to any of several distinct receptors, including the 5-HT, (subdivided into 5-HT1A, 5-HT1B, 5-HT1c), 5-HT2, 5-HT3, and 5-

HT4 receptors, each having distinct agonists (see Table 3). The antagonist pharmacology is still in its early stages, and few selective antagonists are known 57

Table 3. Pharmacological Classification of 5-HT Receptors

Receptor Agonist Antagonist

5-HT1A 8 -OH-DPAT NAN-190 m-CPP

5-HT1b 5-Carboxy-AT ICS 21-009 m-CPP

CGS-12066B • TFMPP

5-HT1c (+)S-a-Methyl-5-HT DOI

5-HT2 (+)S-a-Methyl-5-HT DOI Ritanserin Pizotifen

5-HT3 2-Methyl-5-HT ICS 205-930 1 - MDL72222

5-HT4 BIMU8 DAU 6285 SD2205557 5-methoxytryptamine

Abbreviations: (5-HT) 5-hydroxytryptamine; (CGS-12066B) (±)-12,5-dimethoxy-4-iodo)-2-aminopropane; (DOI) (±)-1 -(2,5-dimethoxy-4- i o d o p h e n y I) - 2 - a m i n o p r o p a n e HCI; (8 -OH-DPAT) 8 -hydroxy- 2 -(di-n-propylamino)-tetralin HBr; (mCPP) 1 -(3-Chlorophenyl) HCI; (MDL-7222) 3-tropanyl-3,5-dichlorobenzoate; (NAN-190) 1-(2-methoxyphenol)- 4-[4-(2-phthalimido)butyl]piperazine hydrobromide; (TFMPP) m- trifluoromethylphenylpiperizine. 58

(for review see Peroutka, 1988; Sanders-Bush, 1989). The 5-HT1t 5-HT2, and 5-

HT4 receptors all appear to act through a G protein linked to second messenger

systems and mediate slow modulatory responses. The 5-HT1A, 5-HT1c, and 5-HT 2

receptors have been cloned and display the seven transmembrane domains typical

of receptors in the G-protein coupled superfamily (see Hartig, 1989). While the 5-

HT1A, 5-HT1b, and 5-HT 4 receptors appear to be linked to adenylate cyclase, 5-

HT1C and 5-HT 2 receptors (which display great homology) are linked to the phosphoinositide second messenger system (Hartig, 1989).

In contrast, the 5-HT 3 receptor - cloned recently (Maricq etal, 1991) - activates fast depolarizing currents in neurons. While only one subunit has thus far been cloned (termed 5-HT 3R-A), it is likely that there are others, characteristic of the excitatory -gated ion channel superfamily. It is also likely that these subunits form a heteromeric complex; however, 5-HT 3R-A alone expressed in oocytes was able to form a functional receptor-ionophore with pharmacology and electrophysiology consistent with the 5-HT 3 receptor. 5-HT 3R-A has 27% homology with the nicotinic acetylcholine receptor and has four transmembrane domains (termed M1 through M4), a large amino-terminal extracellular domain, and a large intracellular domain connecting M3 to M4 (Maricq et al, 1991).

Behavioral Functions of the System

Serotonin is thought to play a role in many behavioral and physiological events, such as the regulation of body temperature, appetite and affective state. 59

Serotonin neuronal systems have also been shown to modulate locomotor activity mediated by the dopaminergic system (for review see Gersen and Baldessarini,

1980; Soubrie etal, 1984; see also chapter IV).

Serotonin systems may also be involved in the reward pathway. Lysergic acid diethylamide (LSD), a hallucinogen which decreases the firing of raphe neurons

(see Aghajanian and Vandermaelen, 1986) possibly by acting as a 5-HT autoreceptor agonist, is used by humans to induce a state of altered perception

(Jaffe, 1985). LSD and related indolamines may be agonists at 5-HT1A and 5-HT 2 receptors (Aghajanian etal, 1988; Rasmussen and Aghajanian, 1990). While this drug is not rewarding in animal models (Jaffe, 1985), it does have discriminitive stimulus properties. Simply, although animals will not work to get LSD, they can tell the difference between LSD and saline (Broadbent and Appel, 1990). The nucleus accumbens may be involved in the subjective effects of LSD. Animals cannot discriminate between systemic injections of LSD and injections of LSD in the nucleus accumbens, but can discriminate between systemic injection and injection into the caudate nucleus (Neilsen and Scheel-Kruger, 1986).

As is quite often the case, literature involving serotonin and drug reward in animal models is contradictory and confusing. Serotonin antagonists block amphetamine self-administration (Lecesse and Lyness, 1984; see also Lyness and

Moore, 1983) and both amphetamine- and morphine-induced place preference

(Nomikos and Spyraki, 1988). On the other hand, increasing serotonin transmission also decreases self-administration of (Lecesse and Lyness, 1984) and 60 conditioned place preference elicited by amphetamine (Kruszewska etal, 1986).

5 -HT3 antagonists block morphine- and nicotine-induced place preference, but not amphetamine-induced place preference (Carboni et al, 1989). Lastly, a recent study has proposed that the serotonin system may play a role in setting limits on the "value" of certain reinforcers (Woger etal, 1991). These studies suggest that

5-HT plays a complex role in drug reward, and more study is required to understand the substrates at which serotonin exerts its effects.

STATEMENT OF THE PROBLEM

Recent research involving the neurobiology of drug reward has suggested that abused drugs trigger activity in a common reward circuit (Koob and Bloom, 1988;

Wise and Bozarth, 1984). This common reward circuit involves neurons which form a ventral tegmentum-nucleus accumbens-ventral pallidum-frontal cortex pathway (Koob and Bloom, 1988). Elements of this circuit also appear to comprise the neural substrate of locomotor activity. The overlap of the neural substrates for both locomotor activity and reward has led to the tempting hypothesis that this common substrate may mediate goal-directed behavior (Swerdlow etal, 1986).

The positive reinforcement model of addiction holds that it is drug reward which motivates an addict to use a drug. The nucleus accumbens, an integral part of the proposed reward substrate, appears to be an interface between limbic and motor areas of the brain (Mogenson and Yim, 1981). A pathway through the nucleus accumbens and incorporating dopamine (Kuczenski, 1983) forms the neural 61 substrate of opiate and psychostimulant action (Swerdlow, 1986; Evenden and

Ryan, 1990) and seems to mediate both the rewarding and locomotor effects of several classes of drugs. Thus, the purpose of the present research was to study the chemoanatomy of the reward substrate (with emphasis on the nucleus accumbens) by examining the effects on locomotor activity and reward (as measured by conditioned place preference) of various classes of drugs which might interact with this pathway.

While a great deal is known about the role of dopamine systems in the nucleus accumbens, relatively little is known about the role of other systems in reward and locomotor activity. Thus the specific aims of this project are trifold:

1 ) While several studies have evaluated the role of excitatory amino acid receptors in locomotor activity, few have examined their role in drug reward. The first aim of this set of experiments was therefore to examine the role of excitatory amino acid receptors in drug reward, using the conditioned place preference paradigm as a measured response.

2) In contrast to the above, the role of opiate receptors in the nucleus accumbens in drug reward has received much , while the role of these receptors in locomotion has not. Thus the aim of this set of experiments was to examine the role of opiate receptors, and especially the p receptor, in the expression of locomotor activity. 62

3) The role of serotonin systems in the nucleus accumbens in locomotor activity

has been studied in the past. However, recent advances in the characterization of different serotonin receptors, and the development of selective ligands for each,

makes a reevaluation of the role of serotonin necessary. Thus the aim of this set of experiments was to determine the contribution of the different serotonin receptor subtypes in the nucleus accumbens in the regulation of locomotor activity.

Together, these studies shed new light on the roles and chemoanatomy of the various neurotransmitter systems in the nucleus accumbens. Thus, these studies may contribute to the growing understanding of reward substrates. It seems likely that a better understanding of the reward substrate will lead to better treatments for the tragic problem of drug addiction. In addition, a better understanding of the reward substrates will ultimately lead to an understanding of affective states. CHAPTER II

THE ROLE OF EXCITATORY AMINO ACID RECEPTORS IN DRUG REWARD

The mesolimbic dopamine system and especially its terminal fields within the

nucleus accumbens (NA) comprise a critical substrate for the locomotor activating

and rewarding properties of amphetamine (Carr and White, 1983; Swerdlow etal,

1986; Koob and Bloom, 1988). The NA, described as a functional interface

between limbic and motor systems, may mediate goal directed behavior

(Mogenson and Yim, 1981; Swerdlow et al, 1986). Recent studies suggest that

glutamatergic and dopaminergic systems may interact in the nucleus accumbens

(Arnt, 1981; Donzanti and Uretsky, 1983). Furthermore, glutamatergic afferents from limbic structures such a s amygdala and hippocampus may modulate within the nucleus accumbens the behavioral effects of psychostimulants (Mogenson and

Yim, 1981).

The following sections present the results of studies designed to examine the role of NMDA receptors and AMPA/kainate receptors in a measure of reward, conditioned place preference. Additionally, effects on locomotor activity were assessed.

63 64

THE NMDA RECEPTOR ANTAGONIST MK-801 ELICITS CONDITIONED PLACE

PREFERENCE IN RATS

Introduction

The N-methyl-D-aspartate (NMDA) receptor, the best characterized of the

excitatory amino acid receptors, has been implicated in such physiological

functions as learning, memory, and locomotor activity. Overstimulation of this

receptor has been implicated in neuronal and associated neurological

conditions (for review see Wroblewski and Danysz, 1989). MK-801 [(+)-5-methyl-

10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] is a potent

noncompetitive antagonist of central NMDA receptors (Wong etal. 1986), which

attenuates neurological damage induced by (Gill etal, 1987) and from

high doses of (Sonsalla et al, 1988). Such observations

suggest that drugs like MK-801 might be useful in minimizing neurological damage

associated with certain .

Even though MK-801 prevents biochemical changes induced by high doses of

methamphetamine, the NMDA antagonist produces behavioral effects that

resemble the effects of amphetamine. Thus, after systemic injection, MK-801, like amphetamine, stimulates locomotor activity (Clineschmidt et al, 1982) and

increases the release of dopamine in limbic areas (Lbscher etal. 1991; Rao etal.

1990). As these effects are often associated with rewarding drugs, it follows that

MK-801, like amphetamine, might also be rewarding. Indeed, it has recently been 65

demonstrated that MK-801 facilitates brain stimulation reward (Corbett, 1989;

Herberg and Rose, 1989) and is self-administered in rhesus monkeys (Beardsley

etal. 1990).

One complication of self-administration studies with drugs which stimulate

hypermotility, such as amphetamine or MK-801, is separation of the influence of

motility from the reward process. One technique that separates these effects is the conditioned place preference paradigm. In this model, animals are injected with drug during the conditioning procedure but are tested for conditioning in the

absence of drugs. Consequently, the conditioned place preference would not be

influenced by the direct motor effects of drugs. In the present study we have

evaluated the ability of MK-801 to produce a conditioned place preference to determine if it is similar to amphetamine in its ability to elicit a place preference.

Furthermore, the effect of MK-801 on locomotor activity was assessed during the training period. Amphetamine was administered as a positive control for both behavioral activation and conditioned place preference.

Methods

Male rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 250 to 350 g, were housed 4 to a cage in a temperature controlled (23 ± 1° C) room with a 12 hour light-dark cycle. A 3-compartment conditioned place preference apparatus with a small central compartment (10 x 10 cm and 56 cm high) separating 2 larger compartments (38 x 76 cm and 56 cm high) was used. Guillotine doors separated 66

the compartments. The small compartment was painted grey while the two larger

compartments were painted either white or black. The white compartment had a

floor composed of steel rods (1.5 cm apart), and the black compartment had a

floor composed of wire mesh. Distance traveled and time spent in each

compartment were monitored with a Videomex-V tracking system (Columbus

Instruments, Columbus, OH). All training or test sessions were done between 8:00

am and 4:00 pm in an isolated environmental room maintained at a temperature

of 23±1°C.

Place preference conditioning occurred over a 4 day period. On the day prior

to training animals were allowed to freely explore the apparatus for 15 minutes with

the guillotine doors open, thus establishing a baseline preference, with the

chamber they spent the least time in considered the non-preferred side. On days

1 and 3 of training, rats were injected with drugs or saline and confined to the non­

preferred side for 35 minutes, while on days 2 and 4 all groups were injected with

saline and confined to the preferred side for 35 minutes. Rats were tested for

place preference on day 5. Rats were placed in the small grey compartment with the doors closed. The doors were then opened, and the amount of time spent in

each compartment during a 15 minute test period was recorded.

Place preference data are expressed as the difference in minutes between time

spent in the drug paired side (CS+; least preferred initially) and the time spent in the saline paired side (CS-). Values from both pretest baseline and test conditions

are shown, with animals that acquire a place preference shifting their preference 67 to the drug paired side. Locomotor data from the first and third training days (in which the animals received the drug) are given as distance traveled (cm). Ail data are expressed as the mean and the standard error of the mean (SEM). The data were evaluated statistically using the nonparametric Kruskal-Wallis one-way analysis of variance followed by the one-tailed Mann-Whitney U-test, with p<0.05 accepted as significant.

Results

Figure 5 demonstrates that both MK-801 (0.1 mg/kg s.c.) and d-amphetamine

(1 .0 mg/kg s.c.) produced a conditioned place preference when paired against saline. Animals injected with saline on the least preferred side did not change their place preference. The administration of both MK-801 and d-amphetamine at these doses resulted in the stimulation of locomotor activity, expressed as total distance traveled (Figure 6 ). The locomotor activity was significantly higher on day 1 for both drugs; however, it was more variable on day 3 for the MK-801 animals which displayed a greater intensity of stereotyped behavior. No differences in locomotor activity were observed between any group on days 2 and 4 when all rats were given saline in the preferred side.

