AN ASSESSMENT OF THE EFFECTS OF

PSYCHOACTIVE DRUGS AND ELECTRICAL STIMULATION

OF THE VENTRAL TEGMENTAL AREA

ON THE STIMULUS PROPERTIES OF AMPHETAMINE

By

JONATHAN PETER DRUHAN

B.Sc, McGill University, 1983 M.A., The University of British Columbia, 1985

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(Department of Psychology)

We accept this thesis as conforming

to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

January 1989

(c) Jonathan Peter Druhan , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of PSYCHOLOGY

The University of British Columbia Vancouver, Canada

Date JANUARY 20, 1989

DE-6 (2/88) ABSTRACT

The discriminative stimulus properties of amphetamine are thought to result from the facilitatory actions of this drug on dopamine neurotransmission within the nucleus accumbens. As such actions within the nucleus accumbens also are hypothesized to be responsible for amphetamine's rewarding effects, the stimulus properties of amphetamine may be related to the hedonic effects of the drug. If these conclusions are correct, then tests for generalization with the stimulus properties of amphetamine might be useful either to determine the dopaminergic actions of drugs, or to screen newly developed compounds for their abuse potential.

In the present thesis rats were trained to discriminate

1.0 mg/kg amphetamine from saline, and then tested for stimulus generalization to a range of amphetamine doses

(0.0, 0.25, 0.50 and 1.0 mg/kg) injected either alone or in combination with either cocaine, apomorphine, haloperidol, nicotine, morphine, midazolam, ethanol or electrical stimulation of the ventral tegmental area (VTA). Comparisons were then made between the amphetamine stimulus generalization functions obtained in the presence and absence of the test stimuli, to determine whether the functions were altered in a manner consistent with the known dopaminergic actions or hedonic effects of the drugs and VTA stimulation. It was predicted that test stimuli that could enhance dopamine neurotransmission or produce positive hedonic effects might augment the stimulus properties of 111 amphetamine and elevate stimulus generalization functions relative to a control curve. Conversely, test stimuli that inhibited dopamine neurotransmission or reduced positive affect might interfere with the amphetamine stimuli and lower the generalization functions.

The results indicated that amphetamine stimulus generalization functions were altered in a manner that generally reflected the known actions of each test stimulus on dopamine neurotransmission. Thus, the generalization functions were elevated by stimuli that enhanced dopamine neurotransmission (cocaine, a dose of apomorphine affecting post-synaptic dopamine receptor sites, nicotine and VTA stimulation) and lowered by stimuli that interfered with dopamine neurotransmission (haloperidol, midazolam, and a dose of apomorphine that acts preferentially at presynaptic dopamine autoreceptors). Ethanol, which has not been found to consistently affect dopamine neurotransmission, did not generalize with the stimulus properties of amphetamine. Only morphine was found to affect amphetamine stimulus generalization functions (a lowering) in a manner that was inconsistent with the drug's facilitatory actions on dopamine neurotransmission.

The amphetamine stimulus generalization functions were not affected in a manner consistent with the hedonic actions of each test stimulus. Certain drugs that could produce positive hedonic effects (morphine, midazolam and ethanol) failed to elevate the generalization functions. In fact, the iv functions were elevated only by stimuli that appear to produce most of their rewarding effects by enhancing mesoaccumbens dopamine neurotransmission (cocaine, apomorphine, nicotine, and VTA stimulation). Two additional experiments suggested that this property could have been responsible for the ability of VTA stimulation to elevate amphetamine stimulus generalization functions. In one experiment, the ability of the VTA stimulation to substitute for the stimulus properties of amphetamine was found to be correlated positively with its rewarding efficacy measured during ICSS tests. A subsequent experiment indicated that dopamine neurons could indeed mediate discriminative stimuli produced by VTA stimulation, as the brain stimulation cues were augmented by amphetamine and attenuated by the dopamine receptor antagonist, haloperidol.

Together, the findings of this thesis indicated that amphetamine stimulus generalization paradigms might be useful for detecting the dopaminergic actions of certain psychoactive drugs. However, such procedures may not detect the abuse potential of all compounds. This latter result indicates that certain drugs of abuse do not produce amphetamine-like stimulus properties, and that this may be due to differences in the neural mechanisms that mediate their positive hedonic effects. V

TABLE OF CONTENTS

Abstract ii

Table of Contents v

List of Tables ix

List of Figures xi

Acknowledgment xiii

Introduction 1

Procedures for Measuring the Rewarding Effects

of Drugs 4

Drug Discrimination Procedures: General Overview .. 9

The Relation of Drug Discrimination Performance

to the Stimulus Properties of Drugs 11

Relations Between the Stimulus Properties and

Hedonic Actions of Drugs 16

Pharmacological Specificity of Amphetamine

Stimuli 18

Neurochemical Substrates for Amphetamine Stimuli .. 19

The Role of Specific Dopamine Projections in

Mediating the Stimulus Properties of Amphetamine .. 21

Common Neurochemical Substrates for the Stimulus

Properties and Rewarding Effects of Amphetamine ... 23

Issues to be Addressed in the Present Thesis 24

Discrimination Training Procedures 27

The Amphetamine Stimulus Generalization Paradigm .. 28

Predicted Effects of Different Test Stimuli on

Amphetamine Stimulus Generalization Functions 33 vi

General Methods 40

Subjects 40

Surgery and Histology 40

Apparatus 41

Drug Discrimination Training 43

Generalization Tests 44

Locomotor Activity Tests 45

Drugs 46

Statistical Analyses 47

Experiment 1: The Effects of Cocaine, Apomorphine, and

Haloperidol on Amphetamine Stimulus

Generalization Functions 50

Methods 52

Results 53

Discussion 62

Experiment 2: The Effects of Nicotine on Amphetamine

Stimulus Generalization Functions 67

Methods 69

Results 69

Discussion 76

Experiment 3: The Effects of Morphine on Amphetamine

Stimulus Generalization Functions 81

Methods 83

Results 83

Discussion 89 vii

Experiment 4: The Effects of Midazolam on Amphetamine

Stimulus Generalization Functions 94

Methods 95

Results 96

Discussion 101

Experiment 5: The Effects of Ethanol on Amphetamine

Stimulus Generalization Functions 105

Methods 106

Results 107

Discussion 113

Experiment 6: The Effects of Electrical Stimulation

of the VTA on Amphetamine Stimulus

Generalization Functions 117

Methods 118

Results 119

Discussion 128

Experiment 7: The Relationship Between the Rewarding

Effects of VTA Stimulation and its

Ability to Generalize with the Stimulus

Properties of Amphetamine 131

Methods 132

Results 133

Discussion 147

Experiment 8: The Effects of Amphetamine and Haloperidol

on Discriminative Stimuli Produced by

Electrical Stimulation of the VTA 151

Methods 153 viii

Results 156

Discussion 162

General Discussion 165

Implications of the Present Findings for a Theory

of the Stimulus Properties of Amphetamine 177

The Utility of Amphetamine Stimulus Generalization

Paradigms as Screening Procedures for Assessing the

Dopaminergic and Hedonic Properties of Drugs 184

Implications of the Present Findings for Theories

of Drug Abuse 188

References 192 ix

LIST OF TABLES

Table 1: The actions on dopamine neurotransmission and

the hedonic effects of the test stimuli

employed for Experiments 1 through 6 35

Table 2: Percentages of responses on the initially

selected lever after cocaine, apomorphine

and haloperidol 59

Table 3: Total number of responses after cocaine,

apomorphine and haloperidol 61

Table 4: Percentages of responses on the initially

selected lever after nicotine 73

Table 5: Total number of responses after nicotine 75

Table 6: Percentages of responses on the initially

selected lever after morphine 87

Table 7: Total number of responses after morphine 88

Table 8: Percentages of responses on the initially

selected lever after midazolam 99

Table 9: Total number of responses after midazolam .... 100

Table 10: Percentages of responses on the initially

selected lever after ethanol 111

Table 11: Total number of responses after ethanol 112

Table 12: Percentages of responses on the initially

selected lever during VTA stimulation 126

Table 13: Total number of responses durinng VTA

stimulation 127 X

Table 14: Percentages of responses on the initially

selected lever during substitution tests with

different parameters of VTA stimulation 140

Table 15: Total number of responses during substitution

tests with different parameters of VTA

stimulation . 141 LIST OF FIGURES

Figure 1: Theoretical outcomes of stimulus

generalization experiments 31-32

Figure 2: Effects of cocaine, apomorphine and

haloperidol on amphetamine stimulus

generalization functions 54-55

Figure 3: Effects of nicotine on amphetamine

stimulus generalization functions 71-72

Figure 4: Effects of nicotine on locomotor

activity 77-78

Figure 5: Effects of morphine on amphetamine

stimulus generalization functions 84-85

Figure 6: Effects of morphine on locomotor

activity 90-91

Figure 7: Effects of midazolam on amphetamine

stimulus generalization functions 97-98

Figure 8: Effects of midazolam on locomotor

activity 102-103

Figure 9: Effects of ethanol on amphetamine

stimulus generalization functions 108-109

Figure 10: Effects of ethanol on locomotor

activity 114-115

Figure 11: Electrode placements for the rats

employed in Experiment 6 120-121

Figure 12: Effects of VTA stimulation on amphetamine

stimulus generalization functions 123-124 Figure 13: Stimulus generalization between

amphetamine and VTA stimulation during

drug-free substitution tests 134-135

Figure 14: Relationship between electrode placements

and stimulus generalization with

amphetamine for the rats employed in

Experiment 7 137-138

Figure 15: Scattergrams showing the correlations

between stimulus generalization with

amphetamine and ICSS rates obtained

with VTA stimulation 143-144

Figure 16: Relationship between electrode placements

and ICSS rates for the rats employed in

Experiment 7 145-146

Figure 17: Electrode placements for the rats

employed in Experiment 8 158-159

Figure 18: Effects of amphetamine and haloperidol on

stimulus generalization to a range of

current intensities in rats trained to

discriminate high and low intensities of

VTA stimulation 160-161 xi i i

ACKNOWLEDGMENT

I would like to extend my gratitude to Dr. Anthony

Phillips for the support and supervision that he provided me throughout my graduate studies. His influence has been critical in keeping my thoughts focussed and my research 'on track'. I also would like to thank Dr. Donald Wilkie for his helpful advice at several stages of my research, from the construction of equipment to the writing of this thesis. I also am grateful to Dr. Peter Graf for his involvement on my departmental thesis committee and his helpful comments concerning the preparation of this thesis.