Discussion

These data demonstrate that MK-801, at a dose which stimulates locomotor activity, produces a conditioned place preference. The stimulation of locomotor 68

[=□ PRETEST BASELINE ^ 9 TEST

SALINE d-AMPH MK-801

Figure 5. Place preference elicited by MK-801 and d-amphetamine. MK-801 (0.1 mg/kg s.c.) and d-amphetamine ( 1.0 mg/kg s.c.) were paired with the less preferred compartment. Data are expressed as the mean ± SEM of the difference in time spent in the drug paired side (CS+) and the vehicle paired side (CS-) for both pretest and test conditions. 69

□ SALINE E 3 d —AMPHETAMINE E 5000 o M K-801 CO U i t j 4000

£ 3000 Q LU _ i 2000 i

1“ u 1000 i o i Ii Q DAY 1 DAY 3

Figure 6 . Locomotor activity elicited during conditioning in the same rats by MK- 801 , d-amphetamine , or saline. Doses used were: MK-801 (0.1 mg/kg s.c., n= 6 ), d-amphetamine (1.0 mg/kg s.c., n=5), or saline (n=7). Locomotor activity is expressed as the mean distance traveled (cm) ± SEM for the number of observations indicated. *p<0.05 with respect to saline control. 70 activity elicited by MK-801 is similar to that elicited by a dose of d-amphetamine which similarly elicits a place preference. Only two conditioning periods with the drugs were necessary to produce a conditioned place preference. This finding is consistent with the results of recent studies demonstrating that MK-801 facilitates lever pressing for brain stimulation reward and is self administered by rhesus monkeys (Beardsley etal, 1990; Corbett, 1989; Herberg and Rose, 1989). Other noncompetitive antagonists of the NMDA channel, such as ketamine and phencyclidine, are self administered by animals (Marquis and Moreton, 1987) and are abused by humans (Lerner and Burns, 1978). Furthermore, MK-801 has phencyclidine-like discriminitive properties (Beardsley etal, 1990).

While d-amphetamine appears to produce its rewarding properties by causing the release of dopamine from nerve terminals in the nucleus accumbens, the site of action of MK-801 in producing conditioned place preference is unclear. In one recent study of male rats, MK-801 did not appear to increase dopamine turnover in the nucleus accumbens (Rao etal, 1990), while in a separate study using a higher dose of MK-801 in female rats an increase in dopamine turnover was observed (Loscher etal, 1991). MK-801 has been shown to increase the firing of dopaminergic neurons from the ventral tegmental area (French and Ceci, 1989) and increase dopamine turnover in the mesocortex (Loscher et al, 1991; Rao et al, 1990), which contains terminal fields of these neurons. It is therefore possible that these dopaminergic neurons, which have been proposed to play a significant role in the rewarding action of cocaine, may mediate the place preference produced by MK-801.

Alternatively, it has also been shown that MK-801 elicits locomotor activity after direct injection into the nucleus accumbens (Raffa, 1989). This effect appears to be independent of dopamine as it is seen even after monoamine depletion

(Carlsson and Svensson, 1990). Further studies will be required to resolve this issue.

In contrast to this finding, another noncompetitive NMDA antagonist which is abused by humans, PCP, has been shown to elicit a place aversion in rats at several doses (Acquas etal, 1989; Barr etal, 1985). While both MK-801 and PCP act as noncompetitive antagonists of the NMDA receptor and MK-801 produces many PCP-like effects, PCP is also a ligand at sigma receptors. It is therefore possible that the aversive effects of PCP as measured in conditioned place preference paradigms are not due to its ability to antagonize the NMDA receptor but may be related to its actions at the .

The conditioned place preference paradigm has been used as a means of evaluating the rewarding properties of drugs. A variety of drugs of abuse can produce conditioned place preferences. The finding that MK-801 can establish a conditioned place preference suggests that this drug may have abuse potential. 72

A ROLE FOR AMPA/KAINATE RECEPTORS IN THE NUCLEUS ACCUMBENS

IN DRUG-INDUCED REWARD

Introduction

The nucleus accumbens, a region of the basal forebrain, has been implicated as a critical substrate in the expression of locomotor activity and drug reward

(Koob and Bloom, 1988; Swerdlow et al, 1986). A role for dopaminergic in the nucleus accumbens in the expression of locomotor activity and drug reward is well established (Phillips and Fibiger, 1979; Wise 1978;

Evenden and Ryan, 1990; Beninger, 1983; Dunnett et al, 1984; Fink and Smith,

1980; Mithani etal, 1986; Le Moal and Simon, 1991; Carr and White, 1983).

Recent studies have suggested an interaction between glutamatergic and dopaminergic systems within the nucleus accumbens. Several lines of evidence support this role. First, anatomical data suggest that the nucleus accumbens receives glutamatergic inputs from several cortical and thalamic structures, including the prefrontal and olfactory cortex, the subiculum, various nuclei of the amygdala (most prominent the lateral and basolateral nuclei), and the midline nucleus of the thalamus (Fuller et al, 1987). Furthermore, the three functionally defined glutamatergic, ionotropic receptors, AMPA, kainate, and NMDA, have been reported in the nucleus accumbens (Cotman et al 1987). Dopaminergic and glutamatergic neurons have been suggested to synapse on the same cell bodies in the striatum and nucleus accumbens (Freund et al, 1984; Kemp and Powell, 1971; Doucet, et al, 1986; Totterdell and Smith, 1989; see also Wilson and

Groves, 1980; Smith and Bolum, 1990). In addition, glutamatergic interneurons

have been demonstrated in the nucleus accumbens (Fonnum and Walaas, 1981).

Second, electrophysiological data suggests that within the nucleus accumbens

dopamine may modulate glutamatergic inputs from areas such as hippocampus

and amygdala (Yang and Mogenson, 1984; Yang and Mogenson, 1986; Yim and

Mogenson, 1982; Yim and Mogenson, 1986; Yim etal, 1991). Third, a reciprocal

relationship between dopamine and glutamate seems to exist (Chesselet, 1984)

such that dopamine inhibits glutamate release (Mitchell and Doggett, 1980;

Rowlands and Roberts, 1980) and glutamate acting at either NMDA (Mount etal,

1990; Marien et al, 1983; Jhamandas and Marien, 1987; Jones et al, 1987) or

AMPA/kainate receptors (Imperato et al, 1990; Desce et al, 1991) increases

dopamine release (also see Roberts and Andersen, 1979; Chdramy et al, 1986;

Romo et al, 1986; Kalivas etal, 1990). Fourth, dopamine may directly modulate the activity of excitatory amino acid-gated ion channels by increasing the open-time of the channel, thereby increasing the current transmitted through the channel

(Knapp and Dowling, 1987; Knapp etal, 1990).

A role for glutamatergic NMDA and AMPA/kainate receptors in the nucleus accumbens in mediating locomotor activity is emerging. Injection of glutamatergic agonists into the nucleus accumbens stimulates coordinated locomotor activity

(Donzanti and Uretsky, 1983; Boldry and Uretsky, 1988) that is inhibited by treatments which impair dopaminergic transmission, such as antagonism by 74

neuroleptics (Arnt, 1981) or inhibition of tyrosine hydroxylase with a-methyl-p-

tyrosine (Boldry et al, 1991). Injection of glutamatergic antagonists into the

accumbens inhibits locomotor activity elicited by either novelty (Mogenson and

Nielsen, 1984), or by psychostimulants such as amphetamine or cocaine which

require dopamine (Willins etal, 1992; Pulvirenti etal, 1989; Pulvirenti etal, 1991;

Kaddis etal, 1991).

As many drugs abused by humans both activate locomotor activity in and are

self-administered by animals, it has been proposed that a high degree of overlap

exists between neural substrates mediating the locomotor activating and positively

reinforcing effects of drugs (Swerdlow et al, 1986; Koob and Bloom, 1988).

Furthermore, treatments which inhibit the increased locomotor activity elicited by these drugs similarly reduce their positively reinforcing effects. For example, intra-

accumbens neuroleptic treatment or specific 6 -hydroxydopamine lesions reduce

both the locomotor activating and rewarding effects of a psychostimulant

(amphetamine), and intra-accumbens methylnaloxonium administration reduces

both the locomotor activating and rewarding effects of an opiate (heroin).

Thus, while a role for glutamatergic neurotransmission in the nucleus accumbens as a mediator of locomotor activity is emerging, it is unclear whether glutamatergic transmission plays a role in drug reward. In light of the recent findings that AMPA/kainate excitatory amino acid receptor antagonists block amphetamine elicited locomotion, the purpose of the present study was to determine whether the positively reinforcing effect elicited by d-amphetamine 75

similar requires activation of the AMPA/ in the nucleus accumbens.

Additionally, a s it has been proposed that opiates may interact with a reward

substrate in the nucleus accumbens which is distinct from that of amphetamine,

the role of AMPA/kainate receptors in the nucleus accumbens in mediating the

positively reinforcing effect elicited by morphine is assessed.

The conditioned place preference (CPP) paradigm, in which animals increase their contact with an environment (the conditioned stimulus) previously paired with

rewarding stimuli (the unconditioned stimulus), is an effective means for assessing the effects of drugs on the neural substrates of motivation and reward (Hoffman,

1989). This paradigm is useful for assessing the effects of drugs on both the

acquisition of reward, in which a drug is administered concurrently with the

rewarding (unconditioned) stimulus during training, and the expression of reward,

in which the drug is administered in the presence of the conditioned stimulus (the environment) on the test day. The purpose of the present study, therefore, was to determine if the AMPA/kainate receptor antagonist DNQX could inhibit the acquisition or expression of a place preference elicited by rewarding drugs, specifically either d-amphetamine or morphine.

Methods

Animals

Male rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 250 to 350 g, were housed 4 to a cage in a temperature controlled (23 ± 1° C) room with a 12 76 hour light-dark cycle.

Surgery

Each rat was anesthetized with ketamine ( 10 mg/kg i.p.) and xylazine (90 mg/kg i.p.) and placed in a stereotaxic frame (David Kopf Instruments, Tajunga,

CA) with the toothbar set at -3.3 mm. The rats were then implanted with a 22 gauge stainless steel guide cannula (Plastic Products Co., Roanoke, VA) aimed at a 10° angle 2 mm above the nucleus accumbens at the coordinates (A 10.2; L -

2 .0 ; H 1 .2 ; measured from the interaural line) using the stereotaxic guide of

Paxinos and Watson (1982). Cannulae were secured with dental acrylic cement anchored to the skull by two stainless steel screws. Animals then received

Chloramphenicol (1 0 0 mg/kg, i.m.) to protect against infection and were allowed at least five days to recover.

All drugs were administered via a 28 gauge cannula (Plastic Products) projecting 2 mm beyond the guide cannula. Drugs or vehicle were injected in a 0.5

|il volume at a rate of 0.5 pl/min.

Drugs

Morphine sulphate and d-amphetamine sulphate were purchased from Sigma

Chemical Co. (St. Louis, MO). 6,7-dinitroquinoxaline-2,3-dione (DNQX) was obtained from TocrisNeuramin (Essex, England). 6-amino-7-fluoroquinoxaline-2,3- dione (AFQX) was generously provided by Dr. Duane D. Miller (The Ohio State 77

University, College of Pharmacy, Division of Medicinal Chemistry). DNQX and

AFQX were dissolved in 0.1 N NaOH and adjusted to the appropriate volume with

water, and the solutions were adjusted to pH 9.0 with 0.1 N HCI. The doses of

DNQX and AFQX shown refer to the amount injected on each side of the nucleus

accumbens; control animals received an equal volume (0.5 pi) of vehicle.

Morphine sulphate and d-amphetamine sulphate were dissolved in normal saline.

Conditioned Place Preference Procedure

A 3-compartment conditioned place preference apparatus with a small central compartment (10 x 10 cm and 56 cm high) separating 2 larger compartments (38 x 76 cm and 56 cm high) was used. Guillotine doors separated the compartments.

The small compartment was painted grey while the two larger compartments were painted either white or black. The white compartment had a floor composed of steel rods (1.5 cm apart), and the black compartment had a floor composed of wire mesh. Distance traveled and time spent in each compartment were monitored with a Videomex-V tracking system (Columbus Instruments, Columbus, OH). All training or test sessions were done between 8:00 am and 4:00 pm in an isolated environmental room maintained at a temperature of 23 ± 1 0 C.

Place preference conditioning occurred over an 8 day period. On the day prior to training, animals were allowed to freely explore the apparatus for 15 minutes with the guillotine doors open, thus establishing a baseline preference, with the chamber they spent the least time in considered the non-preferred side. On days 78

1,3,5, and 7 of training, rats were injected with drugs (amphetamine, 1 mg/kg s.c.; or morphine, 1 0 mg/kg s.c.) or saline and confined to the non-preferred side for 35 minutes, while on days 2, 4, 6 , and 8 all groups were injected with saline and confined to the preferred side for 35 minutes. Rats were tested for place preference on day 10. Rats were placed in the small grey compartment with the doors closed. The doors were then opened, and the amount of time spent in each compartment during a 15 minute test period was recorded.

Place preference data are expressed as the difference in minutes between time spent in the drug paired side (CS+; least preferred initially) and the time spent in the saline paired side (CS-). Values from both pretest baseline and test conditions are shown, with animals that acquire a place preference shifting their preference to the drug paired side. Locomotor data from the training days (the days in which the animals received the drug) are given as distance traveled (cm). All data are expressed as the mean and the standard error of the mean (SEM).

Catalepsy Test

Catalepsy was measured using a 9-cm cork test immediately after the training period during the morphine-DNQX acquisition experiment. Rats were placed with both forepaws on the cork, and the time of maintaining both forepaws on the cork was measured. The test was terminated at 180 s, even if rats had not moved the forepaws. The time in which rats remained in this position was recorded, and catalepsy was scored in a graded fashion: rats remaining cataleptic for up to 30 79

s were scored a 1; 30 to 60 s were scored a 2; 60 to 90 s were scored a 3; 90 to

120 s were scored a 4; 120 to 150 s were scored a 5; 150 to 180 s were scored

a 6 ; and over 180 s were scored with a 7.

Histology

After each experiment, animals were decapitated, and their brains were removed and fixed in a 10% solution of formalin for 48 hours. Frozen sections (40 pm) were cut using a Cryo-Cut microtome (American Optical Corp., Buffalo, NY) to check the location of the tip of the injection needle track. When the tips of the needle tracks were found to lie outside the nucleus accumbens, the data for that animal were excluded from the study.

Statistics

The amphetamine place preference and locomotor data were evaluated statistically with an analysis of variance (ANOVA) followed by the Newman-Keuls multiple range test for comparison of individual groups, with p<0.05 accepted as significant. Morphine place preference and locomotor data were evaluated with the students t test, and catalepsy data were evaluated using the Mann-Whitney U test.

Results

The first experiment was done to determine whether DNQX would affect amphetamine-induced place preference after two pairings between the least preferred side and amphetamine. Under these conditions, d-amphetamine (1 mg/kg s.c.) elicited a place preference after only two pairings in non-canualted animals. However, when injections of either DNQX or vehicle were administered simultaneously into the nucleus accumbens of cannulated animals, the expression of place preference was not seen (Figure 7). The administration of d-amphetamine resulted in the stimulation of locomotor activity, expressed as total distance traveled (Figure 8 ). Consistent with a previous report (Willins et al, 1992), the locomotor activity was significantly inhibited on day 1 by DNQX. However, DNQX did not inhibit locomotor activity on day 3 (Figure 8 ).

In the next experiment, due to the failure of cannulated controls to form a place preference after two amphetamine pairings (with the least preferred side), four amphetamine-least preferred side pairings were done. d-Amphetamine (1 mg/kg s.c.) did elicit a place preference in cannulated control animals after four pairings

(Figure 9) even after vehicle injection. Simultaneous intra-nucleus accumbens administration of DNQX (1.0 pg/0.5 pi) significantly inhibited the acquisition of amphetamine-induced place preference (Figure 9). Similar to the previous experiment, DNQX inhibited locomotor activity on day 1, but not on days 3,5, and

7 (Figure 10). In a separate experiment, administration of DNQX (1.0 pg/0.5 pi) after training, on the test day, significantly inhibited the expression of amphetamine-induced place preference (Figure 11). This dose of DNQX did not inhibit the basal locomotor activity of the animals during the test (Figure 12). In experiments to test the specificity of DNQX, administration of a structural analog of DNQX which competes poorly or not at all for AMPA receptors, AFQX (Supko

et al, 1990), did not inhibit the acquisition or expression of amphetamine-induced

place preference.

In the next experiment, to test the specificity of this response, a different

rewarding drug was used. Morphine has also been shown to elicit a place

preference, but the anatomical substrates for this preference may be different than

those of d-amphetamine. Morphine (10 mg/kg s.c.) elicited a place preference in

cannulated control animals after four pairings (Figure 13). Administration of DNQX

(1.0 pg/0.5 pi) after training, on the test day, significantly inhibited the expression

of morphine-induced place preference (Figure 13). in contrast, pairing of intra­

nucleus accum bens administration of DNQX (1.0 pg/0.5 pi) with systemic morphine

during the training did not significantly inhibit the acquisition of morphine-induced

place preference (Figure 14). Locomotor activity of rats during the acquisition

experiment with the combination of morphine and DNQX was significantly-lower than morphine alone controls (Figures 15). In addition, the dose of morphine used

resulted in a mild catalepsy (Figure 17). Interestingly, the catalepsy elicited by

morphine was potentiated by simultaneous intra-nucleus accumbens administration of DNQX (1.0 pg/0.5 pi) (Figure 17). However, while the DNQX-induced inhibition of d-amphetamine elicited locomotor activity was only seen on the first day, DNQX potentiated morphine catalepsy on all days tested. In both cases, while rats were cataleptic, the righting reflex was unaffected. The catalepsy assessment occurred immediately after the training period. Rats appeared cataleptic in the training 82

CHI PRETEST BASELINE ESJ TEST 4

2 co o 0 -2 L d - 4

I - 6 I- + GO - 8 o -10 I-

L d -12 d-AMPH SALINE d-AMPH d-AMPH + + + VEH VEH DNQX

Figure 7. The effect of DNQX on place preference elicited by only two pairings with d-amphetamine. The place preference elicited by only two pairings with d- amphetamine (d-amph; 1.0 mg/kg s.c.) in the less preferred compartment (CS+) is inhibited by both DNQX (1.0 pg/0.5 pi) or vehicle simultaneously administered into nucleus accumbens. Data are expressed as the mean ± SEM of the difference in time spent in the d-amph paired side (CS+) and the saline paired side (CS-) for both pretest and test conditions. *p<0.05 with respect to saline control. 83

CD SALINE (n = 4 )

ZZA d-AMPH + VEH (n=7)

d-AMPH + DNQX (n=9)

£ 6000 0 v_' (/) h-Ll I 5000 3 Z ZS 4000 m rO \ Q 3000 a 2000 1H- Ui O 1000

1 (/) 0 a I DAY 1 DAY 3

Figure 8 . The effect of DNQX simultaneously administered into nucleus accum bens on the locomotor activity elicited by d-amphetamine. DNQX (1.0 |ig/0.5 pi) simultaneously administered into nucleus accumbens inhibits the locomotor activity elicited by d-amphetamine ( 1.0 mg/kg s.c.) on day 1 , but not day 3. Locomotor activity is expressed as the mean distance traveled (cm) ± SEM for the number of observations indicated. *p<0.05 with respect to saline control. 84

C O PRETEST BASELINE E229 TEST

I co o

I + CO o

L d

CONTROL d-AMPH d-AMPH d-AM PH

Figure 9. Effects of DNQX and AFQX on the acquisition of an amphetamine- induced conditioned place preference. DNQX (1.0 pg/0.5 pi), but not AFQX (1.0 pg/0.5 pi), simultaneously administered into nucleus accumbens inhibits the acquisition of a place preference elicited by d-amphetamine (d-amph; 1.0 mg/kg s.c.) after 4 pairings with the non-preferred compartment (CS+). Data are expressed as the mean ± SEM of the difference in time spent in the d-amph paired side (CS+) and the saline paired side (CS-) for both pretest and test conditions. *p<0.05 with respect to saline control. 85

□ SALINE (n = 6 ) Em d-AMPH + VEH (n= 8 ) E 3 d-AMPH + DNQX (n= 6 ) E 6000 d-AMPH + AFQX (n=4) o u 5000 3 4000 - in ro \ Q 3000 ■ L d

2000

Ld 0 1000 Z

1Q DAY 1 DAY 3 DAY 5 DAY 7

Figure 10. Effects of DNQX and AFQX on the locomotor activity induced by amphetamine during conditioned place preference training. DNQX (1.0 pg/0.5 pi), but not AFQX (1.0 pg/0.5 pi), simultaneously administered into nucleus accumbens inhibits the locomotor activity elicited by d-amphetamine ( 1 .0 mg/kg s.c.) on day 1. Locomotor activity was not inhibited by either drug on training days 3, 5, or 7. Locomotor activity is expressed as the mean distance traveled (cm) ± SEM for the number of observations indicated. *p<0.05 with respect to saline control. 86

CD PRETEST BASELINE E23 TEST 6

I 4 if) O 2

0 i - L d -2

I - 4 + if) —6 O - 8

-1 0 CONTROL d-AMPH d-AM PH d-AM PH + + DNQX AFQX

Figure 11 . Effects of DNQX and AFQX on the expression of an amphetamine- induced conditioned place preference. DNQX (1.0 pg/0.5 pi), but not AFQX (1.0 pg/0.5 pi), administered into nucleus accumbens immediately prior to the test inhibits the expression of place preference elicited by d-amphetamine ( 1.0 mg/kg s.c.). Data are expressed as the mean ± SEM of the difference in time spent in the d-amph paired side (CS+) and the saline paired side (CS-) for both pretest and test conditions. *p<0.05 with respect to saline control. 87

CD SALINE (n = 6 )

VZD d-AMPH + VEH (n=6)

175 ES d-AMPH + DNQX (n=5) ESS d-AMPH + AFQX (n=5) z 150 2 \ 125 E o Q 100 Ld Lul Q. t o 75 s U Q 50 Ld 5 25

(C S+) (CS-)

Figure 12. Effects of DNQX and AFQX on locomotor activity during the expression of an amphetamine-induced conditioned place preference. Neither DNQX (1.0 pg/0.5 pi) nor AFQX (1.0 pg/0.5 pi), administered into nucleus accum bens immediately prior to test inhibits normal locomotor activity in either the CS+ (white) or CS- (black) compartment. Locomotor activity is expressed as the mean distance traveled (cm) per time spent (min) in each chamber ± SEM for the number of observations indicated. *p<0.05 with respect to saline control. 88

CD PRETEST BASEUNE E & TEST 4 2 I 0 o -2 LU - 4 2E F -6 + -8 (/) o -1 0 -12 UJ - 1 4 MORPHINE MORPHINE + DNQX

Figure 13. Effect of DNQX on the expression of morphine-induced place preference. DNQX (1.0 jj.g/0.5 pi) administered into nucleus accumbens immediately prior to the test inhibits the expression of place preference elicited by morphine (MS; 10 mg/kg s.c.). Data are expressed as the mean ± SEM of the difference in time spent in the d-amph paired side (CS+) and the saline paired side (CS-) for both pretest and test conditions. *p<0.05 with respect to morphine control. 89

C D PRETEST BASEUNE Effl TEST

Ld _ 2 - 2 *

2 - 1 0 - Ld

MORPHINE MORPHINE + DNQX

Figure 14. Lack of effect of DNQX on the acquisition of morphine-induced place preference. Pairing of intra-accumbens DNQX (1.0 pg/0.5 pi) with systemic morphine during the training did not inhibit the acquisition of a place preference elicited by morphine (MS; 10 mg/kg s.c.) after 4 pairings with the non-preferred compartment (CS+). Data are expressed as the mean ± SEM of the difference in time spent in the d-amph paired side (CS+) and the saline paired side (CS-) for both pretest and test conditions. □ MORPHINE EZa MORPHINE + DNQX E 1500 o (/) 1200

«n fO 900 \ Q a 600 * s T LU 300 O

a 0 I DAY 1 DAY 3 DAY 5 DAY 7

Figure 15. Effects of DNQX on the locomotor activity of rats given morphine during conditioned place preference training. DNQX (1.0 (j.g/0.5 pi), simultaneously administered into nucleus accumbens significantly reduces the already low level of locomotor activity of morphine treated (10 mg/kg s.c.) rats on day 1, 3, and 5, but not day 7. Locomotor activity is expressed as the mean distance traveled (cm) ± SEM. *p<0.05 with respect to morphine control. 91

!=□ MORPHINE

ZZZ1 MORPHINE + DNQX 175

✓“N z 150 2 \E 125 0 Q 100 111 u 8 ) 75 Ld

1 5 0

§ 25

(CS+) (CS-)

Figure 16. Effects of DNQX on locomotor activity during the expression of a morphine-induced conditioned place preference. DNQX (1.0 pg/0.5 pi) was administered into nucleus accumbens immediately priorto test. Animals were free to interact with either the CS+ (white) or CS- (black) compartment. Locomotor activity is expressed as the mean distance traveled (cm) per time spent (min) in each chamber ± SEM for the number of observations indicated. Speeds for the two groups were not different in each compartment. However, speed for the DNQX group in the CS- was significantly lower than in the CS+. 92

[=□ MORPHINE EZ3 MORPHINE + DNQX 8

L d OH o 0 6 CO >- (/) Q_ Ld 4

1 2

0 DAY 3 DAY 5 DAY 7

Figure 17. Effects of DNQX on the catalepsy induced by morphine during conditioned place preference training. DNQX (1.0 pg/0.5 pi), simultaneously administered into nucleus accumbens potentiates the catalepsy elicited by morphine (10 mg/kg s.c.) on days 3, 5, and 7. Catalepsy is expressed as the mean score ± SEM. *p<0.05 with respect to morphine control. 93

co d ] Saline

Figure 18. Unilateral administration of AMPA into the nucleus accum bens does not elicit a conditioned place preference. Acquisition of a place preference was elicited by d-amphetamine (1.0 mg/kg s.c.) but not AMPA (0.5 pg/0.5 pi, administered unilaterally into the nucleus accumbens) after 4 pairings with the non­ preferred compartment (CS+). Data are expressed as the mean ± SEM of the difference in time spent in the drug paired side (CS+) and the saline paired side (CS-) during the test. *p<0.05 with respect to saline controls. 94

chamber, however, they would try to elude the experimenter when he reached for

them. Cataleptic appearance would subsequently resume while rats were being

handled and transferred to the cork for catalepsy analysis.

DNQX did have an effect on locomotor activity in the test for expression of

morphine-induced place preference. While speeds of DNQX treated rats were not

different from saline treated rats in either compartment, the speed of DNQX treated

rats in the CS- (black) compartment was lower then their speed in the CS+ (white)

compartment (Figure 16).

Since the AMPA/kainate receptor antagonist DNQX appears to block some

aspects of drug reward, the question arose as to whether an agonist would elicit

reward. Although the glutamate agonist elicits pronounced locomotor activity when

injected into the nucleus accumbens, unilateral administration of AMPA (0.5 pg/0.5 pi) into the nucleus accumbens did not result in a place preference (Figure 18).

The AMPA was administered four times; the procedure was the same as above

(data were collected manually, however).

Discussion

These data suggest that non-NMDA receptors within the nucleus accumbens may play a role in the mediation of reward elicited by conditioned stimuli. In addition, these data suggest that acquisition of place preference elicited by d- amphetamine and morphine may utilize different neural elements within the nucleus accumbens. 95

Effects of DNQX on Place Preference Acquisition

The acquisition of d-amphetamine induced conditioned place preference has

been shown to depend on dopaminergic transmission in the nucleus accumbens

(Hiroi and White, 1989; Carr and White, 1983). In the present study, DNQX

administration into the nucleus accumbens inhibits the acquisition of d-

amphetamine induced conditioned place preference. These results suggest that

activation of both AMPA/kainate receptors and dopamine receptors in the nucleus

accumbens is required for the acquisition of d-amphetamine induced conditioned

place preference.