I am grateful to a number of research colleagues who deserve special mention for their contributions to my work.

In particular, Dr. Chuck Blaha provided me with valuable instruction in the preparation and administration of various drugs, and he also directed my attention to several of the references reported in this thesis. Fred LePiane, Shayne

Kardell, Lonn Myronuck and Chris Yamakura also provided their assistance at various stages of this research.

Finally, I would like to express my deepest appreciation to my wife, Lorraine for the consistent emotional, intellectual, and practical support that she provided me

(and Josh) throughout one very intense year. 1

INTRODUCTION

As was the case with many drugs of abuse, the psychomotor stimulant amphetamine was first used for medical purposes, specifically, for its peripheral actions as a bronchodilator and nasal decongestant. Subsequently, amphetamine was shown to be a central nervous system (CNS) stimulant capable of producing behavioral activation and subjective feelings of well-being and euphoria. The drug soon became subject to widespread non-medical use, and it became apparent that amphetamine might have powerful addictive properties (Cox, Jacobs, Leblanc, & Marshman,

1983).

Studies on the etiology of amphetamine abuse have played a major role in the development of recent theories of drug addiction. Traditional theories (Wikler, 1973; Seigel, 1983) often emphasized the importance of physiological dependence factors in maintaining patterns of habitual drug use. Drug- taking behaviors were thought to be maintained primarily by a need to alleviate the aversive withdrawal effects associated with drug abstinence. This model of drug addiction was based primarily on evidence that withdrawal from opiates, barbiturates or ethanol could result in severe abstinence syndromes that could intensify an individual's urge to self-administer these drugs (Cox et al., 1983).

However, such abstinence syndromes did not appear to be a prime motivator for the continued use of amphetamines or other psychomotor stimulant compounds (Griffith, 1977). The 2 symptoms associated with stimulant withdrawal were found to be relatively mild in comparison with those of the depressant drugs, and the patterns of stimulant self- administration were not consistent with what might be predicted from dependence models. The use of psychomotor stimulant drugs typically was found to occur in "runs", wherein an individual would repeatedly administer high doses of a stimulant over a period of a few days and then "crash".

During this crash phase, the individual might experience withdrawal symptoms characterized by fatigue and depression.

However, drug-taking behaviors often did not resume until well after this withdrawal phase, when the physiological abstinence syndrome had dissipated (Jaffe, 1987; Villarreal

& Salazar, 1981) .

In 1964 the World Health Organization Expert Committee on Addiction-Producing Drugs indicated that the central characteristics of amphetamine addiction included:

"...(1) a desire or need to continue taking the drug; (2) consumption of increasing amounts to obtain greater excitatory and euphoric effects or to combat more effectively depression and fatigue, accompanied in some measure by the development of tolerance; (3) a psychic dependence on the effects of the drug related to a subjective and individual appreciation of the drug's effects; and (4) general absence of physical dependency so that there is no characteristic abstinence syndrome when the drug is discontinued." (cf. Griffiths, 1977).

This statement emphasized the importance of direct subjective effects of amphetamine as the basis for its addictive properties, and downplayed the role of physiological dependence factors. Acceptance of this view of amphetamine addiction lead researchers to develop a variety 3 of psychometric scales for quantifying the subjective effects of drugs in humans, and the application of these measures to the study of non-stimulant drugs revealed that a wide range of addictive compounds shared an ability to elevate mood and induce euphoria (Bozarth, 1988). In recent years, attempts to provide a unified theory of drug addiction have focused primarily on the positive affective consequences of drug intake as the fundamental common denominator between drugs of abuse, and physiological dependence factors have been given a secondary role in maintaining pharmacological addictions (Baker, Morse, &

Sherman, 1986; Stewart, de Wit, & Eikelboom, 1984; Wise,

1987; Wise & Bozarth, 1987).

This change in theoretical perspective has been accompanied by a shift in focus for empirical investigations into the physiological basis for drug addiction. Instead of investigating the potential mechanisms for drug tolerance and dependence, studies over the past 20 years have attempted to identify the neural processes that give rise to the positive affective properties of various compounds. To facilitate this latter goal, a number of experimental procedures have been developed to measure the hedonic effects of drugs in laboratory animals. For the most part these procedures have involved measurements of the capacity for drugs to produce rewarding effects in operant conditioning paradigms. 4

Procedures for Measuring the Rewarding Effects of Drugs

The most commonly employed techniques for measuring the rewarding effects of drugs have been the self- administration, conditioned place preference and intracranial self-stimulation (ICSS) procedures (Bozarth,

1988; Phillips, Broekkamp, & Fibiger, 1983). With the self- administration procedure, animals are given the opportunity to perform an operant response which results in intravenous or intracerebral infusions of a drug (Brady, Griffiths,

Heinz, Ator, Lukas, & Lamb, 1988). The capacity of the infused drug to exert rewarding effects is reflected by selective increases in the performance of responses that result in the infusion, but not in the performance of inconsequential responses. To date, there has generally been a strong correspondence between the range of drugs that are self-administered by animals and compounds that are abused by humans (Brady et al., 1988). Moreover, the patterns of drug intake observed in animals often parallel those seen in human drug addicts. For example, experimental animals given access to amphetamine or other psychomotor stimulant compounds show the same pattern of "runs" and "crash" phases seen with human users (Yokel, 1988). In contrast, animals provided with opiate compounds generally exhibit the steady, continuous patterns of drug intake that are characteristic of human opiate addictions. These parallels between human and animal drug taking behaviors indicate that the self- administration paradigm may provide a valuable model for 5 investigating the neurobehavioral basis of drug addictions in infrahuman species.

The conditioned place-preference procedure measures the tendency for neutral environmental stimuli to acquire conditioned incentive value as a consequence of repeated association with a rewarding drug (Carr, Fibiger, &

Phillips, in press; White, Messier, & Carr, 1988). Animals tend to approach and spend more time in a chamber previously paired with injections of rewarding drugs relative to a chamber paired with saline injections. This preference for the previously neutral environment appears to reflect the development of an association between the environment and the positive hedonic properties of the drug (White et al.,

1988).

The intracranial self-stimulation paradigm represents a third means of assessing the rewarding properties of drugs

(Esposito & Kornetsky, 1978; Wise & Bozarth, 1984; Phillips et al., 1983). In this paradigm, animals are trained to respond for brain-stimulation reward and the effects of addictive drugs on self-stimulation are determined. Numerous studies have found that drugs which have abuse potential in humans also enhance the rewarding effects of electrical brain-stimulation (Bozarth, 1988). This enhancement usually is reflected as increases in response rates for the brain- stimulation reward or as decreases in the current intensity required to maintain threshold levels of responding. These effects are attributed to the capacity of addictive drugs to 6 enhance the excitability of neural processes that give rise to positive affect (Esposito & Kornetsky, 1978; Wise &

Bozarth, 1984).

Two other paradigms that have been employed to assess the rewarding effects of drugs in animals include the conditioned reinforcement and the self-administration reinstatement procedures (Beninger, Hanson, & Phillips,

1981; Davis & Smith, 1988; Robbins, Watson, Gaskin, & Ennis,

1983; Stewart & de Wit, 1988). Two variations of the conditioned reinforcement procedure exist. In one procedure

(Davis & Smith, 1988), animals are given repeated presentations of a neutral external stimulus (e.g. a buzzer) paired with the non-contingent administration of a rewarding drug. After such pairings, the animals will respond for presentations of the drug-paired stimulus in the absence of the drug, suggesting that the stimulus has acquired rewarding effects. In the second procedure (Beninger et al.,

1981; Robbins et al., 1983), a neutral stimulus is paired with a natural reward (e.g. food) in the absence of any drug treatment. The animals are then given tests in which they may perform a novel response to receive presentations of the conditioned reinforcer after injections of either a rewarding drug or its vehicle solution. Studies involving this procedure have indicated that a few addictive compounds will increase responding for the conditioned reinforcer, but the paradigm does not provide a reliable measure of the abuse potential of drugs. 7

The final procedure to be discussed in this section is an extension of the drug self-administration paradigm discussed above. In this paradigm, rats are trained to self- administer a rewarding drug intravenously and then placed on extinction for as many trials as are required for the cessation of operant responding. Following extinction, the rats are given a single non-contingent priming injection of either the training drug or another rewarding compound. The priming injection usually results in the reinstatement of operant responding even when responses do not produce further infusions of the drug. This reinstatement effect has been attributed to the capacity of the injected drug to activate appetitive motivational processes that usually mediate the drug's rewarding effects. As a consequence, the rats again become responsive to drug-related stimuli in the environment (i.e. the response lever; Stewart & de Wit,

1988; Stewart et al., 1984).

The procedures outlined above have been extremely useful for investigating the nature of drug addiction. On a theoretical level, studies employing the abovementioned procedures have provided firm evidence that addictive drugs can produce rewarding effects in non-dependent rats.

Furthermore, studies of the reinstatement phenomenon have indicated that rats will reinitiate self-administration responding for certain compounds after priming injections of the training drug, but not after injections of an antagonist to the drug. Thus, it is the presence of a drug in the body 8 that appears to determine the resumption of drug taking behaviors after a period of abstinence, not the absence of the drug (Stewart et al., 1984). These findings provide strong support for theories maintaining that the positive hedonic properties of drugs are important determinants of drug addictions in humans.

On a more practical level, these procedures have provided both a means of assessing the abuse potential of various compounds and a tool for investigating the neural basis for drug addiction. As noted, there has been a strong correlation between a drug's potential for abuse by humans and its capacity to produce rewarding effects in animals

(Brady et al., 1988; Weeks & Collins, 1988). With respect to determining the neural basis for drug addiction, many of these procedures have been used to assess the effects of lesions and transmitter manipulations on the rewarding effects of drugs (for reviews, see Carr et al., in press;

Phillips et al., 1983; Wise, 1983). These procedures also have been employed to assess the potential rewarding effects of drugs administered directly into discrete brain regions

(Bozarth & Wise, 1981; Broekkamp, 1988; Carr & White, 1986;

Phillips & Lepiane, 1980; Phillips, Mora, & Rolls, 1981;

Stewart & de Wit, 1988). Accordingly, such studies have helped to identify brain regions and neurotransmitter systems involved in mediating the hedonic properties of drugs.