In contrast, the acquisition of a place preference elicited by morphine was not

blocked by intra-accumbens DNQX. Morphine may elicit reward by acting on two

substrates, the ventral tegmental area and the nucleus accumbens (see Koob and

Bloom, 1988; Wise, 1990). While dopamine release in the nucleus accumbens

(elicited by disinhibition of ventral tegmental dopaminergic neurons) probably does

contribute to the rewarding effects of morphine (Phillips and LePiane, 1980; Phillips

and LePiane, 1982; Bozarth and Wise, 1981; Bozarth, 1987; Britt and Wise, 1983;

Vaccarino et al, 1985), a direct action of morphine at an , independent of dopamine, in the nucleus accumbens may also elicit reward (Olds,

1982; van der Kooy etal, 1982; Vaccarino etal, 1985; Mackey and van der Kooy,

1985). If dopamine release were the sole mediator of reward, it seems likely that

DNQX administration would have blocked the acquisition of morphine-induced place preference as it blocked the acquisition of amphetamine-induced place preference. The present results are consistent with a model incorporating at least two substrates for morphine reward, one dependent on dopamine, the other

independent. Alternatively, in the present study DNQX may have only affected a

subpopulation of dopaminergic synapses, leaving dopaminergic transmission

stimulated by morphine at the level of the ventral tegmental area undisturbed. In either case, it seems likely that the substrates of both amphetamine- and

morphine-induced place preference converge, as lesions of the tegmental pedunculopontine nucleus, a brain structure located "down stream" in the proposed locomotor pathway, block the rewarding effects of both amphetamine and morphine in a conditioned place preference paradigm (Bechara and van der Kooy,

1989).

Effects of DNQX on Place Preference Expression

DNQX administration on the test day inhibits the expression of both d- amphetamine- and morphine-induced conditioned place preference. It has been demonstrated previously that dopaminergic transmission in the nucleus accumbens is stimulated in response to a conditioned reinforcing stimulus (Blackburn et al,

1987; Blackburn et al, 1989a; Blackburn et al, 1989b; Migler, 1975; Clody and

Carlton, 1980; Phillips etal, 1991; Taylor and Robbins, 1984; Taylor and Robbins,

1986), that is, a neutral stimulus which gains rewarding properties by being paired to a rewarding stimulus. Stimuli such as those found in the drug paired side of the conditioned place preference chamber are conditioned stimuli. The expression of 97

amphetamine-induced place preference depends in part on dopaminergic

transmission, as it is blocked by D 1 and higher doses of D2 antagonists (Hiroi and

White, 1991). The expression of d-amphetamine-induced place preference was

similarly inhibited by DNQX in the present study. As the expression of morphine-

induced place preference was also inhibited by DNQX, our results further suggest

that the expression of a morphine-induced place preference may be mediated by

the same dopaminergic mediated substrates as d-amphetamine, which are

activated by a conditioned stimulus and possibly impaired by intra-accumbens

DNQX. It is unlikely that DNQX resulted in an inhibition of the expression of place

preference by impairing locomotion, as DNQX did not significantly inhibit locomotor

activity during the test, and previous work in our laboratory has shown that DNQX

does not inhibit spontaneous locomotion (Willins etal, 1992).

Interestingly, lesions of the lateral nucleus of the amygdala, (but not lesions of

other amygdala nuclei, hippocampus, or cortex), similarly inhibit the acquisition and

expression of amphetamine-induced place preference (Hiroi and White, 1991). It

is therefore tempting to speculate that glutamatergic fibers projecting from the

lateral nucleus of the amygdala to the nucleus accumbens and acting via

AMPA/kainate receptors are required for amphetamine-induced place preference.

Effects of DNQX on Amphetamine Locomotor Activity

These data confirm the finding that intra-accumbens administration of the

AMPA/kainate receptor antagonist DNQX inhibits the locomotor response to systemic d-amphetamine (Wiliins etal, 1992). However, this inhibition is lost after one pairing with amphetamine. These data further suggest that the interaction between dopaminergic and glutamatergic neurons in the nucleus accumbens may change with exposure to amphetamine. Although this phenomena cannot be explained in the context of the present study, it may relate to the phenomenon of amphetamine sensitization, as sensitization has been demonstrated after one dose of amphetamine (Brown and Segal, 1977; Robinson, 1984). Furthermore, non-

NMDA excitatory amino acid receptors have been implicated as mediators of the effect (Karler etal, 1991). Further studies will be required to resolve this issue.

The effects of DNQX in all of the above studies appear to be receptor mediated effects rather than non-specific effects. AFQX, a chemical analogue of DNQX, has been shown not to inhibit the binding of [ 3H]-AMPA to its receptor in brain membranes (Supko et al, 1990). In the present study, the effects of intra- accumbens AFQX were not different from vehicle controls.

Effects of DNQX on Morphine Motor Effects and Catalepsy

Catalepsy, a condition characterized by immobility, elicited by morphine appears to be mediated by opiate receptors in the nucleus accumbens (Winkler etal, 1982; Havemann and Kuschinsky, 1985; Dauge etal, 1988). As morphine is reasonably selective for the p opioid receptor (Chang, 1984), catalepsy is probably a p mediated effect. Interestingly, the catalepsy elicited by morphine was potentiated by simultaneous intra-nucleus accumbens administration of DNQX. 99 While morphine catalepsy and akinesia are probably not due to effects on

dopamine release and are probably related to post-synaptic actions (see Layer et

al, 1991; Chapter III), inhibitory effects on glutamate release may also be involved.

DAGO, an agonist of the p opioid receptor, inhibits the release of glutamate in the

striatum (Jiang and North, 1992), and high doses of AMPA, an excitatory amino

acid agonist, appear to partially reverse morphine-induced akinesia. Thus, if

morphine is preventing glutamate release from afferent glutamatergic projections

in the nucleus accumbens, and this is related to catalepsy, the DNQX should

potentiate this effect. Catalepsy has never been observed in our laboratory after a 1 pg dose of DNQX alone in the nucleus accumbens, which, in fact, does not

seem to inhibit normal locomotor activity (Willins etal, 1992). Furthermore, unlike the DNQX-induced inhibition of d-amphetamine elicited locomotor activity, which was only seen on the first day, the potentiation of morphine catalepsy was observed on all days tested, and did not appear to be diminished in any way.

The data described above support the hypothesis that AMPA/kainate excitatory amino acid receptors within the nucleus accumbens are involved in the regulation of locomotor activity in the rat. These data further suggest that AMPA/kainate excitatory amino acid receptors in the nucleus accumbens are involved in the expression of the conditioned place preference reward elicited by amphetamine and morphine. However, while these data suggest a common substrate within the nucleus accumbens in mediating expression of both amphetamine and morphine- induced place preference, these data also suggest the presence of possibly two 100 distinct substrates within the nucleus accumbens which mediate the acquisition of place preference. CHAPTER III

EFFECTS OF MORPHINE IN THE NUCLEUS ACCUMBENS

ON STIMULANT-INDUCED LOCOMOTION

Introduction

The nucleus accumbens is a brain region which has been described as a functional interface between limbic and motor system s (Mogenson and Yim, 1981).

One of the procedures used to study the role of the nucleus accumbens in the

regulation of motility has been to measure locomotor activity after micro-injection of compounds directly into the region. The results of such studies indicate that several different neurotransmitter systems in the nucleus accumbens are involved in the regulation of motor responses. The most extensively studied is the dopaminergic system. Activation of dopaminergic receptors either indirectly via amphetamine-induced release of dopamine or directly with injected dopamine or produces a marked increase in locomotor activity (Fog, 1970; Kelly etal, 1975; Kuczenski, 1983; Pijnenburg etal, 1976). Agonists for glutamatergic excitatory amino acid receptors also stimulate an increase in locomotor activity

(Arnt, 1981; Boldry et al, 1991; Donzanti and Uretsky, 1983). Among these, activation of the quisqualic acid or a-amino-3-hydroxy-5-methylisoxazole-4-

101 102

propionic acid (AMPA) receptor elicits the largest response (Donzanti and Uretsky,

1983). There appears to be an interaction between the glutamatergic and

dopaminergic systems as a low level of activation of dopaminergic receptors is

required for the locomotor stimulating effects of the quisqualic receptor agonist

AMPA (Boldry etal, 1991,Donzanti and Uretsky, 1983). Picrotoxin, which blocks

the chloride channel associated with the GABA-A receptor, also stimulates a

marked increase in locomotor activity (Morgenstern et al, 1984). However, this

response appears to be independent of the dopaminergic system as it is not

inhibited by dopaminergic blockers (Morgenstern et al, 1984). •

In contrast to the stimulation of locomotor activity by dopaminergic and

glutamatergic agonists, activation of some other neurotransmitter receptors in the

nucleus accum bens results in a decreased level of motor activity. Thus, injection

of morphine into this region results in akinesia and catalepsy (Costall etal, 1978;

Winkler et al, 1982). This effect appears to be the result of a specific action on

p-receptors as the response is achieved by selective p-agonists (Dauge et al,

1988). The specificity of this effect is further evidenced by observations that

specific 5-agonists increase locomotor activity (Daug& etal, 1988; Havemann and

Kuschinski, 1985) and specific kappa-agonists have little effect (Havemann and

Kuschinski, 1985).

The fact that both p opiate receptor agonists and direct and indirect acting

dopaminergic agonists act within the nucleus accumbens to produce opposite effects on motility suggests that dopaminergic and opioid systems have an antagonistic interaction in the nucleus accumbens. The specific anatomy and

biochemistry that contribute to this interaction have not been conclusively

determined. Some studies suggest that morphine induced akinesia may be the

result of a decrease in dopaminergic transmission in areas such as the striatum and nucleus accumbens (Carenzi et al, 1975; Carlson and Seeger, 1982;

Kuschinsky and Hornykiewicz, 1973; Pollard et al, 1977; Pollard et al, 1978;

Schwartz, 1979; Slater etal, 1980; Yonehara and Clouet, 1984). This hypothesis is supported by several observations. First, both morphine and drugs which impair dopaminergic transmission such as can produce catalepsy and akinesia after systemic administration (Babbini and Davis, 1972; Kuschinsky and

Hornykiewicz, 1973); second, morphine inhibits stereotypy induced by amphetamine but not apomorphine (Seeger and Carlson, 1980); third, opiate receptors are localized on both nucleus accumbens (Pollard et al, 1977) and nigrostriatal dopaminergic neurons (Pollard etal, 1978); fourth, an opiate degrading enzyme, , has been localized to nigrostriatal dopaminergic neurons

(Malfroy et al, 1978); fifth, opiates can decrease dopamine release from striatal slices (Carenzi etal, 1975; Pollard etal, 1978; Yonehara and Clouet, 1984); and sixth, opiates can inhibit d-amphetamine-induced turning behavior of rats with unilateral 6 -hydroxydopamine-induced lesions of nigrostriatal dopaminergic neurons

(Slater et al, 1980). One direct test of dopaminergic-opiate interactions in the nucleus accumbens has been reported. Morphine coinjected into the nucleus accumbens with dopamine blocks the locomotor stimulating effect of the 104 catecholamine (Costall et al, 1978b). Thus, morphine may inhibit dopaminergic synapses in the nucleus accumbens by acting presynaptically at the dopaminergic nerve terminal as well as postsynaptically.

The observations reported above argue strongly for dopaminergic-opiate interactions within striatum and nucleus accumbens. However, the role of such interactions within the nucleus accumbens on the regulation of motility has not been well characterized. Furthermore, morphine interaction with non-dopaminergic stimulators of hypermotility has not been reported. The goal of the present work was to determine how morphine sensitive opiate mechanisms in the nucleus accumbens might interact with neuronal pathways that control locomotor stimulation. Towards this end, we have coinjected into the nucleus accumbens morphine along with compounds that elicit hypermotility and have m easured the resultant locomotor activity. Using this procedure, the interaction of morphine with compounds that activate dopaminergic and glutamatergic neurotransmission and that inhibit GABAergic neurotransmission were compared. In addition, effects of intra-accumbens morphine on hypermotility elicited by systemic injection of caffeine and scopolamine were studied.

Methods

Measurement of Locomotor Activity

Male rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 250 to 350 g, were housed 4 to a cage in a temperature controlled (23 ± 1° C) room with a 12 105

hour light-dark cycle. For direct injection into the nucleus accumbens, the rats

were anesthetized with a /oxygen mixture and placed in a stereotaxic

frame (David Kopf Instruments, Tajunga, CA) with the toothbar set at -3.3 mm. A

midline incision was made in the scalp, and holes were drilled bilaterally into the

skull at the coordinates: 10.2 mm anterior to the intraaural line and 1 .2 mm lateral

to the sagittal suture (Paxinos and Watson, 1982). The needle of a 10 pi Hamilton

syringe (Hamilton Co., Reno, NE) was then inserted into the holes to a position 2.0

mm above the intraaural line. Drugs or vehicle were injected in a 0.5 pi volume

at a rate of 0.5 pl/min. The needle was left in place for an additional minute to

allow for diffusion of the solution. After removal of the needle, the incision was

closed with wound clips and swabbed with 5% (w/v) lidocaine ointment.

After injections into the nucleus accumbens, anesthesia was discontinued, and the animals were removed from the stereotaxic frame. Animals recovered from the

anesthesia within 5 minutes, after which they were placed in motor activity cages

(Opto Varimex-Minor, Columbus Instruments, Columbus, OH) where they were allowed 10 minutes to adapt to the cages. The motor activity cages contained a grid (12 X 12) of infrared beams 3.5 cm apart and 5.0 cm from the bottom of the cage in a ventilated plexiglass box measuring 42 cm square and 20 cm high.

Ambulatory activity was measured as the number of times 2 consecutive beams were interrupted. Measurement of locomotor activity was done between 8:00 am and 4:00 pm in an isolated environmental room maintained at a temperature of 23

± 1° C. 106

Histology

After each experiment, animals were decapitated, and their brains were

removed and fixed in a 4% solution of formalin for 48 hours. Frozen sections (40

urn) were cut using a Cryo-Cut microtome (American Optical Corp., Buffalo, NY)

to check the location of the tip of the injection needle track. When the tips of the

needle tracks were found to lie outside the nucleus accumbens, the data for that

animal were excluded from the study. The histological examination showed that

the majority of injections were localized to a relatively small subsection of the

nucleus accumbens just medial and ventral to the anterior commissure at an

anterior-ventral level corresponding to 10.2 mm anterior to Bregma according to the atlas of Paxinos and Watson (1982).

Drugs

The following compounds were purchased from Sigma Chemical Co. (St. Louis,

MO): morphine sulphate, d-amphetamine sulphate, nialamide, dopamine, ascorbic acid, caffeine HCIi scopolamine, and [D-Thr^-leucine enkephalin-Thr (DTLET). a-

Amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and (+)-MK-801 hydrogen maleate were purchased from Research Biochemicals Inc. (Natick, MA).

Morphine sulphate and AMPA were dissolved in distilled H 20; all other drugs were dissolved in sterile saline. The doses of morphine refer to the free base; for all other drugs the doses refer to the weights of the salts. The doses shown refer to the amount injected on each side of the nucleus accumbens. 107

Statistics

Data were expressed as the mean and the standard error of the mean (SEM).

The data were evaluated statistically using the one-tailed Mann-Whitney U-test with

p<0.05 accepted as significant.

Results

Effects of Morphine and DTLET on Normal Locomotor Activity in the Rat

The bilateral injection of DTLET (1.0 pg), a specific 8 agonist, into the nucleus

accumbens produced a significant increase in locomotor activity. Conversely, administration of morphine (5.0 pg) into the nucleus accumbens markedly inhibited

locomotor activity (Figure 19). However this degree of akinesia did not impair performance on a rotorod. Animals placed thirty minutes after injection (time of

maximum akinetic response) on a 45 mm diameter rod rotating at 1.25 rpm

maintained themselves on the rod for the entire five minute testing period.

Effects of Morphine on Amphetamine- and AMPA-Stimulated Locomotor Activity in the Rat

d-Amphetamine, which induces hyperlocomotion in the rat primarily by releasing dopamine from dopaminergic nerve terminals and by preventing its reuptake, induced a pronounced locomotor effect in the present study (Figure 20).

The locomotor effect was characterized by the animals running along the periphery of the cage. They would typically stop at the corner and sniff for a moment before 108

resuming ambulatory movement. Rats did not bump into the walls of the cage,

and they would avoid obstacles placed in their path. However, when morphine

(5.0 pg) was co-injected with d-amphetamine (10 pg) into the nucleus accumbens,

the d-amphetamine-induced increase in locomotor activity was abolished (Figure

20). Rats given morphine along with amphetamine tended to stay in the middle

of the activity cages, in contrast to animals receiving only saline which stayed in the corner of the cage.

AMPA, an excitatory amino acid and quisqualate receptor agonist which

appears to require dopamine to induce locomotion, produced a large increase in

locomotor activity after bilateral injection (0.5 pg) into the nucleus accumbens

(Figure 21). The locomotion elicited by AMPA was qualitatively similar to that elicited by amphetamine; however, the amount of ambulatory movement after

AMPA was greater. Co-administration of morphine (1.5 pg and 5.0 pg) into the

nucleus accumbens inhibited AMPA-induced locomotor activity in a dose dependent manner, with the dose of 5 pg almost totally abolishing locomotor activity (Figure 21).

Data presented above demonstrate that 5 pg of morphine completely inhibits hypermotility elicited by either 10 pg d-amphetamine (Figure 20) or 0.5 pg of

AMPA (Figure 21). To determine whether this interaction is competitive in nature, the ability of higher doses of d-amphetamine and AMPA to overcome morphine inhibition was studied. The dose of each stimulant was increased three-fold.

While this resulted in a partial reversal of the morphine inhibitory effect when 109

AMPA was the locomotor stimulant (Figure 22), the locomotor responses to higher

doses of amphetamine (30 pg and 50 pg) were still completely blocked by

morphine (Figure 22).

Effects of Morphine on other agents that stimulate Locomotor Activity in the Rat

The observation that morphine attenuated both normal locomotor activity and the hypermotility elicited by amphetamine or AMPA suggests that morphine

injected into the nucleus accumbens can nonspecifically inhibit locomotor activity.

Therefore, the effects of morphine injected into the nucleus accumbens were tested against several agents that stimulate hypermotility. Bilateral administration of MK-801 (5.0 pg), an antagonist of the NMDA receptor, into the nucleus accumbens stimulated locomotor behavior in the rat, and this effect was abolished by coadministration of morphine (5.0 pg) into the nucleus accumbens (Figure 23).

Similarly, the increases in locomotor activity induced by systemic administration of both the methylxanthine caffeine (1 0 mg/kg s.c.) and the cholinergic antagonist scopolamine (0.5 mg/kg s.c.) were abolished by simultaneous administration of morphine (5.0 pg) into the nucleus accumbens (Figure 23). In addition, direct administration of dopamine (2 0 pg) into the nucleus accumbens two hours after pretreatment with nialamide (2 0 0 mg/kg i.p.) produced an increase in locomotor activity (Figure 24), which was abolished by coadministration of morphine (5 pg and 10 pg), (Figure 24). The dopamine hypermotility effect was long-lasting, as the rats were still hyperactive after 3 hours. The inhibition of this behavior by 110

morphine was long-lasting as well, as rats were akinetic as long as three hours

after drug injection (data not shown). The dose of each of these agents that was

tested was chosen to produce a submaximal response, and a reliable response

would have been obtained from both the dose used and double that dose. Thus

the morphine effect is not likely to result from a shift to the right of a compound

with a bell-shaped dose response curve. No overt differences in the pattern of

locomotion were observed for the ambulatory movement elicited by these agents.

In agreem ent with previous studies (Morgenstern et al, 1984), administration

into the nucleus accumbens of picrotoxin (0.15 pg and 0.5 pg), a blocker of the

chloride channel associated with the GABA-A receptor, also markedly stimulated

locomotor activity. However, in sharp contrast to the other stimulants investigated

in this study, the picrotoxin-induced stimulation of locomotor activity was not

attenuated by coadministration of morphine (5 pg or 10 pg) into the nucleus

accumbens (Figure 25).

Discussion

The results of the present study clearly demonstrate that morphine injected into the nucleus accumbens blocks the locomotor stimulation elicited by amphetamine, which acts indirectly by releasing endogenous dopamine, and dopamine (in the

nialamide-pretreated rat), which directly activates dopaminergic receptors. This

effect of morphine was not due to a general impairment in motor function, since

morphine treatment did not interfere with performance of the animals on a rotorod, 111

2000

1500

1000

500

SALINE MORPHINE DTLET

Figure 19. Effects of morphine and DTLET on locomotor activity in the rat after bilateral injection into the nucleus accumbens. Animals received 5.0 pg of morphine (n= 8 ), 1.0 pg of DTLET (n=5) or saline (n=4). After injection, the animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. *p<0.05 with respect to vehicle control. 112

4000

C d ID ^ 3000 co I- z D 8 2000 Cd o

o 1000 o o

Morphine Amphetamine Amphetamine + Morphine

Figure 2 0 . Morphine inhibits d-amphetamine-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens. Animals received 5.0 pg of morphine (n= 8 ), 10 pg of amphetamine (n= 6 ), or both morphine and amphetamine (n=7). The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the m eant S. E.M. *p<0.05with respect to stimulant alone control. The data for the morphine group are the same as shown in Figure 19. 113

5000 r

g 12500 - x co i- 0000 - z: 3 8 7500 - QL O o 5000 - 2 O o o 2500 _ l

AMPA: MORPHINE: 1.5jug 5 .0 /zg

Figure 21. Effect of morphine on AMPA-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens. A solution of morphine (1.5 pg or 5.0 pg) or vehicle was co-injected with AMPA (0.5 pg) in a 0.5 pi volume. The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for 4 rats in the AMPA- treated groups or 5 rats in the control group; *p<0.05, **p<0.01 with respect to stimulant alone control. 114

100 o z£ o o 80 - Lul z § h- 60 • z 3 3 2 H" 40 ■ (/) Ll . o 20 o a: Ll I Q.

0.5 1.5 10 30 50 AMPA d-AMPHETAMINE

Figure 22. Effect of morphine on locomotor activity induced by high concentrations of AMPA or d-amphetamine in the rat after bilateral injection into the nucleus accumbens. A solution of morphine (5.0 pg) was co-injected with AMPA (0.5 or 1.5 pg) or d-amphetamine (10,30 or 50 pg) in a 0.5 pi volume. The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Data are expressed as a percentage of the stimulant control mean. Each point represents the mean ± S.E.M. of the percentage values for the number of rats shown in parenthesis. Control locomotor responses to AMPA; 0.5 pg (10930 ± 1781) and 1.5 pg (14053 ± 5737 counts), were not statistically different. Control responses to d-amphetamine; 10 pg (3202 ± 663 counts), 30 pg (4946 ± 586 counts), and 50 pg d-amphetamine (8214 ± 526); significantly increased with each successive dose (Mann-Whitney U-test). Asterisk indicates stimulant counteracted morphine effect (p<0.05 compared to stimulant alone control). 115

4000 CHI Stimulant cr z> ■ ■ Stimulant -f Morphine ^ 3000 to i—

Z) O O 2000 a: o h— O 2 O 1000 o o

MK—801 Caffeine Scopolamine

Figure 23. Effect of morphine on hypermotility elicited by bilateral injection of MK- 801 into the nucleus accumbens or by systemic administration of caffeine or scopolamine. A solution of morphine (5.0 pg) or vehicle was co-injected with MK-801 (0.5 pg) in a 0.5 pi volume or administered simultaneously with caffeine (10 mg/kg s.c.) or scopolamine (0.5 mg/kg s.c.). The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for 4 rats in each group. *p<0.05 with respect to stimulant alone control. 116

4000

3000

CO c o 2000 o

o E 1000 - o o o

Vehicle Morphine Morphine (5 Mg) (10 ms)

Figure 24. Effect of morphine on dopamine-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens. Rats were pretreated with nialamide (200 mg/kg IP), and two hours later a solution of morphine (5.0 pg, n=5, or 10.0 pg, n=6) or vehicle (n=6) was co-injected with dopamine (20 pg) in a 0.5 pi volume. The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for 5 or 6 rats in each group. *p<0.05 with respect to dopamine control. 117

9000 □ PTX OL EZ3 PTX + Morphine (5 fig) Z> o ES23 PTX + Morphine (10 fig) X \ CO 6000 =) o o Od O f- o 3000 2 O o o

PTX (0.15 fig) PTX (0.5 fig)

Figure 25. Lack of effect of morphine on picrotoxin-induced locomotor activity in the rat after bilateral injection into the nucleus accumbens. A solution of morphine (5.0 pg or 10 pg) or vehicle was co-injected with picrotoxin (0.15 pg or 0.5 pg) in a 0.5 pi volume. The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parenthesis. 118

Figure 26. Possible sites of action of morphine within the nucleus accumbens; the "downstream" model. 119

Nuo. Aoo.

Figure 27. Possible sites of action of morphine within the nucleus accumbens; the "multiple pathways" model. 12Q

g 7000 x > 6000 C O 5000 (J ^ 4000 > H- 3000 O < a: 2000 O i— o 1000 o o o PTX PTX -F DNQX

Figure 28. DNQX ( 1 .0 ng/0.5 pi) inhibits the locomotor activity elicited by picrotxin (0.5 pg/0.5 pi) when both are administered simultaneously into the accumbens. nucleus 121 and morphine did not inhibit the locomotor stimulation produced by picrotoxin. The fact that morphine is equally effective against both the direct and the indirect dopaminergic stimulants strongly implies that the morphine inhibition is exerted at a locus at or "downstream" from the dopaminergic receptors in the nucleus accumbens (see Figure 26). It is unlikely that morphine is acting directly on dopaminergic receptors as morphine does not compete for dopaminergic receptors in binding assays (Carlson and Seeger, 1982), and important differences between morphine- and neuroleptic-induced catalepsy have been reported (De Ryck etal,

1980; Teitelbaum, 1982).

The inhibition of amphetamine-induced stimulation of locomotion is compatible with the results of several studies suggesting that morphine impairs motor function by inhibiting the depolarization-induced release of dopamine. (Carenzi etal, 1975;

Carlson and Seeger, 1982; Kuschinsky and Hornykiewicz, 1973; Pollard et al,

1977; Pollard etal, 1978; Raffa etal, 1989; Seeger and Carlson, 1980; Slater et al, 1980; Yonehara and Clouet, 1984). However, several observations do not support this hypothesis. Thus, while opiate receptors have been localized on dopaminergic nerve terminals in the nucleus accumbens (Pollard et al, 1977), opiate receptors have been found in greater quantity on cell bodies and on terminals in the nucleus accumbens that are not dopaminergic (Esposito et al,

1987; Unterwald et al, 1989). In addition, while activation of opiate receptors by morphine has been shown to inhibit the depolarization-induced release of dopamine (Carenzi et al, 1975; Yonehara and Clouet, 1984), the amphetamine- 122

induced release of dopamine is produced by a different mechanism, involving the

release of dopamine from a cytoplasmic pool and the inhibition of dopamine

reuptake (Kuczenski, 1983). It is, therefore, unlikely that the inhibitory effect of

morphine on the depolarization-induced release of dopamine is responsible for the

antagonism of the amphetamine response. In our study the intraaccumbens

injection of morphine was able to inhibit the hypermotility response to dopamine

(Figure 23). These observations suggest that the ability of morphine to counteract the effects of amphetamine resides in a postsynaptic rather than a presynaptic action.