Although the procedures outlined above have been 9 extremely useful for investigating the hedonic properties of drugs, there are certain limitations associated with each paradigm that sometimes have made interpretation of experimental results difficult (Bozarth, 1988). For example, the self-administration, self-stimulation, conditioned reinforcement and reinstatement procedures each measure animals' response level, and this dependent variable may be confounded by drug or lesion effects on response capacity.

On the other hand, place preference learning might be influenced by drug or lesion effects on associative learning, or by effects of state-dependent learning. In acknowledging these limitations, investigators have gone to great lengths either to control for such confounds, or to develop additional methods for assessing the hedonic effects of drugs. The remainder of this thesis will be devoted to reviewing and assessing one such alternative method which involves measuring the discriminative stimulus properties of drugs.

Drug Discrimination Procedures: General Overview

Another technique that could be employed to measure the hedonic actions of drugs in animals involves using drug sensations as discriminative stimuli in operant conditioning procedures. Animals may be trained to emit one response to obtain reinforcement (e.g. food) after receiving a specific dose of a drug, and an alternative response after saline injections. After several training sessions animals learn to discriminate accurately between the drug and saline states 10 and make the appropriate response at the start of each session. Thereafter, the initial response may be used to index the presence or absence of the drug state following experimental manipulations (see Colpaert, 1977b, 1978b, for reviews of these procedures).

The main advantage of employing drug discrimination procedures to investigate the hedonic properties of drugs is that the dependent measure is largely unaffected by the types of confounds that interfere with measures of the rewarding effects of drugs. For example, discrimination measures may be less affected when lesions or drug manipulations dramatically reduce operant performance capacity. Although such effects might decrease the level of responding observed during a discrimination trial, they would not be expected to influence animals' choices of which response is appropriate for the particular stimulus conditions. Discrimination measures might be affected by drugs that alter cognitive performance, however this latter influence can be distinguished from selective effects on stimulus perception by employing appropriate control procedures (see pages 28 to 33).

The major limitation of the drug discrimination procedure is that its face validity for the study of drug abuse has not yet been established. Recently, Overton (1988) indicated that the establishment of such face validity requires that three conditions be satisfied. First, drug abuse must be related to the sensory (subjective) effects of 11 abused compounds. Second, operant responses observed in drug discrimination experiments must reflect the control of behavior by sensory properties of the drug. Thirdly, the sensory effects that are discriminated by animals must be the same as those that determine the abuse potential of the substance.

As indicated above (pp. 3), there is now good reason to believe that most drugs of abuse are self-administered by humans for their euphoria-producing properties. Thus, the requirement that drug abuse be related to the subjective effects of drugs appears to be satisfied. The following two sections will briefly evaluate: 1) whether drug discrimination procedures measure sensory properties of drugs; and 2) whether such sensory properties are related to the euphoria-producing effects of the drugs.

The Relation of Drug Discrimination Performance to the

Stimulus Properties of Drugs.

Unlike externally generated stimuli, drug cues generally do not arise from actions on pre-established, peripheral sensory pathways (Overton, 1988). Therefore, drugs cannot be attributed stimulus properties by virtue of an ability to impinge on normal sensory transduction mechanisms. Instead, the capacity of drugs to function as stimuli must be inferred from similarities between drugs and externally generated events in the stimulus control of conditioned responses.

Although extensive comparisons between discriminations 12 involving drug or external stimuli have not yet been conducted, certain experimental observations suggest that the control of operant responses by drugs is similar to that obtained when conventional stimuli are employed. For example, the development of stimulus control appears to be similar regardless of whether animals are trained to discriminate drugs or exteroceptive stimuli (Overton, 1988).

In either case, operant responses during initial training trials tend to reflect the unconditioned biases of the animals. Response accuracy then increases gradually over several trials so that responses come to reflect the stimulus conditions for the trial rather than unconditioned biases (Colpaert, 1978b; Overton, 1988). The exact rate of acquisition may increase as a function of both increasing stimulus intensity when exteroceptive cues are employed, and increasing drug dosage when interoceptive cues are used.

High levels of asymptotic performance may be obtained in either case, depending on the particular qualitative and quantitative characteristics of stimulus or drug employed

(for further discussion of these comparisons see Overton,

1988). Interestingly, a recent study has shown that prior learning of a light versus dark discrimination could partially block the acquisition of a superimposed drug discrimination task (Jarbe, Svensson, & Laaksomen, 1983; c.f. Overton, 1988). Conversely, prior learning of a drug discrimination could block acquisition of discriminative control by differing illumination conditions. Thus, it 13 appeared that interoceptive drug stimuli could compete with exteroceptive stimuli for associative processing mechanisms.

Animals trained to discriminate a drug from saline may show a graded decrease in drug-appropriate responding when tested with progressively lower doses of the training compound (Colpaert, Niemegeers, & Janssen, 1979; Jarbe &

Swedberg, 1982). Such dose-response gradients are analogous to the stimulus generalization functions obtained when animals are tested with progressively lower intensities of an exteroceptive discriminative stimulus (Mackintosh, 1974).

Similar functions may be obtained when animals are tested with different doses of novel compounds that have pharmacological properties similar to those of the training drug. For example, animals trained to discriminate a psychomotor stimulant drug will emit drug-appropriate responses when tested with other stimulants (Colpaert et al., 1979; Silverman & Ho, 1977), and animals trained with an anxiolytic compound will emit drug appropriate responses when tested with other anxiolytics (Garcha, Rose, &

Stolerman, 1985). In contrast, drug-appropriate responses do not occur when animals are tested with compounds that lack pharmacological actions of the training drug (Colpaert et al., 1979; Garcha et al., 1985; Silverman & Ho, 1977). In such instances, animals consistently emit responses appropriate for the saline-condition.

These findings are consistent with the hypothesis that responses measured in drug discrimination experiments are 14 controlled by the stimulus properties of the training compound. Animals may learn to discriminate the presence versus absence of specific pharmacological actions exerted by a drug. Injections of other compounds with similar actions may reproduce the stimulus effects and result in drug-appropriate responses. In contrast, drugs that lack the necessary pharmacological actions do not reproduce the stimulus effects, and under these conditions animals emit responses appropriate for the absence of the training cue.

Although the present thesis is concerned primarily with the use of operant conditioning procedures to study the stimulus properties of drugs, it is worth mentioning here that drugs also appear capable of functioning as conditional stimuli in Pavlovian learning paradigms. Much of this work has involved analyses of the effects of pairing the stimulus properties of one drug with illness produced by a second drug (e.g., lithium chloride). In these procedures, the first drug acts as a conditional stimulus whereas the illness-producing drug functions as an aversive unconditional stimulus. Following the drug-drug pairings, subsequent injections of the conditional drug alone result in a variety of apparent conditioned responses. The conditioned responses that have been found to occur include bradycardia (Wilkin, Cunningham, & Fitzgerald, 1982), adipsia (conditioned sickness; Revusky, Taukulis, & Peddle,

1979) and various conditioned opponent processes that counteract the aversive properties of the illness-producing 15 drug (conditioned antisickness response; Lett, 1983; Revusky

& Harding, 1986). Such responses were not observed in control groups that received the two drugs either alone or in the reverse injection order (backward pairings). These findings suggest that drugs may have conditional stimulus attributes in addition to the discriminative stimulus properties described above.

Alternative explanations for drug discrimination learning invoke state-dependent mechanisms to account for the differentiation of responses under different injection conditions. During drug discrimination training, animals are required to perform one response alternative while in a drugged state or another response alternative while in an undrugged state. Such training could conceivably result in the independent learning of two state-dissociated responses, the performance of which would require the operation of state-dependent retrieval or response initiation mechanisms

(Bindra & Reichert, 1966; 1967; Bliss, 1974; Overton, 1978).

Indeed, numerous studies have shown that responses learned in a drugged state often are not performed in an undrugged state, whereas responses acquired in the absence of a drug are often not emitted following drug treatments (for a review see Overton, 1988). However, the drug doses usually required to demonstrate such state-dependent phenomena are usually much higher than those employed during drug discrimination training (Overton, 1988). Furthermore, state- dependency models for drug discrimination learning do not 16 predict the occurrence of saline-appropriate responses when animals are given substitution tests with compounds that differ pharmacologically from the training drug. Rather, such models would predict responses to be randomly distributed and dramatically reduced after injections of these latter compounds.

Relations Between the Stimulus Properties and Hedonic

Actions of Drugs

At present, there is little evidence to confirm or refute a relationship between the discriminative stimulus properties and hedonic actions of addictive drugs. Evidence in support of a general relation between the cueing and hedonic effects of drugs has been limited primarily to casual observations that all drugs of abuse can be discriminated by animals (Overton, 1988). However, animals can also discriminate a number of non-addictive drugs

(Overton, 1982; 1988), and there appears to be only a weak correlation between the discriminability of different compounds and their abuse potential (Overton & Batta, 1977).

These findings indicate that animals are capable of discriminating non-hedonic actions of drugs, and that such actions may contribute to the discriminative stimulus properties of certain addictive compounds.

The relative contributions of hedonic and non-hedonic drug actions to the control of discriminated responding may vary as a function of the particular compound employed during training. For example, animals trained to 17 discriminate narcotic compounds from saline appear to respond primarily to non-hedonic properties of the drugs. In one study, midbrain lesions that blocked the rewarding actions of morphine did not prevent this drug from acting as a discriminative stimulus (Martin, Bechara, & van der Kooy,

1987). Additional studies have indicated that the cueing potencies of several narcotic compounds are positively correlated with their analgesic actions (Colpaert, 1978a).

Drug discrimination studies involving psychomotor stimulant compounds present a different picture. For example, Colpaert et al. (1979) found that rats trained to discriminate cocaine from saline generalized to a variety of compounds that supported intravenous self-administration. In contrast, generalization was not observed when the rats were tested with compounds that lacked such rewarding actions.

These results suggested a possible hedonic basis for the discriminative stimulus properties of cocaine. Indeed, other studies have found that the cueing effects of cocaine may be mediated by the same neural processes that give rise to the hedonic properties of this compound (for reviews see Ho &

Silverman, 1978; Silverman & Ho, 1977).

An important goal for future drug discrimination research should be to provide independent assessments of the extent to which the stimulus properties of individual substances reflect hedonic actions of the drugs. One means of accomplishing this task might be to compare the neural substrates for the cueing and hedonic actions of drugs. A 18 relationship between these actions of a drug would be indicated if the separate functional properties were found to be derived from the same neuropharmacological actions.

To date, only a few compounds have been studied extensively enough that the neural substrates for both the stimulus properties and rewarding effects of the drug are known. Perhaps the most extensively studied compound in this regard is amphetamine. The following four sections will review evidence that the stimulus properties of amphetamine are derived from pharmacological actions on specific neural processes, and that these processes may be the same ones which mediate the hedonic properties of amphetamine.

Pharmacological Specificity of Amphetamine Stimuli

In an earlier section it was mentioned that drug stimuli tend to be pharmacologically specific, such that stimulus generalization may occur only between drugs with similar pharmacological properties. The stimulus properties of amphetamine are consistent with this rule. Thus, animals trained to discriminate amphetamine from saline emit drug appropriate responses following injections of other psychomotor stimulant drugs such as 1-amphetamine, methyl- amphetamine, cocaine, methylphenidate, cathinone and amfonelic acid (Aceto, Rosencrans, Young, & Glennon, 1984;

Colpaert, Niemegeers, & Janssen, 1978; Ho & McKenna, 1978;

Huang & Ho, 1974a; Huang & Wilson, 1986; Schecter &

Rosencrans, 1973). In contrast, amphetamine-trained animals respond primarily on the saline-appropriate lever when 19 tested with compounds that lack psychomotor stimulant actions (Jarbe, 1982; Schecter & Rosencrans, 1973; Silverman

& Ho, 1977). Among the more important of these latter compounds are the peripherally acting stimulant, para- hydroxyamphetamine (Jarbe, 1982; Jones, Hill, & Harris,

1974), and the general CNS stimulants nikethamide, picrotoxin and strychnine (Huang & Ho, 1974b). Para- hydroxyamphetamine is an amphetamine analogue which does not cross the blood-brain barrier, therefore it exerts only peripheral actions. The lack of generalization to this drug indicates that the stimulus properties of amphetamine involve actions within the CNS3-. The absence of generalization to the CNS stimulants nikethamide, picrotoxin and strychnine further suggests that animals do not simply discriminate a general increase in CNS excitability or arousal. Rather, animals appear to discriminate specific psychomotor stimulant properties of amphetamine that result from the CNS actions of this drug.

Neurochemical Substrates for Amphetamine Stimuli

Although amphetamine is capable of enhancing neurotransmission at dopaminergic, noradrenergic, and serotonergic synapses throughout the CNS, it is the actions at dopaminergic synapses that appear to be responsible for

1. The stimulus properties of low amphetamine doses (0.125 mg/kg) may be mediated by peripheral processes, as they have been found to generalize with parahydroxyamphetamine (Colpaert, Kuyps, Niemegeers, & Janssen, 1976). However, the use of such low training doses is rare, and thus the present review will refer only to stimulus properties associated with higher training doses (i.e. > 0.5 mg/kg). 20 the drug's discriminative stimulus properties. Rats trained to discriminate amphetamine from saline responded on the drug-appropriate lever when injected with the direct dopamine receptor agonists apomorphine, piridebil, and N- propylnoraporphine (Schecter, 1977). Amphetamine-appropriate responses also were observed following injections of the anti-Parkinson drug amantidine (Schecter, 1977), the antidepressant compounds deprenyl, tranycypromine, amineptine, and bupropion (Porsolt, Pawelec, & Jalfre, 1982) and as indicated above (pp. 18), the psychomotor stimulants cocaine, methylphenidate, cathinone, nomifensine, and amfonelic acid. All of these compounds are capable of enhancing dopamine neurotransmission, either by stimulating the release of dopamine or by blocking its reuptake (Wagner,

Preston, Ricaurte, Schuster, & Seiden, 1982; Westerink,

1979). Importantly, rats did not emit amphetamine- appropriate responses when injected with compounds that did not affect dopaminergic neurotransmission (see Silverman and

Ho, 1977; Porsolt et al., 1982).

Studies involving pharmacological interference with monoaminergic neurotransmission also support the hypothesis that amphetamine stimuli result from drug actions at dopaminergic synapses. Rats trained to discriminate amphetamine from saline responded primarily on the saline- appropriate lever when the amphetamine injections were preceded by pretreatments with the DA receptor antagonists haloperidol, pimozide, spiroperidol, cis(Z)-flupenthixol, 21 trifluperazine, perphenazine or chlorpromazine (Ho & Huang,

1975; Nielsen & Jepsen, 1985; Schecter & Cook, 1975).

Similar results were obtained when dopamine synthesis was impaired temporarily by pretreatment with the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine (Ho & Huang,

1975; Schecter & Cook, 1975) or when CNS dopamine neurons were destroyed by an intraventricular injection of the neurotoxin 6-hydroxydopamine (6-OHDA; Woolverton & Cervo,

1986). In contrast, pretreatment with the noradrenaline antagonists phentolamine, phenoxybenzamine, and propranolol

(Ho & Huang, 1975; Schecter & Cook, 1975) or the serotonin antagonists methysergide and cinanserin (Ho & Huang, 1975) had no effect on amphetamine stimuli. The drug cues also were not affected when noradrenaline or serotonin synthesis was disrupted by treatment with the dopamine-B-hydroxylase inhibitor disulfiram or the tryptophan hydroxylase inhibitor, p-chloro-phenylalanine (Schecter & Cook, 1975).

These latter results indicate that the amphetamine stimuli do not depend on drug actions at noradrenergic or serotonergic synapses.

The Role of Specific Dopamine Projections in Mediating the

Stimulus Properties of Amphetamine

Many of the behavioral effects of amphetamine appear to be mediated by either the mesostriatal or mesocortical dopamine neurons. Both of these neural projections arise from cell bodies within the ventral mesencephalon and terminate either within the striatal nuclei or within 22 various limbic and cortical regions of the forebrain (Fallon

& Moore, 1978). Recent studies have indicated that the mesocortical dopamine neurons may mediate the stimulus properties of amphetamine. In one study, amphetamine stimuli were attenuated by the atypical dopamine receptor antagonists sulpiride, clozapine, thioridazine, and molindone (Nielsen & Jepsen, 1985). These antagonists exhibited relatively selective binding at mesocortical dopamine receptor sites, and exerted limited activity at striatal dopamine receptors (Lane & Blaha, 1986). Although the amphetamine cue also was antagonized by the striatal dopamine receptor antagonist metoclopromide, the doses required for this antagonism were quite high (7.5 & 10.0 mg/kg). At these doses metoclopramide may be less selective for striatal receptors and the attenuation of the amphetamine cue may have reflected additional actions at mesocortical receptors.

The component of the mesocortical dopamine pathways that terminates in the nucleus accumbens appears to play a particularly important role in mediating the stimulus properties of amphetamine. Rats trained to discriminate systemic injections of amphetamine from saline emitted drug appropriate responses when amphetamine was injected directly into the nucleus accumbens (Nielsen & Scheel-Kruger, 1986).

This generalization between systemic and central nucleus accumbens injections was blocked when the dopamine receptor antagonist, sulpiride was coinjected into the nucleus 23 accumbens along with amphetamine. Importantly, rats in this study did not generalize to injections of amphetamine into the dorsomedial and lateroventral caudate. This latter result indicates that the stimulus properties of amphetamine are not mediated by mesostriatal dopamine neurons.

Common Neural Substrates for the Stimulus Properties and

Rewarding Effects of Amphetamine

The studies reviewed above indicate that the stimulus properties of amphetamine may be mediated by dopamine projections to the nucleus accumbens. These mesoaccumbens dopamine neurons also appear to mediate the rewarding effects of amphetamine. Rats will self-administer amphetamine directly into the nucleus accumbens (Monaco,

Hernandez, & Hoebel, 1980) and show conditioned place preferences for environments that have been associated with intra-accumbens injections of this drug (Aulisi & Hoebel,

1983; Carr & White, 1983, 1986). Injections of amphetamine into the nucleus accumbens also can enhance the behavioral control exerted by conditioned reinforcers (Taylor &

Robbins, 1984). In contrast, self-administration and place preference conditioning maintained by peripheral injections of amphetamine can be attenuated by 6-OHDA lesions of the mesoaccumbens dopamine projections (Lyness, Friedle, &

Moore, 1979; Spyraki, Fibiger, & Phillips, 1982). Given the evidence that mesoaccumbens dopamine neurons may mediate both the stimulus and rewarding properties of amphetamine, it is possible that the stimulus properties may reflect 24 hedonic actions of this drug.

Issues to be Addressed in the Present Thesis

From the findings reviewed in the previous four sections, it is possible to formulate two hypotheses about the stimulus properties of amphetamine. One hypothesis is that the stimulus properties of amphetamine result from the capacity of this drug to enhance mesocortical dopamine neurotransmission, particularly at synapses within the nucleus accumbens. To the extent that this neurochemical action produces rewarding effects, a second hypothesis may be formulated regarding the functional nature of the amphetamine stimuli. Specifically, it is suggested that the stimulus properties of amphetamine may reflect the hedonic actions of this drug.

If these hypotheses are correct, then tests for generalization with the stimulus properties of amphetamine might be useful either as a general behavioral assay for determining the dopaminergic actions of drugs or as a screening procedure to evaluate the abuse potential of newly developed compounds. Generalization with the stimulus properties of amphetamine might indicate that a drug could enhance dopamine neurotransmission or produce positive hedonic effects, whereas an absence of generalization might indicate that the compound lacked these actions. To date, these predictions have been satisfied consistently by experiments showing that amphetamine stimuli generalize to other psychomotor stimulants, direct dopamine agonists, and 25 psychomotor stimulant-like antidepressants, but amphetamine cues do not generalize to a variety of non-stimulant drugs that lack dopaminergic or positive hedonic actions (Porsolt et al., 1982; Silverman & Ho, 1977). Nevertheless, there are potential limitations of this screening procedure which should be evaluated before it is accepted as a valid paradigm for assessing the neurochemical or affective properties of drugs.

One potential limitation of the amphetamine generalization paradigm as a general screening procedure is that the stimulus properties of amphetamine may not generalize to drugs other than psychomotor stimulants and direct dopamine agonists. In general, there appear to be limitations on the ability of drugs to generalize to compounds from different pharmacological classes (Colpaert,

1978a; Lai, 1977; Overton, 1988; Silverman & Ho, 1977).