In addition to its ability to block locomotion elicited by activation of the

dopaminergic system in the nucleus accumbens, morphine also attenuated

locomotion elicited by the excitatory amino acid agonist AMPA. The hypermotility effects of AMPA are dependent upon intact dopaminergic neurotransmission within the nucleus accumbens (Arnt, 1981; Boldry et al, 1991; Donzanti and Uretsky,

1983). Thus, the ability of morphine to block effects of AMPA may result from its action which counteracts dopaminergic stimulation.

Although morphine inhibited the hypermotility induced by either AMPA or amphetamine, increasing the dose of AMPA overcame the morphine induced inhibition, while increasing the dose of amphetamine did not. There are several possible explanations for this difference. The ability of AMPA to counteract the inhibition by morphine could be related to the greater intensity of the locomotor response to this drug as compared to amphetamine. It is also possible that the 123

high dose of AMPA used (1.5 fxg) may stimulate locomotion in a manner unrelated

to the effects of morphine. Recently, we have compared the abilities of D1 and

D2 dopaminergic receptor antagonists to attenuate hypermotility responses to

amphetamine and AMPA. Higher doses of antagonists were needed to inhibit the

effects of AMPA than were required to inhibit the response to amphetamine. Thus

results with both dopaminerigic antagonists and morphine suggests that AMPA is

more powerful than amphetamine in stimulating nucleus accumbens locomotor

circuits.

In contrast to the above findings, coadministration into the nucleus accumbens of morphine did not antagonize the locomotor response to picrotoxin, an inhibitor of the chloride channel associated with the GABA A receptor complex. This observation indicates that morphine does not inhibit the locomotor response to all drugs that stimulate locomotion by acting in the nucleus accumbens. One explanation for this observation is that picrotoxin acts at a site in the nucleus accumbens that is located "downstream" from the morphine site (Figure 26).

Alternatively, it is possible that there are at least two divergent neuronal pathways in the nucleus accumbens involved in the regulation of motility (see Figure 27).

Regardless of which hypothesis is correct, our results are consistent with the observation that picrotoxin-induced hypermotility does not require dopaminergic receptor activation, since haloperidol injected into the nucleus accumbens does not attenuate the locomotor activity elicited by picrotoxin (Morgenstern et al, 1984).

We observed that morphine in the nucleus accumbens blocked hypermotility 124 elicited by systemic administration of scopolamine and caffeine or by injection of

MK-801 into the nucleus accumbens. We originally did these experiments to determine whether morphine exerts a non-specific locomotor depressant effect.

It has been hypothesized that these compounds stimulate hypermotility by mechanisms other than dopaminergic activation in the nucleus accumbens. Thus, monoamine depletion or blockade of dopaminergic receptors do not abolish the locomotor effect of MK-801 (Carlson and Carlson, 1989; Raffa et al, 1989).

Furthermore, destruction of dopamine neurons with 6 -hydroxydopamine does not significantly inhibit hypermotility elicited by caffeine or scopolamine (Joyce and

Koob, 1981), and the dopamine receptor antagonist, alpha-flupenthixol, does not block hypermotility elicited by caffeine (Swerdlow et al, 1986). However, our results suggest that all three compounds activate a morphine sensitive pathway in the nucleus accumbens, or that all three compounds activate a pathway elsewhere in the brain that is inhibited as a result of morphine action in the nucleus accumbens. If caffeine, scopolamine and MK-801 activate pathways in the nucleus accumbens, the focus of activity must be downstream from the dopaminergic receptor or be on a morphine-sensitive pathway that does not contain dopaminergic receptors;

The present results are consistent with the hypothesis that the p-opiate receptor subtype mediates akinesia in the nucleus accumbens (Daug 6 etal, 1988;

Havemann and Kuschinski, 1985). In the striatum, p receptors mediate rigidity

(see Havemann etal, 1980; Havemann etal, 1981). Administration of morphine, 125

which is about 125 times more active at p-sites then at 5-sites (Chang, 1984),

resulted in akinesia, while administration of DTLET, a highly selective 5-receptor

agonist, resulted in stimulation of locomotor activity. It has been demonstrated

elsewhere that administration of other specific p-agonists such as DAGO into the

nucleus accumbens results in akinesia (Daugd et al, 1988), whereas specific 5-

agonists into the nucleus accumbens result in hyperactivity (Daugd et al, 1988;

Havemann and Kuschinski, 1985).

One limitation that must be considered in interpreting our results is that residual

anesthetic and/or the stress from surgery might cause release of endorphins or

other compounds that might affect behavior. Our experience is that injection of amphetamine, morphine, AMPA, and saline via implanted cannula into awake

animals and injection into anesthetized animals (as used in the present study)

produce identical locomotor responses. Furthermore, our results with MK-801,

picrotoxin, and dopamine agree with results obtained by injecting these compounds via implanted cannula (Morgenstern et al, 1984; Pijnenburg, 1976; Raffa et al,

1989). Thus complications from the method of drug administration are thought to be minimal.

Based on our results and observations previously reported by others, we propose an explanation for the biphasic effect of systemic morphine and heroin on locomotion in rodents. After systemic administration, lower doses of the compounds exert locomotor stimulation by acting primarily on p-receptors in the ventral tegmental area (Holmes and Wise, 1985; Joyce and Iversen, 1979). This produces a disinhibition of dopaminergic neurons, resulting in activation of

dopaminergic neurotransmission in the nucleus accumbens. Higher doses of the compounds may act primarily on p-receptors in the nucleus accum bens to produce akinesia which overcomes the increased activity in the dopaminergic system. This same argument would also explain why drug abusers sometimes use morphine or heroin in combination with amphetamine or cocaine to decrease the hyperactivity and agitation of the latter drugs.

Interestingly, while the locomotor activity elicited by picrotoxin was not blocked by morphine, it was blocked by the AMPA/kainate excitatory amino acid antagonist

DNQX (Figure 28). Thus it appears that DNQX is able to block this morphine insensitive locomotor pathway. CHAPTER IV

EFFECT OF SEROTONERGIC AGONISTS IN THE NUCLEUS ACCUMBENS

ON d-AMPHETAMINE-STIMULATED LOCOMOTION

Introduction

Systemic administration of d-amphetamine elicits increases in locomotor activity in the rat. The mesolimbic dopaminergic projection from the ventral tegmental area to the nucleus accumbens, a possible interface between limbic and motor system s (Mogenson and Yim, 1981), is a critical substrate for this effect of amphetamine (Pijnenburg etal, 1976). Behavioral and biochemical studies indicate that d-amphetamine acts primarily by releasing dopamine from dopaminergic nerve terminals and by preventing its reuptake (Kuczenski, 1983). Serotonin

(5-hydroxytryptamine, 5-HT) neuronal systems have been shown to modulate locomotor activity mediated by the dopaminergic system (for review see Gersen and Baldessarini, 1980; Soubrie etal, 1984). The nucleus accumbens receives serotonergic projections from the medial and dorsal raphe nuclei (Parent et al,

1981). Several lines of evidence indicate that these serotonergic neurons may exert an inhibitory tone overthe mesolimbic dopaminergic neurons which innervate the nucleus accumbens and mediate d-amphetamine stimulated locomotor activity.

127 Amphetamine-stimulated locomotor activity is potentiated by a) the 5-HT

antagonists and (Hollister etal, 1976); b) treatments

which reduce endogenous 5-HT concentrations such as pretreatment with

p-chlorophenylalanine, an inhibitor of 5-HT synthesis (Hollister etal, 1976; Mabry

and Campbell, 1973; Segal, 1976; Breese et al, 1974), or maintenance on a free diet (Hollister et al, 1976); and c) electrolytic lesions of serotonergic cell bodies (Geyer etal, 1976; Lucki and Harvey, 1974; Green and Harvey, 1974;

Carey, 1976; Neill et al, 1972; Costall et al, 1979) or lesions of serotonergic

neurons with 5,7-dihydroxytyptamine (Hollister et al, 1976; Breese et al, 1974).

Furthermore, prior administration of the 5-HT precursors tryptophan or

5-hydroxytryptophan inhibits d-amphetamine-stimulated locomotor activity in animals that are food deprived or maintained on a tryptophan deficient diet

(Hollister etal, 1976); and d-amphetamine-stimulated locomotor activity is inhibited by infusions of 5-HT into the nucleus accumbens (Carter and Pycock, 1978;

Costall etal, 1979) or cerebral ventricles (Warbritton etal, 1978).

Multiple 5-HT receptor subtypes have now been characterized, and specific agonists for these subtypes have been developed. Furthermore, various subtypes of 5-HT receptors are localized within the nucleus accumbens (Palacios and Dietl,

1988). Thus, the goal of the present work was to determine which 5-HT receptor subtypes in the nucleus accumbens are important in the modulation of d-amphetamine-stimulated locomotor activity. Towards this end, we have simultaneously administered systemic d-amphetamine and injected into the nucleus 129 accumbens various classes of 5-HT receptor agonists and have measured the resultant locomotor activity. Using this procedure, the effects of various 5-HT receptor subtype agonists on d-amphetamine-stimulated locomotor activity were evaluated.

Methods

Measurement of Locomotor Activity

Male rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 250 to 350 g, were housed 4 to a cage in a temperature controlled (23±1 °C) room with a 1 2 hour light-dark cycle. For direct injection into the nucleus accumbens, the rats were anesthetized with a halothane/oxygen mixture and placed in a stereotaxic frame

(David Kopf Instruments, Tajunga, CA) with the toothbar set at -3.3 mm. A midline incision was made in the scalp, and holes were drilled bilaterally into the skull at the coordinates: 1 0 .2 mm anterior to the intraaural line and 1 .2 mm lateral to the sagittal suture (Paxinos and Watson, 1982). The needle of a 1 Ojj.1 Hamilton syringe (Hamilton Co., Reno, NE) was then inserted into each of the holes to a position 2.0 mm above the intraaural line. Drugs or vehicle were injected in a

0.5pl or 1.Opl volume at a rate of 0.5pl/min. The needle was left in place for an additional minute to allow for diffusion of the solution. After removal of the needle, the incision was closed with wound clips and swabbed with 5% (w/v) lidocaine ointment. d-Amphetamine (0.5 mg/kg s.c.) was administered after removal of the

Hamilton syringe, while the animal was still anesthetized. After injections into the nucleus accumbens, anesthesia was discontinued, and

the animals were removed from the stereotaxic frame and were placed in motor

activity cages (Opto Varimex-Minor, Columbus Instruments, Columbus, OH).

Animals recovered from the anesthesia and were upright and mobile within 5

minutes, after which they were allowed 10 minutes to adapt to the cages. At this

point, collection of locomotor data began. The motor activity cages contained a grid (12 X 12) of infrared beams 3.5 cm apart and 5.0 cm from the bottom of the cage in a ventilated plexiglass box measuring 42 cm square and 20 cm high.

Ambulatory activity was measured as the number of times 2 consecutive beams were interrupted. Measurement of locomotor activity was done between 8:00 am and 4:00 pm in an isolated environmental room.

Histology

After each experiment, animals were decapitated, and their brains were removed and fixed in a 10% solution of formalin for 48 hours. Frozen sections

(40mm) were cut using a Cryo-Cut microtome (American Optical Corp., Buffalo,

NY) to check the location of the tip of the injection needle track. When the tips of the needle tracks were found to lie outside the nucleus accumbens, the data for that animal were excluded from the study. 131

Drugs

Morphine sulphate, serotonin creatinine sulphate (5-HT), and d-amphetamine

sulphate were purchased from Sigma Chemical Co. (St. Louis, MO).

1-(3-Chlorophenyl)piperazine HCI (mCPP), CGS-12066B,

(±)-12,5-dimethoxy-4-iodo)-2-aminopropane (DOI),

8-hydroxy-2-(di-n-propylamino)-tetralin HBr ( 8 -OH-DPAT), 1-phenylbiguanide,

, 3-tropanyl-3,5-dichlorobenzoate (MDL-7222), and 1-(2-methoxyphenol)-

4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) were purchased from

Research Biochemicals Inc. (Natick, MA). d-Amphetamine sulphate was dissolved in 0.9% saline. Morphine sulphate, DOi, 1-phenylbiguanide, and quipazine were dissolved in distilled H 20 . 5-HT and mCPP were dissolved in dilute (0.01 N) acetic acid. CGS-12066B was dissolved in 0.1 N HCI, and 8 -OH-DPAT was dissolved in 0.1 N HCI with 0.1 % w/v ascorbic acid. NAN-190 and MDL-7222 were dissolved in dilute (0.01 N) acetic acid with mild heating on a hot plate. Appropriate vehicle controls were run simultaneously. The doses shown refer to the amount injected on each side of the nucleus accumbens.

Statistics

Data were expressed as the mean and the standard error of the mean (SEM).

The data were evaluated statistically with an analysis of variance (ANOVA) followed by the Newman-Keuls multiple range test for comparison of individual groups, with p<0.05 accepted as significant. 132 Results

Systemic d-amphetamine (0.5 mg/kg s.c.) induced a pronounced locomotor stimulation (Figure 29,30,31). In agreement with previous studies (Carter and

Pycock, 1978; Costall et al, 1979), 5-HT (50 and 100 nmol) injected into the nucleus accumbens inhibited-amphetamine-stimulated locomotion (Figure 29).

Although 5-HT was able to inhibit d-amphetamine stimulated locomotion, the

5-HT agonist quipazine, the general 5-HT, agonist mCPP, and the 5-HT 1c/5-HT2 agonist DOI produced no inhibition at the highest dose tested, (1 0 0 nmol) (Figure

30). In contrast, the 5-HT 3 agonist 1-phenylbiguanide (100 nmol) potentiated d-amphetamine-stimulated locomotion, (Figure 30). The potentiation of d-amphetamine-stimulated locomotion by 1-phenylbiguanide was partially reversed by the 5-HT 3 antagonist MDL-7222 (Figure 30). Statistical analysis revealed that while the d-amphetamine- stimulated locomotor activity of animals recieving 1- phenylbiguanide and MDL-7222 was not significantly higher than those receiving vehicle, the d-amphetamine- stimulated locomotor activity of animals receiving 1- phenylbiguanide alone was not significantly different from animals receiving 1- phenylbiguanide and MDL-7222.