These limitations may vary depending on the particular class of training drug employed. For example, generalization with the stimulus properties of narcotic agonists appears to be highly selective (Colpaert, 1978a). In contrast, anesthetics, benzodiazepines, muscle relaxants and anticonvulsants readily generalize to one another, suggesting that drugs from these individual classes may generate common stimuli related to their depressant actions

(Overton, 1988). The stimulus properties of psychomotor stimulant compounds appear to be fairly specific, although cocaine has been found to generalize partially to the 26 narcotic analgesic fentanyl, the psychotomimetic phencyclidine, the noradrenergic receptor blocker propranalol, and the muscarinic acetylcholine receptor blockers dexetimide and benztropine.

A second possible limitation of the amphetamine generalization paradigm relates specifically to its utility for detecting the abuse potential of drugs. As yet, it has not been determined whether the stimulus properties of amphetamine reflect a general state of positive affect which could generalize to all positively hedonic compounds.

Stimulus generalization with amphetamine may be restricted to drugs that produce their hedonic effects by acting on those processes that mediate the rewarding (and cueing) actions of amphetamine. If this latter case were true, then the screening procedure might only be useful for identifying the abuse potential of a limited range of compounds2.

The present thesis determined whether these constraints on generalization between drug stimuli would limit the utility of an amphetamine stimulus generalization paradigm for screening either the dopaminergic actions or the abuse potential of psychoactive drugs. To accomplish this goal, rats were trained to discriminate amphetamine from saline and then given stimulus generalization tests with drugs from

2. Wise and Bozarth (1987) have suggested that all drugs of abuse may produce their hedonic effects by acting either on the mesoaccumbens dopamine projection, or processes afferent or efferent to these neurons. If this hypothesis is correct, then the second limitation presented here would not restrict the utility of the amphetamine generalization paradigm as a procedure for screening the abuse potential of drugs. 27 diverse pharmacological classes and with a non- pharmacological stimulus produced by electrical stimulation of the VTA. The drugs and brain-stimulation employed for these experiments were chosen on the basis that their actions on both dopamine neurotransmission and affective processes were already known. Thus, it was possible to predict the probable outcomes that should be obtained in the absence of any of the abovement ioned constraints on generalization. Differences between the actual and predicted outcomes would indicate that the generalization paradigm might have limits to its utility as a screening procedure.

Discrimination Training Procedures

A wide variety of procedures have been employed to assess the discriminative stimulus properties of drugs. For example, animals may be trained to discriminate either a fixed dose of a drug from saline, one dose from a second dose of the same compound or a specific dose of one drug from a specific dose of a different compound (Jarbe and

Swedberg, 1982). Animals also may be trained to perform a variety of other discriminations that can involve three or more drug stimuli (Overton, 1988). To date, most studies of the stimulus properties of amphetamine have involved discriminations between a set dose of amphetamine (usually between 0.75 and 1.0 mg/kg) and saline. This type of discrimination procedure also was employed for the present experiments, with rats being trained to discriminate 1.0 mg/kg amphetamine from saline. 28

Drugs may exert stimulus control over a wide variety of operant responses. These responses may be appetitively or aversively motivated, may involve position or go/no-go tasks, and they may be maintained by a range of reinforcement schedules. The particulars of these procedures, as well as their advantages and disadvantages have been reviewed by Colpaert (1977b, 1978b) and by Overton and Hayes (1984). In general, most investigators have come to prefer a two-lever, appetitively motivated choice procedure in which animals are required to respond on one of two levers to obtain reinforcement after receiving a drug injection, or on the alternative lever after receiving a saline injection. Reinforcement is usually delivered on a fixed ratio (FR) schedule that requires the animals to emit at least ten responses on the appropriate lever to obtain each reward. The present experiments employed a similar two- lever choice procedure with an FR-32 reinforcement schedule.

The Amphetamine Stimulus Generalization Paradigm

In most drug discrimination experiments, stimulus generalization between compounds is assessed by giving animals tests in which a novel compound is substituted for the usual training drug. Generalization is assumed when animals emit responses appropriate for the training-drug state following injection of the novel compound (Colpaert,

1977b). However, this assumption is valid only when drugs induce strong generalization such that the animals consistently emit responses appropriate for the drug 29 condition. In cases where only partial generalization occurs, animals will often alternate their responses between the two available choices. This behavior pattern may be difficult to interpret as the random lever-choices may reflect either partial generalization or a general disruption of performance (Colpaert, 1988). In these circumstances, it is important to demonstrate further that stimuli associated with the test compound can summate with the cueing effects produced by low doses of the training compound (e.g., see Reavill & Stolerman, 1988). Such summation would be reflected as a more complete generalization with the combination of the two drugs than is obtained with either drug alone.

In order to provide for the unambiguous interpretation of the stimulus generalization experiments3 in the present thesis, the individual psychoactive drugs and VTA stimulation were given in combination with a range of amphetamine doses (0.04, 0.25, 0.50 and 1.0 mg/kg). Under

3. The present experiments represent a departure from traditional approaches to generalization testing. Generalization tests usually involve the presentation of simple stimuli that differ quantitatively from the training stimulus along a particular sensory dimension (Mackintosh, 1974). In the present experiments, animals are tested with complex stimuli that result from the super imposition of drug cues. The tests require that the animals detect the extent of summation of familiar cue properties upon a background of novel stimuli that may differ qualitatively from the training stimulus. Despite these differences, the procedure will continue to be referred to as a generalization test to reflect the fact that animals are being tested for the extent to which the test stimuli evoke the responses appropriate for the training cue. 4. The 0.0 mg/kg amphetamine "dose" is actually a saline injection. Although this injection normally is used as the reference stimulus for the amphetamine discrimination, it 30 baseline conditions, each successive dose of amphetamine was expected to elicit increasing amounts of responding on the drug-appropriate lever, resulting in an orderly stimulus generalization function (Figure 1). The combination of amphetamine with test stimuli that possessed amphetamine• like cueing actions might result in increased drug-lever responding at the low amphetamine doses (eg. 0.0, 0.25 and

0.5 mg/kg in Figure 1) due to a summation of the common stimulus properties. This would be reflected as an elevation of the stimulus generalization function relative to the vehicle control curve. In contrast, test stimuli that antagonized the cueing potency of amphetamine might decrease drug-lever responses at most amphetamine doses (0.25, 0.5 and 1.0 mg/kg in Figure 1) and lower this function relative to the vehicle curve. Test stimuli that produced general disruptions of discriminative performance would not produce such uniform additive or subtractive effects on the generalization functions. Instead, these stimuli should result in generalization functions that reflect a more

will be described throughout the thesis in terms of its position along a continuum of amphetamine doses when the results of generalization tests are being considered. This will facilitate discussions of the data. Nevertheless, the reader should bear in mind that increases in drug- appropriate responding observed when a test stimulus is combined with this amphetamine "dose" actually reflect generalization with the stimulus properties of amphetamine rather than summation between drug stimuli. 31

Figure 1: Theoretical outcomes of stimulus generalization experiments. The curves reflect the percentage of responses that rats might emit on the lever appropriate for an amphetamine test (y-axis) as a function of increasing amphetamine doses (x-axis). The solid curve represents the theoretical baseline generalization function that might be obtained following vehicle control tests. Under these conditions, the percentage of responses emitted on the amphetamine (drug) lever should increase as a monotonic function of increasing amphetamine dose. Experimental manipulations (drugs or brain-stimulation) that augment the stimulus properties of amphetamine should result in increased responding on the drug-lever at the lower amphetamine doses and an elevation of the amphetamine generalization function relative to the control curve. In contrast, manipulations that interfere with the amphetamine stimuli should reduce drug-lever responses at the higher amphetamine doses and lower the generalization function.

Extreme disruptions of discriminative performance would cause responses to be randomly distributed between levers regardless of the amphetamine dose, and thus the generalization function would lie flat around the 50% response level. 32

• DISRUPTION

0.0 0.25 0.50 . 1.0 AMPHETAMINE DOSE (mg/kg) 33 random distribution of responses. Figure 1 shows a hypothetical example of the most extreme case in which a drug produces random responding at each dose of amphetamine.

Other cases may exist where the amount of disruption varies as a function of the amphetamine dose. Under such circumstances the test and control functions would overlap at one end of the amphetamine dose continuum and diverge at the other end, with responses at the divergent end being somewhat random.

Predicted Effects of Different Test Stimuli on Amphetamine

Stimulus Generalization Functions

In the previous section it was suggested that test stimuli that possess amphetamine-like stimulus properties might elevate amphetamine stimulus generalization functions due to the summation of common stimulus elements. In contrast, test stimuli that were antagonistic to amphetamine cues might lower the stimulus generalization functions due to subtractive interactions between the stimuli. To the extent that the stimulus properties of amphetamine may be related to either the dopaminergic and or hedonic actions of the drug, it would be predicted that:

1) Test stimuli that can enhance mesocortical dopamine neurotransmission might summate with the stimulus properties of amphetamine and elevate the stimulus generalization functions relative to curves obtained with amphetamine alone. In contrast, test stimuli that interfere with mesocortical dopamine neurotransmission might antagonize the 34 cueing effects of amphetamine and lower the stimulus generalization functions.

2) Test stimuli that exert positive hedonic actions might summate with the stimulus properties of amphetamine and elevate the stimulus generalization functions relative to curves obtained with amphetamine alone. In contrast, test stimuli with anhedonic actions might antagonize the cueing effects of amphetamine and lower the stimulus generalization functions.

The particular test stimuli to be employed in the present thesis are presented in Table 1. As indicated above

(pp. 26) the drugs were chosen from a wide range of pharmacological classes to determine whether the stimulus generalization paradigm could be used to screen a diverse selection of compounds for their dopaminergic actions and hedonic effects. Thus, each drug listed in Table 1 is a representative either of a major class of abused compounds or of a dopamine receptor agonist (apomorphine) or antagonist (haloperidol). Electrical stimulation of the VTA also was employed as a test stimulus, to represent a major class of non-pharmacological hedonic stimuli. The particular stimuli selected as representative of each class were chosen on the basis that their dopaminergic actions and hedonic effects have been determined previously. As indicated in

Table 1 the range of test stimuli employed includes agents that either can enhance, attenuate or have no effects on dopamine neurotransmission, and that may produce either 35

TABLE 1

The actions on dopamine neurotransmission and the hedonic effects of the test stimuli employed for Experiments 1 through 6.

DRUG DOPAMINERGIC ACTION HEDONIC EFFECT

Expt 1: Cocaine Enhances Positive

Apomorphine Low Dose Attenuates

High Dose Positive Mimicks

Haloper idol Attenuates Anhedoni

Expt 2: Nicotine Enhances Pos itive

Expt 3: Morphine Enhances Positive

Expt 4: Midazolam Attenuates Positive

Expt 5: Ethanol No Effects Positive

Expt 6: Stimulate VTA Enhances Positive 36 positive hedonic or anhedonic effects. The selection of test stimuli with these different actions allowed an assessment of the capacity for the present generalization paradigm to detect both additive and subtractive interactions with the stimulus properties of amphetamine.

The first experiment assessed the effects of cocaine, apomorphine, and haloperidol on amphetamine stimulus generalization functions. Cocaine and high doses of apomorphine can both augment dopamine neurotransmission and produce rewarding effects in laboratory animals (Anden,

Rubensson, Fuxe, & Hokfelt, 1967; Ritz, Lamb, Goldberg, &

Kuhar, 1987). Either of these actions might result in elevations of the amphetamine stimulus generalization functions relative to control curves. In contrast, the generalization functions might be lowered by haloperidol, which can both interfere with dopamine neurotransmission and attenuate the rewarding effects of various drugs (Anden,

Butcher, Corrodi, Fuxe, & Ungerstedt, 1970; Carr et al., in press) and by low doses of apomorphine that decrease the impulse-dependent release of dopamine (Gonon & Buda, 1985;

Grace & Bunney, 1985; Lane & Blaha, 1986; Zetterstrom &

Ungerstedt, 1984). Such results would confirm the sensitivity of the generalization paradigm for detecting the additive or subtractive effects of other psychomotor stimulants and dopaminergic receptor agonists and antagonists.

The subsequent four experiments examined the effects on 37 amphetamine stimulus generalization functions of non- psychomotor stimulant drugs including nicotine (a CNS stimulant), morphine (an opiate), midazolam (a benzodiazepine), and ethanol (a sedative hypnotic). Nicotine and morphine both can enhance mesoaccumbens dopamine neurotransmission (Di Chiara & Imperato, 1986; Gysling &

Wang, 1983; Imperato, Mulas, & Di Chiara, 1986; Mereu et al., 1987; Moleman, van Valkenburg, & van der Krogt, 1984 ) and can exert positive hedonic actions in laboratory animals and humans (Henningfield & Goldberg, 1983; Jasinski, 1977;

Martin & Jasinski, 1977). Accordingly, these drugs were predicted to augment the stimulus properties of amphetamine and elevate the generalization functions relative to control curves.

Midazolam and ethanol each produce differential effects on dopamine neurotransmission and on affective processes.

Both of these drugs can produce rewarding effects in laboratory animals (Ator & Griffiths, 1987; Mello, 1981;

Szostak, Finlay, & Fibiger, 1987). However, midazolam has been found to decrease dopamine neurotransmission (Finlay et al., 1987; Haefely, Pieri, Pole, & Shaffner, 1981) whereas ethanol has been shown to have no effect on dopamine function (Hellevuo & Kiianmaa, 1988; Kalant, 1975; Nutt &

Glue, 1986; Smith, 1977; but see Di Chiara & Imperato,

1986). These differences in the neurochemical and functional actions of midazolam and ethanol necessitate separate predictions for the stimulus generalization experiments. If 38 the dopaminergic actions of a drug determine its effects on amphetamine stimuli, then midazolam should lower generalization functions whereas ethanol should have no effects. If the hedonic actions of a drug determine its effects on amphetamine stimuli, then both drugs should elevate the generalization functions. The outcomes of these experiments may indicate the relative importance of either the hedonic properties or the dopaminergic actions of drugs in determining interactions with amphetamine stimuli.

The four abovementioned experiments also examined the effects of nicotine, morphine, midazolam, and ethanol on locomotor activity. Changes in activity levels often reflect parallel changes in dopaminergic neurotransmission within the nucleus accumbens. Thus, drugs which decrease mesoaccumbens dopamine neurotransmission consistently reduce activity levels, whereas compounds that enhance dopamine function within this region often increase locomotion.

Accordingly, the locomotor activity tests provided an independent behavioral means of assessing the dopaminergic actions of the drugs employed in experiments 2 through 5.

Experiment 6 evaluated the effects of electrical stimulation of the VTA on amphetamine stimulus generalization functions. As stimulation of this region can both activate mesoaccumbens dopamine neurons and produce rewarding effects in rats (Blaha, Phillips, & Fibiger, 1988;

Fibiger, Lepiane, Jakubovic, & Phillips, 1987), this stimulation might augment the cueing effects of amphetamine 39 and elevate the stimulus generalization functions. Such a result would indicate that the amphetamine stimulus generalization function could detect the dopaminergic or hedonic actions of non-pharmacological stimuli.

Experiments 7 and 8 were designed to investigate the roles of both the hedonic effects and dopaminergic actions of VTA stimulation in determining the interactions of this test stimulus with the stimulus properties of amphetamine observed in Experiment 6. Experiment 7 assessed whether individual differences in the amount of generalization obtained between the stimulus properties of amphetamine and

VTA stimulation might be related to differences in the efficacy of the stimulation for producing rewarding effects during ICSS tests. A correlation between the extent of stimulus generalization and ICSS rates would suggest that the generalization was related to the hedonic effects of the stimulation. Experiment 8 determined whether the stimulus properties of VTA stimulation could be modulated by amphetamine and haloperidol, in an attempt to confirm the dopaminergic mediation of this non-pharmacological stimulus. 40

GENERAL METHODS

Subjects

Male hooded rats (Charles River, Long Evans strain) were used as subjects for these experiments. All rats were housed individually in stainless steel cages with tap water available ad libitum. Access to food was restricted, and 22 to 24 gms of standard rat chow was provided at the end of each day. A 12 hr light-dark cycle was maintained in the rat colony, and the rats were tested either within the last two hrs of the light phase or during the first two hours of the dark phase of this cycle.

Surgery and Histology

Prior to discrimination training, the rats in

Experiments 6 through 8 were anesthetized with 65 mg/kg sodium pentobarbital and a bipolar electrode (Plastic

Products MS303/2) was implanted stereotaxically into the

VTA. With the incisor bar set at -3.2 mm below the interaural line, the coordinates from stereotaxic zero were:

2.8 mm anterior, 0.6 mm lateral (toward the left hemisphere) and 2.1 mm dorsal. Electrodes were anchored to the skull with jewelers screws and dental cement.

Upon completion of the brain-stimulation experiments, rats with electrodes were killed with an overdose of sodium pentobarbital and perfused transcardially with 0.9% saline followed by 10% formol-saline. The brains were removed and stored for at least one week in 10% f ormol-sal ine before being frozen, sliced in 30 um sections, mounted on 41 microscope slides and stained with cresyl-violet for verification of electrode placements.

Apparatus

Most of the discrimination experiments were conducted in six test chambers (24 X 29 X 30 cm), each having Plexiglas walls and ceiling and a wire grid floor. These chambers contained two levers (5X8 cm) mounted 3.5 cm above the floor on opposite walls, a 28 v house-light attached to the center of a third wall and a food-hopper positioned directly below the light (3 cm above the floor). Sound attenuating enclosures (55 X 55 X 60 cm) with ventilation fans were used to contain each chamber and isolate the rats from extraneous environmental stimuli. For Experiment 8, electrode leads

(Plastic Products 303-302) were attached to Mercotac swivel commutators stationed on the ceilings of the outer enclosures and the wires were passed through openings at the top of each chamber. Constant current electrical stimulation was delivered by independent channels of a programmable sine-wave stimulator. A Data General Nova 3 computer with

MANX software was used to control the experimental events and record responses made by the rats.

Experiments 6 and 7 were conducted in four test chambers

(32 X 32 X 40 cm), each having four walls (one aluminum and three Plexiglass), a Plexiglas ceiling and a wire grid floor. A food hopper was positioned 8 cm above the floor in the middle of the aluminum wall of each chamber, with a 28 v house-light stationed 8 cm directly above. A retractable 42

lever (Coulbern Instruments, model E23-05, 4X4 cm) was

stationed 6.5 cm on either side of each food hopper (8 cm above the floor). Sound attenuating enclosures (55 X 55 X 60

cm) with ventilation fans were used to contain the chambers and mask extraneous noise. During generalization tests, the

rats were attached to electrode leads (Plastic Products,

303-302) that were suspended from Mercotac commutators and

passed through openings in the chamber ceilings. Constant

current stimulation was delivered to the rats by a 6

channel, programmable sine-wave stimulator. An INI computer

with MANX software was used to control the experimental

events and record responses made by the rats. Stimulation

currents were monitered continuously on a Tektronix T912

storage oscilloscope.

Locomotor activity tests were conducted in six chambers

(45 X 45 X 30 cm), each having three wooden walls, one

Plexiglas wall, and either a wood or wire mesh floor. Each

chamber had eight photocell emitters stationed 10 cm apart

and 1 or 2.5 cm above the floor on two adjacent walls. The

associated photocell detectors were placed opposite the

emitters on the remaining walls. Interruptions of the

horizontal photocell beams were integrated for each chamber

by a multiplexor and relayed to the abovementioned INI

computer, which recorded the activity scores.

The self stimulation tests in Experiment 7 were

conducted in 5 chambers (23 X 29 X 46 cm), each having

plexiglass walls and a wire grid floor. A single lever (5 X 43

8 cm) was mounted at the middle of one wall (2.5 cm above the floor) and electrode leads were suspended from Mercotac commutators and passed through the open tops of the chambers. Electrical stimulation was delivered by five independent sine-wave stimulators and responses were recorded on mechanical counters.

Drug Discrimination Training

All rats were initially trained to lever-press for food

(45 mg Bioserve pellets) on a continuous reinforcement schedule during four 30 min drug-free sessions.

Subsequently, the rats were given daily 30 min discrimination trials, with either 1.0 mg/kg d-amphetamine sulphate (dissolved in 0.9% saline to a concentration of 1.0 mg/ml) or 1.0 ml/kg saline injected intraperitoneally (IP) immediately prior to each session. During the initial 15 min of the session the house-light was turned off and the pellet dispenser was inactivated. For Experiments 6 and 7, the levers were retracted during this period. Subsequently, the house-light was turned on (and the levers inserted in

Experiments 6 and 7) for the remaining 15 minutes and the rats could obtain food by responding selectively on one of the two levers following injections of 1.0 mg/kg amphetamine or on the alternative lever after saline injections. The lever appropriate for each injection was counterbalanced among rats and responses on the inappropriate lever were recorded but had no programmed consequences. Throughout training the amphetamine and saline trials were intermixed 44

randomly, with the single limitation that neither solution was administered on more than two consecutive sessions.