The specific 5-HT1A agonist 8 -OH-DPAT (100 nmol) did not inhibit d-amphetamine-stimulated locomotion (Figure 31), but a pronounced lateral headweaving behavior was observed. The specific 5-HT1B agonist CGS-12066B

(100 nmol) had no effect on d-amphetamine-stimulated locomotor activity (Figure

31). However, the combination of 8 -OH-DPAT (50 nmol) and CGS-12066B (50 133

L_ 3 4000 O X

■*->0) c 3000 3 O o 2000 > o < L. o -M 1000 o E o o o —I 5 H T 5 H T 5 H T 25 nmol 50 nmol 100 nmol

Figure 29. Effects of 5-HT bilaterally injected into the nucleus accumbens on systemic d-amphetamine stimulated locomotor activity in the rat. A solution of 5-HT or vehicle (V) was injected in a 0.5 |xl volume, and d-amphetamine was given concurrently (0.5 mg/kg s.c.). The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. *p<0.05 with respect to vehicle control. 134

8000

6000

>N 4000 > o < L_ o 2000 o E o o o —I V mCPP DOI QUIP PBG PBG + MDL

Figure 30. Effects of several 5-HT agonists on systemic d-amphetamine-stimulated locomotor activity in the rat after bilateral injection into the nucleus accumbens. A solution of 1-(3-chlorophenyl)piperazine (mCPP), DOI, quipazine (QUIP), 1-phenylbiguanide (PBG), PBG + MDL-7222 (MDL), or vehicle (V) (each 100 nmol) was injected in a 1.0 pi volume, and d-amphetamine was given concurrently (0.5 mg/kg s.c.). The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. *p<0.05 with respect to vehicle control. 135

4000

c 3000 3 o CJ 2000 > o < k. o 1000

( 10)

CGS DPAT CGS + DPAT

Figure 31. Effects of 8 -OH-DPAT (5-HT1A agonist) and CGS-12066B (5-HT1B agonist) on systemic d-amphetamine-stimulated locomotor activity in the rat after bilateral injection into the nucleus accumbens. A solution of vehicle (V), 8 -OH-DPAT (100 nmol), CGS-12066B (100 nmol), or both (each 50 nmol) was injected in a 1.0 \i\ volume, and d-amphetamine was given concurrently (0.5 mg/kg s.c.). The animals were placed in motor activity cages, and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. *p<0.05 with respect to vehicle control. normal locomotor activity. locomotor normal Figure 32. Lack of effect of serotonergic agonists in the nucleus accum bens on on bens accum nucleus the in agonists serotonergic of effect of Lack 32. Figure

LOCOMOTOR COUNTS/ 1 HOUR 0 0 ■ I 1000■ 1400 r r 1400 1200 0 - —. .— - 800 0 ■ 1 ■ 400 0 ■ — 200 ■ 600 o——LJ—U—U—U—U—U—U—U • * o0 5T P DI UP B CS PT CGS DPAT CGS PBG QUIP DOI CPP m 5HT 0 Ho t — t — DPAT 136 137

nmol) produced a significant inhibition of d-amphetamine-stimulated locomotor

activity (Figure 31). Lateral headweaving was still observed with the combination

of drugs. The inhibition of d-amphetamine-stimulated locomotor activity by intra-

accumbens 5-HT (50 nmol), however, was not reversed by systemic (8 mg/kg s.c.)

administration or coinjection into the nucleus accumbens (4 pg) of the 5-HT1A

antagonist NAN-190.

Administration of all agonists alone (in the absence of d-amphetamine) into the

nucleus accumbens (all 100 nmol) did not alter basal locomotor activity (Figure

32).

Discussion

After confirming earlier observations that 5-HT inhibits the locomotor response

elicited by systemic amphetamine (Carter and Pycock, 1978; Costall etal, 1979), the present study presents information on the subtypes of 5-HT receptor in the

nucleus accumbens that may regulate d-amphetamine-stimulated locomotor activity. The effects of agents with specificities for various subtypes of 5-HT

receptor suggest that serotonergic modulation of dopaminergic activity within the

nucleus accumbens is the result of a complex interaction of these receptor subtypes. Concurrent activation of both 5-HT1A and 5-HT1B receptors may be required to inhibit amphetamine stimulated hypermotility, while 5-HT 3 receptor activation appears to potentiate the amphetamine effect (Figure 33).

Although 5-HT injected into the nucleus accumbens attenuated the hypermotility 138

elicited by systemic administration of amphetamine, none of the compounds with

selectivity for various receptor subtypes was able to reproduce this effect.

However, the combination of 8 -OH-DPAT, a 5-HT1A agonist, and CGS-12066B, a

5-HT1B agonist (see Neale et al, 1987; but also Schoeffter and Hoyer, 1989),

mimicked the effects of 5-HT. The concept that this is an interaction between two distinct receptor mechanisms is derived from the observation that neither compound when used alone at double the dose used in the combination experiment was able to attenuate the hypermotility elicited by amphetamine. In support of this hypothesis, another interaction between 5-HT1A and 5-HT1B

receptors has been reported. Thus, a high dose of CGS-12066B potentiates

8 -OH-DPAT stimulated hypothermia in mice (Berensen and Broekkamp, 1990).

This suggests that such interactions might be important in the production of a variety of functional effects mediated by serotonin. On the other hand, the 5-HT1A antagonist NAN-190, in the present study, was unable to reverse the effects of 5-

HT on d-amphetamine-stimulated locomotion. Either the dose of NAN-190 used in the present study was insufficient or 8 -OH-DPAT may be acting at some site other than the 5-HT1A receptor. NAN-190, at the systemic dose used in this study, has previously been shown to block the behavioral syndrome, but not the hypothermia, elicited by 8 -OH-DPAT (Przegalinski etal, 1990).

The 5-HT agonist mCPP, which has affinity for both 5-HT1B and 5-HT1c receptors, has been shown to- inhibit spontaneous ambulatory behavior after systemic administration in the rat (Lucki et al, 1989) even though mCPP when 139

administered into the nucleus accumbens did not decrease hypermotility elicited

by amphetamine in our experiments. The apparent discrepancy between these

observations could be explained if mCPP after systemic administration exerts a

hypomotility effect at a locus other than the nucleus accumbens. Another

possibility is that the hypermotility effect of amphetamine is so powerful that it

readily overcomes a weak hypomotility effect of mCPP.

PBG, a 5-HT3 receptor agonist, had an effect opposite to that of 5-HT. This

finding that activation of the 5-HT3 receptor potentiates the effects of amphetamine

is supported by the results of several recent studies. Direct injection of

2-methyl-5-HT, another 5-HT3 receptor agonist, into the nucleus accumbens

potentiates amphetamine stimulated locomotor activity (Costall et al, 1987). In

addition, both 5-HT3 receptor agonists, 2-methyl-5-HT and PBG, increase the

release of endogenous dopamine from slices of the rat striatum (Blandina et al,

1989; Schmidt and Black, 1989), an area with many biochemical correlates to the nucleus accumbens. The dopamine release induced by 2-methyl-5-HT, but not

PBG, was reversed by the selective 5-HT3 antagonist ICS 205-930 (Blandina etal,

1989; Schmidt and Black, 1989). Thus while it is possible that activation of 5-HT3 receptors in the nucleus accumbens may enhance the stimulant response to amphetamine by enhancing the release of dopamine by amphetamine at this site,

PBG may be acting as an inhibitor of dopamine uptake (Schmidt and Black, 1989).

In the present study, the potentiating effect of PBG on d-amphetamine-stimulated locomotor activity was partially blocked by the 5-HT3 antagonist MDL-7222, 140

suggesting a role for both a direct action mediated by 5-HT3 receptors and an

inhibition of dopamine uptake.

Thus, it seems likely that 5-HT may have opposing effects on regulation of

d-amphetamine-stimulated locomotor activity in the nucleus accumbens by enhancing the release of dopamine from nerve terminals (5-HT3 receptor action) and by counteracting the effects of dopamine receptor activation (possibly 5-HT1A

+ 5-HT1B receptor action), see Figure 33. When 5-HT is infused into the nucleus accumbens it is likely that both effects occur, with the inhibitory effect predominating. Quipazine, unlike 5-HT, had no effect on d-amphetamine-stimulated locomotor activity in the present study. Although quipazine is generally considered a relatively nonselective 5-HT receptor agonist, there is evidence that quipazine acts as an agonist for the 5-HT2 receptor (Lyon and Titeler, 1988) and as an antagonist for the 5-HT1A and 5-HT3 receptors

(Schoeffter and Hoyer, 1989), resulting in the absence of a net change in amphetamine-stimulated locomotion. DOI, a selective agonist at 5-HT1c and 5-HT2 receptors, also had no effect on d-amphetamine stimulated locomotor activity.

These data suggest that the 5-HT2 receptor does not play an important role in modulation of locomotor activity elicited by amphetamine in the rat nucleus accumbens. This is supported by the results of several other recent studies which have implicated 5-HT, receptor subtypes, as opposed to the 5-HT2 subtype, in the inhibition of locomotor activity (Lucki et al, 1989; Kennet and Curzon, 1988;

Klodzinska etal, 1989), although to our knowledge none of these studies have 141

RAPHE

5-HT-3

Nuc. Acc.

Figure 33. Possible sites of action of serotonin in the nucleus accumbens. 142 examined the role of these subtypes in the nucleus accumbens. Furthermore,

5-HT has a much higher affinity for 5-HT, sites than for 5-HT2 sites (Nelson, 1988), and the nucleus accumbens contains a higher concentration of 5-HT, specific binding sites, although 5-HT1A receptors appear to comprise only 3% of 5-HT, specific binding (Palacios and Qietl, 1988).

Interestingly, pronounced repetitive lateral head-weaving was elicited by injection of 8-OH-DPAT into the nucleus accumbens. This behavioral phenomenon is an important component of the serotonin motor syndrome (See Gersen and

Baldessarini, 1980). Other components of the syndrome such as forepaw treading, hind limb abduction, tremor, or Straubs tail were not observed. Several recent studies suggest that activation of the 5-HT,A receptor produces this complex behavioral syndrome (Middlemiss and Fozard, 1983; Sills e ta l, 1984; Lucki etal,

1984). Our data indicate that the 5-HT,A receptors which mediate the head weaving component of the syndrome are located in the nucleus accumbens, while those mediating the other components are probably located elsewhere.

Serotonergic projections from the raphe nuclei have long been thought to regulate locomotor activity in rats (Gerfen and Baldessarini, 1980; Hollister e ta l,

1976; Jones et al, 1981). Furthermore, it has been proposed that some serotonin-mediated behaviors are modulated by multiple subtypes of 5-HT receptors (Glennon etal, 1991). The results of this study are consistent with this hypothesis and suggest that activation of no single 5-HT receptor subtype can mimic the effects of 5-HT. Furthermore, it appears that 5-HT neurons within the 143

nucleus accumbens can differentially modulate the locomotor activity elicited by d-amphetamine, perhaps through interactions between 5-HT1A) 5-HT1B, and 5-HT3 receptors. CHAPTER V

SUMMARY AND CONCLUSIONS

SUMMARY

A reward system has evolved in vertebrates which reinforces biologically useful behaviors like approaching a goal object and subsequently eating, drinking, and reproducing. Thus it feels good to do these things. Additionally, an organism will engage in these rewarding behaviors again. Addicting drugs gain access to this system, and mimic natural reward by artificially stimulating it. By stimulating the reward system, addicting drugs elicit pleasure and result in a compulsion to maintain that state.

The mesolimbic dopamine system and especially its terminal fields within the nucleus accum bens comprise a critical substrate for both the locomotor-activating and rewarding properties of most abused drugs. A vast quantity of evidence suggests that an increase in dopamine release in the nucleus accumbens is required for the expression of reward and positive reinforcement. Several neural system s may interact with the dopaminergic system within the nucleus accumbens and thus play a role in the expression of reward. The studies described in this dissertation have demonstrated a role for excitatory amino acid receptors, opiate

144 145 receptors, and serotonin receptors in the nucleus accumbens in mediating the behaviors mediated by the dopamine system.

Recent studies suggest that glutamatergic afferents (inputs) from limbic structures such as amygdala and hippocampus interact with dopamine afferents within the nucleus accumbens. The purpose of the first set of experiments

(Chapter 2) was to examine the role of NMDA and AMPA/kainate excitatory amino acid receptors in the expression of behavior associated with increased dopamine transmission in the nucleus accumbens. Thus, the results of the first set of experiments demonstrated that MK-801, a potent noncompetitive antagonist of central NMDA receptors, at a dose which produces behavioral activation, is positively reinforcing in a conditioned place preference paradigm (and therefore may have abuse potential). These findings are consistent with reports that MK-801 increases dopamine transmission in the nucleus accumbens. The results of the second set of experiments demonstrated that a non-NMDA glutamate antagonist

(DNQX) within the nucleus accumbens could inhibit the expression of a drug- induced conditioned place preference. In addition it was found that DNQX inhibited the acquisition of a place preference elicited by amphetamine, while it did not significantly inhibit that of morphine. This finding is consistent with the hypothesis that multiple reward substrates may exist within the nucleus accumbens.

While a role for opiates in the nucleus accumbens in reward is well established, their effects on locomotor activity are less clear. The results of the next set of 146 experiments (Chapter 3) demonstrated an interaction between opiate, glutamatergic, and dopaminergic innervation of the nucleus accumbens, using locomotor activity as the measured response. The dopaminergic stimulants d- amphetamine or dopamine induced a large increase in locomotor activity when injected into the nucleus accumbens and were blocked by coadministration of morphine. The hypermotility response elicited by a-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA), an excitatory amino acid agonist that requires dopaminergic receptor activation for the expression of its locomotor activating effect, was also abolished by coadministration with morphine. Intra- accumbens morphine also blocked the hypermotility elicited by systemic caffeine or scopolamine, compounds whose hypermotility responses do not involve the dopaminergic synapses within the nucleus accumbens. However, picrotoxin injected into the nucleus accumbens elicited a hypermotility that was not attenuated by coinjection of morphine. These data demonstrate that opiate and have competing actions on regulation of locomotion in the nucleus accumbens, and that AMPA stimulated locomotor activity is sensitive to inhibition by morphine. The finding that morphine, acting at the p receptor, can inhibit locomotor activity produced by some drugs, but not others, suggests that multiple locomotor substrates exist within the nucleus accumbens.