During the first four discrimination sessions, each response on the appropriate lever resulted in the delivery

of a food pellet. Subsequently, the number of responses required for each food pellet was doubled after every fourth session, until a fixed-ratio-32 (FR-32) reinforcement schedule was in effect. Training then continued with the FR-

32 schedule until the rats responded correctly on eight out

of 10 consecutive sessions. A correct response was defined as the completion of the first FR-32 requirement within a

session on the lever appropriate for the injection received

on that trial.

Generalization Tests

Rats that acquired the discrimination task were given

generalization tests with a range of amphetamine doses (0.00

[i.e., saline], 0.25, 0.50 and 1.0 mg/kg) administered

either alone (i.e. with a vehicle solution where

appropriate) or in combination with a psychoactive drug or

VTA stimulation. These tests were similar to the usual

training sessions except that the rats were injected with a

test compound at an appropriate interval before testing or

administered VTA stimulation beginning 3 minutes after the

start of the session. As usual, the house-light was turned

on after 15 min and the responses on both levers were

recorded. As the appropriate lever could not be determined a

priori for these tests, the rats were reinforced for 45

continuing to respond on the lever on which the first FR-32

requirement was completed. The tests were spaced at least

two days apart, with the usual saline and amphetamine

baseline trials occuring on the intervening days. The order

in which drug doses and stimulation currents were administered was counterbalanced for all rats.

Locomotor Activity Tests

The activity tests were conducted at least two weeks after completion of the generalization tests. During the

interim period the rats remained in their home cages, where

they were fed daily. For the four days prior to the start of activity testing, the rats were given injections of amphetamine and saline on alternate days and returned to

their home cages. On the fifth day the rats were given the

first of three locomotor activity tests. Each rat was

injected with either saline or one of the two doses of the

drug it had received during generalization tests. After the

appropriate delay (nicotine = 20 min; morphine = 60 min;

ethanol = 15 min; midazolam = 5 min), the rats were placed

in the darkened activity chambers for 30 min and their activity was measured. All rats were tested in this manner

on every third day with a different dosage of the drug (i.e.

vehicle, dose 1, or dose 2). Alternating amphetamine and

saline injections were given in the home cages on the

intervening days. 46

Drugs

Amphetamine sulphate (Smith, Kline and French) was dissolved in physiological (0.9%) saline and injected IP at the start of each session. Apomorphine hydrochloride (Sigma) was dissolved in de-oxygenated physiological saline and

injected subcutaneously (SC) 5 min before the start of a

session. Cocaine hydrochloride (BDH Chemicals) was dissolved

in saline and injected IP 5 min before the session.

Haloperidol (Haldol, McNeil Pharmaceuticals) was diluted in distilled water and injected IP 45 min before a session. L-

nicotine bitartrate (BDH Chemicals) was dissolved in saline,

brought to a pH of 7.2 with 0.05 M NaOH, and frozen in

sealed ampules. On test days, individual ampules were thawed

and the nicotine solutions were injected SC 20 min before

the session. Morphine sulphate (BDH Chemicals) was dissolved

in saline and injected SC 60 min before test sessions, and

midazolam maleate (Hoffman-La Roche) was dissolved in

distilled water and injected SC 5 min before the sessions.

Ethanol was prepared by diluting a 95% stock solution down

to either 25% or 12.5% with saline. This yeilded solutions

with concentrations of 0.125 and 0.25 g/ml, each which was

injected IP 15 min before a session at a volume of 4 ml/kg.

With the exception of ethanol, the concentrations of all

drug solutions were varied to maintain an injection volume

of 1 ml/kg. Fresh drug solutions were prepared for all test

sessions. Most reported dosages are expressed in terms of

the weight of the drug salt, except for nicotine which is 47 expressed as the weight of the drug base.

Statistical Analyses

From each test session conducted in Experiments 1 through 6, a calculation was made of the percentage of responses emitted prior to the first reinforcement that occurred on the amphetamine-appropriate lever. These percentages were averaged across rats and dose-response functions were constructed which indicated the tendency for rats to respond on the amphetamine-appropriate lever as a function of the amphetamine dose following either saline injections, different doses of a psychoactive drug or different stimulation currents. These data were then analysed using a two-way repeated measures analysis of variance (ANOVA) with the dose or current intensity of the test stimulus as one factor and the amphetamine dose as the second factor. Occasionally, the disruptive effects of certain dosage combinations prevented rats from completing even the first FR-32 response requirement of the test session. Under these circumstances, analyses were performed only on the data from rats that completed the first response requirement. Consequently, the ANOVA1s for these experiments had fewer degrees of freedom.

The post-reinforcement responses within a session could not be used as a reliable measure of the discriminative stimulus properties of amphetamine, as the distribution of these responses was influenced to a large extent by the delivery of the first food pellet. The rats usually 48 continued to respond selectively (90 to 100% of responses)

on the lever that had produced the first reinforcer within the session (i.e. the lever on which the first FR-32 response requirement was completed). Nevertheless, the percentage of post-reinforcement responses emitted on this

initially selected levera was used as an index of possible disruptive actions of drugs on discriminated responding. If

these responses were found to be less selective for the

reinforced lever after a particular drug treatment, then

this might indicate that the drug was exerting a general disruptive action on discriminative and/or reinforcing

stimulus control of responding. Accordingly, the percentages

of post-reinforcement responses emitted on the selected-

lever were analysed using a two-way, repeated measures ANOVA

with the dose or current intensity of the test stimulus as

one factor and the amphetamine dose as the second factor. A

similar ANOVA was performed on the total number of responses

emitted within the last 15 min of each session, to provide

an additional measure of response facilitation or

disruption.

Activity scores from each locomotion test were recorded

separately for each 5 min period of the 30 min test

sessions. These scores were analysed with a two-way repeated

measures ANOVA with drug dose as one factor and time period

as the second factor.

5. Throughout the thesis, the term "selected lever" will be used to refer to the lever on which a rat completed the first FR-32 response requirement within a particular test session. 49

When significant main effects were found with any of the above ANOVA's, post hoc analyses were performed using

Newman-Keuls test. Differences revealed with both the

ANOVA's and Newman-Keuls tests were considered significant when the probability level was less than 0.05. 50

EXPERIMENT 1

The Effects of Cocaine, Apomorphine and Haloperidol on

Amphetamine Stimulus Generalization Functions

As indicated in the introduction, drug discrimination studies traditionally have employed simple drug-substitution procedures to determine whether generalization can occur between different pharmacological compounds. By comparison, the procedures employed in the present thesis represent a relatively novel approach to the study of drug stimuli. Rats trained to discriminate amphetamine from saline will be given generalization trials with a range of amphetamine doses (0.0, 0.25, 0.5 and 1.0 mg/kg) administered either alone or in combination with different psychoactive drugs or

VTA stimulation. If the drugs or the VTA stimulation produce amphetamine-like stimuli, then these stimuli should summate with the cues produced by each amphetamine dose. This would

be reflected as an increase in the amount of drug- appropriate responses elicited at the lower doses of amphetamine, and an elevation of the amphetamine stimulus

generalization functions relative to curves obtained in the absence of the drugs or brain-stimulation. Subtractive

interactions between amphetamine and a test stimulus would

result in decreases in drug-appropriate responding over the

range of amphetamine doses, and a lowering of the stimulus

generalization functions.

To assess the reliability of the abovementioned

procedure, rats in the present experiment were given 51 generalization tests with a range of amphetamine doses after pretreatment with drugs that previously have been found

either to generalize or interfere with the stimulus properties of amphetamine. These latter drugs included the psychomotor stimulant cocaine, the dopamine receptor agonist apomorphine and the dopamine receptor antagonist

haloperidol. Stimuli produced by cocaine and apomorphine

have been shown to generalize reliably with the stimulus

properties of amphetamine (Colpaert et al., 1978; Huang &

Ho, 1974a; Huang & Wilson, 1986; Schecter, 1977; Schecter &

Cook, 1975). Accordingly, the cocaine and apomorphine

stimuli also might summate with the cueing effects of low amphetamine doses and elevate the stimulus generalization

functions relative to the respective control curves.

Haloperidol has been shown to interfere with the stimulus

properties of amphetamine (Colpaert et al., 1978; Nielsen &

Jepsen, 1985; Schecter & Cook, 1975), and thus should lower

the amphetamine generalization functions relative to the

control curve. Such results would both confirm the

reliability of the procedures employed in the present

thesis, and illustrate how drugs that either generalize or

interfere with the stimulus properties of amphetamine might

affect amphetamine generalization functions.

The present experiment also might confirm previous

reports that the stimulus properties of amphetamine result

from the actions of this drug at dopaminergic synapses (Ho &

Silverman, 1978; Silverman & Ho, 1977). Elevations of the 52 amphetamine stimulus generalization functions after cocaine

and apomorphine, and a lowering of the function following

haloperidol would be consistent with the effects of these drugs on dopamine neurotransmission. Cocaine enhances

dopamine neurotransmission by blocking the reuptake and

metabolic degradation of this neurotransmitter (Ritz et al.,

1987), whereas high doses of apomorphine can mimic

dopaminergic neurotransmission by exerting agonist actions

at post-synaptic dopamine receptors (Anden et al., 1967).

Haloperidol acts as an antagonist at dopamine receptors and

interferes with the function of this neurotransmitter (Anden

et al., 1970). In addition, low doses of apomorphine can act

preferentially at dopamine autoreceptors and decrease the

impulse-dependent release of dopamine (Gonon & Buda, 1985;

Grace & Bunney, 1985; Lane & Blaha, 1986; Zetterstrom &

Ungerstedt, 1984). A lowering of the stimulus generalization

functions at this dose of apomorphine would provide further

evidence of a dopaminergic substrate for the stimulus

properties of amphetamine.