Serotonergic projections from the raphe nuclei to the nucleus accumbens modulate locomotor activity. In recent years, several serotonin receptor subtypes have been discovered and demonstrated in the CNS. Which receptors mediate locomotor activity within the nucleus accumbens, however, is not clear. Thus, the

purpose of the final set of experiments (Chapter 4) was to characterize the effects

of various classes of serotonergic agonists administered into the nucleus

accumbens on d-amphetamine-stimulated locomotor activity in order to determine

which serotonin receptor subtypes are involved. Administration of the nonselective

5-HT agonist quipazine, the 5-HT, agonist mCPP, the 5-HT1A agonist 8-OH-DPAT,

the 5-HT1B agonist CGS-12066B, and the 5-HT1c/5-HT2 agonist DOI did not inhibit

d-amphetamine-stimulated locomotor activity. The combination of the 5-HT1A

agonist 8-OH-DPAT and the 5-HT1B agonist CGS-12066B, however, did inhibit

d-amphetamine-stimulated locomotor activity. Inhibition of

d-amphetamine-stimulated locomotor activity by intra-accumbens 5-HT was not

reversed by the 5-HT1A antagonist NAN-190. In contrast, the 5-HT3 agonist

1 -phenylbiguanide enhanced the locomotor effect of d-amphetamine. This effect

was partially reversed by the 5-HT3 antagonist MDL-7222. These studies suggest that serotonin has complex and multiple effects on the regulation of locomotor

activity within the nucleus accumbens, depending on which receptor subtypes are

involved.

A SIMPLE MODEL

On the basis of the behavioral pharmacology of dopaminergic agonists and antagonists, a general theory proposing a role for dopamine in the nucleus accumbens in reward and locomotion has been advanced. Indeed, reward and 148 locomotion may share a common neural substrate. Thus, increases in nucleus accumbens dopamine transmission both elicit reward and stimulate locomotion, and these behavioral phenomena are inhibited by dopamine antagonists. The k agonist U50.488H both reduces dopaminergic transmission and locomotor activity, and is aversive. Clearly, amphetamine acts on a substrate within the nucleus accumbens which results in the expression of reward and locomotor activity. The findings described in this dissertation are consistent with this theory. However, the present findings further suggest a heterogeneity of function within the nucleus accumbens such that multiple substrates exist which mediate both locomotor activation and reward. For example, morphine in the nucleus accumbens, which is rewarding (van der Kooy et al, 1982), inhibits locomotor activity, while AMPA in the nucleus accumbens, which stimulates locomotion, is not rewarding (Figure 18).

Furthermore, a D, antagonist inhibits the conditioned place aversion to systemic picrotoxin (Di Chiara et al, 1991), which suggests that an increase in dopamine transmission is necessary to associate internal cues signaling aversion and environmental cues. It thus appears that multiple functional substrates within the nucleus accumbens may exist.

If multiple substrates for reward and locomotor activity coexist within the nucleus accumbens, then this may explain the seeming paradox of the involvement of accumbens dopamine in stress. It is well established that stress increases dopamine transmission in the nucleus accumbens (Abercrombie etal, 1989), and that dopaminergic antagonists suppress responding to an aversive conditioned 149

stimulus, such as a tone which precedes a shock (Baldessarini, 1985). This

paradox can be reconciled, however, if one entertains the possibility of multiple

pathways through the nucleus accumbens. Dopamine may modulate distinct

pathways subserving both stress and reward.

While dopamine is involved in reward and stress, it also seems to play an

important role in motivation (Di Chiara etal, 1991) and the anticipation of reward

(Phillips et al, 1991). In the next few paragraphs, I will propose a tentative explanation for these phenomena.

In Chapter 3, the effects of the AMPA/kainate antagonist DNQX on three events were measured; the acquisition of amphetamine reward, the acquisition of morphine reward, and the expression of reward elicited by either. The acquisition of a place preference indicates two things; 1) that the unconditioned stimulus was indeed rewarding, and 2) that the animal was able to associate the unconditioned stimulus (drug) with the appropriate conditioned stimulus (white side of the conditioned place preference apparatus). As DNQX did not inhibit the acquisition of morphine-induced place preference, it does not seem likely that it should have blocked the ability to associate a conditioned stimulus with an unconditioned stimulus, rather it seems more likely that the DNQX inhibited amphetamine-induced reward directly. This suggests a difference between the substrates of morphine and amphetamine induced reward. The expression of a place preference indicates the ability of the conditioned stimulus to elicit reward. That DNQX inhibited the expression of both morphine- and amphetamine-induced place preference 150 suggests that a similar substrate mediates the reward elicited by the conditioned

stimulus in both cases.

I propose a simple model to account for these data (see Figure 34). This model, based on one proposed by Grace (1990), is greatly simplified, but may be of some heuristic value. In order to elicit reward, amphetamine must cause the release of dopamine within the nucleus accumbens, which comes from neuron DA and acts at receptor D (for dopamine). Simultaneously, glutamate must be released from neuron GLU2 and act at receptor A2 (for AMPA). Thus, dopamine antagonists (Mackey and van der Kooy, 1985) or DNQX will inhibit amphetamine reward. For morphine to elicit reward, it need only act at receptor p. Neuron

GLU2 does not have to be active, and DNQX does not inhibit morphine reward.

In order for the conditioned stimulus to elicit reward, neuron GLU1 must be active, and glutamate must act at receptor A1, on neuron DA. This causes an increase in dopamine release (see Imperato etal, 1990; Desce etal, 1991), and therefore dopamine acts at receptor D. Thus both dopamine antagonists (Hiroi and White,

1989) and DNQX will block the expression of place preference. It is possible that neuron GLU1 comes from the amygdala, as the amygdala is required for the establishment of conditioned reinforcement (Cador ef al, 1989). Neuron GLU2 may originate in the prefrontal cortex, as this area has been implicated in mediating cocaine reward (Goeders and Smith, 1983).

The phenomenon of motivation may interact with the proposed model in the following way. Phillips et al (1991) have shown that dopamine levels in the 151

VTA

DA

GLUi AMY

GIU2 PFC ?

GABA

Figure 34. A model of neuronal interactions in the nucleus accumbens, involving phasic and tonic dopamine release. Abreviations: (VTA) ventral tegmental area; (AMY) amygdala; (PFC) prefrontal cortex. 152

nucleus accumbens rise in response to the anticipation of reward. If the

recognition of a conditioned stimulus is mediated by the amygdala, and this causes

activation of neuron GLU1 which releases dopamine through a presynaptic

mechanism, then tonic levels of dopamine will rise in the accumbens, accounting

for the data of Phillips et al (1991). This may act to "prim®" the system. If the

stimulus is rewarding enough, then phasic release will occur following some sort

of positive feedback loop. Thus the difference between general motivation and

reward may be the difference between tonic and phasic dopamine release. A

similar but distinct system may function in parallel within the nucleus accumbens to account for conditioned avoidance responding and stress. It remains to be seen

what effects DNQX in the nucleus accumbens might have in these paradigms.

IMPLICATIONS FOR CLINICAL TREATMENT OF HUMAN DRUG ADDICTION

Patterns of drug intake among addicted persons differ depending on the degree of addiction and drug which is abused. Use patterns range from stable levels of intake to periodic binges or "runs". The hallmark of compulsive, noncontrollable, drug use of any type is the intense craving for the drug, which is predictive of and usually is followed by relapse and repeated drug use.

The negative reinforcement theory of addiction holds that a condition of abnormal depression of the positive reinforcement pathway occurs after repeated administration, possibly by depletion of synaptic dopamine (Dackis and Gold,

1985). Thus while increased dopamine mediates reward, a condition of depleted 153

synaptic dopamine in the nucleus accumbens results in the intense craving which

predicts relapse.

Drug craving, however, appears to be a highly motivated condition. The

condition of drug craving has been likened to a powerful "lust" for the drug.

Addicts become fixated on thinking about the drug and its procurement. Animal

studies reveal that motivated behavior in response to an environmental cue

involves increased dopamine transmission in the nucleus accumbens. This, then,

is seemingly at odds with the dopamine depletion hypothesis.

The positive reinforcement theory, in line with incentive motivation theory, holds that it is the positively reinforcing effects of the drug which result in its reuse. Wise

(1990) suggests that the addict remembers the last reinforcement, and the

reactivation of that memory by associated stimuli is sufficient to elicit drug craving.

Indeed, in one study of cocaine addicts (Gawin and Kleber, 1986), it was reported that, even after 28 weeks of abstinence and lack of craving, intense craving returned on seeing old cocaine-using friends and passing homes in which they had used cocaine. Intravenous users reported experiencing craving after having blood drawn for laboratory tests. In animal self-administration studies, "priming", the delivery of a noncontingent drug stimulus, is effective at starting the animal in responding at the beginning of a session. Thus, in humans, of past drug reinforcements associated with environmental cues may act as priming stimuli. Wise (1990) has further suggested that the biological correlate of craving may lie not in positive reinforcement substrates, but in substrates of memory. 154

Recently, the nucleus accumbens has been implicated in one form of memory, social memory (Ploeger etal, 1991).

The results contained in this dissertation suggest that functional heterogeneity exists within the nucleus accumbens, an important reward substrate. Furthermore, they suggest that modulation of the proposed reward pathway by other systems may occur. While it seems that the best therapy for the addicted person is removal of environmental cues associated with drug use, this may not be feasible, and pharmacological treatment (if possible in the future) may be desirable. The neural systems which modulate the nucleus accumbens may be possible substrates for a pharmacological treatment of drug addiction. In fact, 5-HT3 receptor antagonists have recently been proposed as treatments for drug addiction

(Trickelbank, 1989). While studies in this area are in their infancy, it is fervently hoped that further research will shed light on this important topic.

As a final word, this author feels that recent proposals suggesting the legalizing of drugs such as cocaine, amphetamine, and heroin, would be a mistake of epic proportions. Animal data suggests that these drugs, and especially cocaine and amphetamine, are the most addiction prone drugs known. Human data suggest that increased availability to psychoactive drugs results in increased consumption.

In fact, the incidence of drug addiction among medical professionals who have easier access to psychoactive drugs was found to be much higher than in a matched control population. Furthermore, an epidemiological study done in the

United States (U.S.) before opiates and cocaine were made illegal suggests that the proportion of U.S. adult drug addicts was not different from the proportion of

U.S. adult alcoholics currently (for review see Goldstein and Kalant, 1990).

Recommendations for solving this immense problem are beyond the scope of this dissertation. It is hoped, however, that continued research and public education will help to reduce the magnitude of the current addiction problem. APPENDIX

ABBREVIATIONS USED IN THIS DOCUMENT

5-HT- serotonin; 5-hydroxytryptamine

8-OH-DPAT- 8-hydroxy-2-(di-n-propylamino)-tetralin HBr

AbC- core of nucleus accumbens

AbS- shell of nucleus accumbens

ac- anterior commisure

ACPC- 1-aminocyclopropanecarboxylic acid

ACPD-1-amino-1,3-cyclopentane-dicarboxylic acid

ADCP- cis-1 -amino-1,3-dicarboxycyclopentane

AFQX- e-amino^-fluoroquinoxaline^.S-dione

AMPA- a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

AP-3- 2-amino-3-phosphonopropionic acid

AP-4- 2-amino-4-phosphonobutyric acid

AP-5- 2-amino-5-phosphonovaleric acid

AP-7- 2-amino-7-phosphonoheptanoic acid cc- corpus callosum

CGS-12066B- (±)-(1,2,5-dimethoxy-4-iodo)-2-aminopropane

156 CNQX- 6-cyano-7-nitroquinoxaline-2,3-dione

CP- caudate-putamen or dorsal'striatum

CS+- conditioned stimulus +

CS- conditioned stimulus -

CPP- 3-(2-carboxypiperazin-4-yl)-propyl-1 -phosphonate

CTAP- D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2

CTX- cortex

CTX- cortex

DA- dopamine

DAGO- Tyr-D-Ala-Gly-MePhe-Glyol

DMT- dorsomedial thalamus

DNQX- 6,7-dinitroquinoxaline-2,3-dione

DOI- (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCI

DPDPE- Tyr-D-Pen-Gly-Phe-D-Pen

DTLET- [D-Th^J-leucine enkephalin-Thr

GAMS- D-glutamylamino-methyl sulfonate

GDEE- glutamic acid diethylester

GLU- glutamate

HIP- hippocampus

IC- islands of Calleja

ICI74864 N,N-bisallyl-tyr-Aib-Aib-Phe-Leu-OH {Aib: aminoisobutyrate}

LSD- lysergic acid diethylamide mCPP- 1-(3-chlorophenyl)piperazine HCI

MDL-7222- 3-tropanyl-3,5-dichlorobenzoate mfb- medial forebrain bundle

MK-801- [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]

MS- morphine sulphate

NAN-190- 1-(2-methoxyphenol)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide

NBQX- 2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f)quinoxaline

NDB- nucleus of the diagonal band

NMDA- N-methyl-D-aspartate

OT- olfactory tubercle

PFC- prefrontal cortex

PPN- pedunculopontine nucleus

QUIP- quipazine

RN- raphe nuclei s.c.- subcutaneous injection

SEM- standard error of the mean

TFMPP- m-trifluoromethylphenylpiperizine

U50488H- (trans-(±)-3,4-dichloro-n-methyl-[2-(1-pyrrolidinyl)cyclohexylJ) benzeneacetamide methane sulphonate

VP- ventral pallidum

VTA- ventral tegmental area LIST OF REFERENCES

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