Methods

Twelve male hooded rats were used as subjects for this

experiment. These rats were trained to discriminate 1.0

mg/kg amphetamine from saline using the procedures outlined

in the general methods section. Rats that acquired the

discrimination task (11 out of 12) were tested for

generalization to a range of amphetamine doses (0.0 [i.e.,

saline], 0.25, 0.50 and 1.0 mg/kg) following pretreatment 53 with various doses of apomorphine (0.05, 0.15 and 0.20 mg/kg, injected SC 5 min before the session). After these apomorphine experiments, ten of the rats were given a second set of generalization tests with the same range of amphetamine doses being injected after pretreatment with cocaine (2.5 and 5.0 mg/kg, injected IP 5 min before the session). A third set of tests was then conducted following pretreatment with haloperidol (0.10 and 0.125 mg/kg, given

IP 45 min before the session). Separate control sessions were conducted for each set of generalization tests, in which the amphetamine doses were administered after pretreatment with the respective drug vehicles.

Results

Eleven of the 12 rats trained for this experiment

learned to discriminate 1.0 mg/kg amphetamine from saline, requiring 20 to 30 days to reach the criterion of eight correct responses across 10 consecutive trials. Figure 2 shows the results of the generalization tests in which a

range of amphetamine doses (0.0, 0.25, 0.5 and 1.0) was administered in combination with either cocaine (Figure 2a), apomorphine (Figure 2b) or haloperidol (Figure 2c). Each of

the three graphs reveals that the rats emitted a greater

percentage of their pre-reinforcement responses on the amphetamine-appropriate lever as a direct function of

increasing amphetamine doses. The significance of these dose-response relations was confirmed by separate ANOVAs

performed for the effects of amphetamine-dosage on drug- 54

Figure 2: Effects of cocaine (vehicle, 2.5 and 5.0 mg/kg), apomorphine (vehicle, 0.05, 0.15 and 0.20 mg/kg) and haloperidol (vehicle, 0.10 and 0.125 mg/kg) on amphetamine stimulus generalization functions. A) Both doses of cocaine

(2.5 and 5.0 mg/kg) produced significant increases in drug- lever responses, causing amphetamine generalization functions to be elevated relative to the control curve. B)

Relative to the control curve, generalization functions were lowered by the lowest dose of apomorphine (0.05 mg/kg) and elevated by the highest dose of apomorphine (0.20 mg/kg).

The intermediate dose of apomorphine produced drug- appropriate responding when administered alone (i.e. at the

0.0 mg/kg amphetamine dose), but also decreased the drug- lever responding usually elicited at high amphetamine doses.

However, these changes were not significant. C) The high dose of haloperidol reduced drug-lever responses significantly and lowered the amphetamine stimulus generalization functions relative to the control curve. A COCAINE B. APOMORPHINE C HALOPERIDOL

AMPHETAMINE DOSE (mg/kg) 56 lever responses measured during the experiments with cocaine

(F[3,221 = 15.80; p < .0001), apomorphine (F[3,30] = 27.42; p < .0001) and haloperidol (F[3,27] = 20.67; p < .0001).

Statistical analyses of the influence of test-drug dosage on amphetamine-lever responses revealed significant effects of cocaine (F[2,16] = 20.78; p < .0001), apomorphine

(F[3,27] = 10.16; p < .0001) and haloperidol (F[2,18) =

5.55; p < .025). Post hoc analyses (Newman-Keuls, p < .05)

indicated that the rats emitted significantly more responses on the drug-lever across the range of amphetamine doses after pretreatment with each dose of cocaine relative to tests with the drug vehicle (Figure 2a). In addition, the rats emitted significantly more drug lever responses during tests with the high dose of cocaine (5.0 mg/kg) relative to tests with the lower dose (2.5 mg/kg). These effects resulted in elevations of the amphetamine dose-response gradients relative to the curves obtained during vehicle tests.

After pretreatment with the lowest dose of apomorphine

(0.05 mg/kg) the amount of drug-lever responding was significantly reduced relative to tests with the drug vehicle, so that the amphetamine dose-response gradient was

lowered relative to the control curve (Figure 2b). In contrast, drug-lever responses following pretreatment with the highest dose of apomorphine (0.20 mg/kg) were significantly increased such that the amphetamine response gradient was elevated above the 80% response level. The 57

intermediate dose of apomorphine (0.15 mg/kg) produced trends towards a reduction of drug-lever responses at higher amphetamine doses (0.5 and 1.0 mg/kg), but this dose also resulted in substantial drug-lever responding when it was given alone (i.e., with 0.0 mg/kg amphetamine). The net result of these opposing effects was such that the average

level of drug lever responding at this apomorphine dose was

not significantly different from that obtained during tests with the apomorphine vehicle.

The significant haloperidol effects were due to

reductions in drug lever responding after pretreatment with

the higher dose of this drug (0.125 mg/kg) relative to tests with the lower dose (0.10 mg/kg) or vehicle solution (Figure

2c). This effect resulted in a lowering of the amphetamine dose-response gradient with the high dose of haloperidol.

There also appeared to be a lowering of the response

gradient with the lower dose of haloperidol, however this

effect was not statistically significant.

Analyses of the drug dosage interactions revealed

significant differences in the effects of apomorphine

treatment at the different amphetamine doses (F[9,73] =

4.77; p < .0001). Post hoc analyses indicated that these

differences were due in part to: a) the increase of drug-

lever responses observed after the combined injection of

0.20 mg/kg apomorphine with saline relative to the

combination of the apomorphine vehicle with saline; and b)

the reduction of drug-lever responding after the combined 58 treatment of 0.05 mg/kg apomorphine and 0.25 mg/kg amphetamine relative to tests with this dose of amphetamine injected with either 0.20 mg/kg apomorphine or the apomorphine vehicle. In contrast to these apomorphine effects, there were no significant drug dosage interactions during tests with cocaine (F[6,44] = 2.0; p > .05) or haloperidol (F[6,48] = 2.28; p > .05).

Discriminated response performance was affected to varying degrees by cocaine, apomorphine, and haloperidol.

Analyses of the influences of test-drug dosages on the percentage of post-reinforcement responses emitted on the selected lever revealed significant effects of apomorphine

(F[3,30] = 5.875; p < .005), but not cocaine (F[2,16] =

0.80; p > .05) or haloperidol (F[2,48] = 0.84; p > .05).

Post hoc analyses of the apomorphine dose factor indicated an overall reduction in the percentage of selected-lever responses at the highest dose of apomorphine (0.20 mg/kg;

Table 2) relative to tests with the other doses or vehicle solution. However as may be seen in Table 2, this reduction

in selected lever responding was relatively minor.

Additional significant differences in selected-lever responses were obtained from the analyses of the cocaine X amphetamine dosage interactions (F[6,44] = 3.91; p < .005), the apomorphine X amphetamine dosage interactions (F[9,73] =

2.34; p < .025) and the main effect of amphetamine dosage obtained from tests with apomorphine (F[3,30] =3.09; p <

.05). The significant cocaine X amphetamine dose interaction 59

TABLE 2

The percentages of all responses occurring after the first presentation of reinforcement within a session that were emitted on the initially selected lever. The data represent the means for all rats at each dose combination during tests with cocaine, apomorphine, and haloperidol.

AMPHETAMINE DOSE (mg/kg)

0.0 0.25 0.5 1.0

COCAINE: vehicle 100 98 96 99

2.5 mg/kg 9 8 100 100 100

5.0 mg/kg 91 100 100 100

APOMORPHINE: vehicle 99 91 97 100

0.05 mg/kg 99 97 97 100

0.15 mg/kg 89 100 97 92

0.20 mg/kg 79 86 100 100

HALOPERIDOL:

vehicle 100 96 98 100

0.10 mg/kg 100 98 100 100

0.125 mg/kg 98 99 100 92 60

was due to a slight reduction in the percent of responses emitted on the selected-lever when 5.0 mg/kg cocaine was injected alone (i.e., 0.0 mg/kg amphetamine;

Table 2). The significant effects obtained during the apomorphine tests were due to slight reductions in the selected-lever response percentages when apomorphine was

injected alone.

Analyses of the total number of responses emitted during the last 15 min of the generalization sessions (Table

3) revealed significant differences between dosages of apomorphine (F[3,30] = 93.4; p < .0001) and haloperidol

(F[2,18] = 14.70; p < .0005), but no effects of the cocaine dosages (F[2,16] = 1.58; p > .05). Post hoc analyses

indicated that responses were decreased significantly after pretreatment with each dose of apomorphine and haloperidol relative to responses emitted during vehicle control tests.

In addition, the reduction in responding was greater with each successive dose, such that all doses were significantly different from each other.

Significant differences in responding also were revealed by analyses of the amphetamine dosage and drug-dosage

interaction effects obtained during tests with haloperidol

(F[3,27] = 5.68; p < .005; and F[6,54] = 3.97; p < .005,

respectively). Post hoc analyses of these effects indicated

that response levels were significantly lower during tests

in which haloperidol was injected alone or with 0.25 mg/kg amphetamine relative to tests either with the haloperidol 61

TABLE 3

The total number of responses occurring during the final 15 minutes of each session. The data represent the means for all rats at each dose combination during tests with cocaine, apomorphine, and haloperidol.

AMPHETAMINE DOSE (mq/kq)

0.0 0.25 0.5 1.0

COCAINE; vehicle 1255 1401 1249 980

2.5 mg/kg 1275 1359 1032 919

5.0 mg/kg 1324 1323 1101 865

APOMORPHINE; vehicle 1056 1140 1143 1257

0.05 mg/kg 657 783 978 893

0.15 mg/kg 303 404 605 549

0.20 mg/kg 221 210 212 96

HALOPERIDOL: vehicle 1619 1608 1380 1377

0.10 mg/kg 674 968 1457 1328

0.125 mg/kg 327 760 934 798 62 vehicle or with this drug injected along with the higher doses of amphetamine. The response suppressing effects of apomorphine also appeared to be greater when injected alone or with 0.25 mg/kg amphetamine (see Table 3), however analyses of the amphetamine dosage and drug-dosage

interaction effects did not reach statistical significance

(F[3,30] = 2.54; p = .07; and F[9,90] = 1.90; p = .06, respectively).

Discussion

In the present experiment rats were trained to discriminate 1.0 mg/kg amphetamine from saline and then tested with a range of amphetamine doses after pretreatment with either cocaine, apomorphine, haloperidol or the

respective drug vehicles. The results of the vehicle control

tests indicated that responses on the drug-appropriate lever

increased as a direct function of increasing amphetamine dose. To the extent that the drug lever responses were determined by the discriminative stimulus properties of amphetamine, the dose-response gradients likely reflected

varying degrees of stimulus generalization between the

training and tests doses of the drug.

The results of tests with cocaine, apomorphine, and

haloperidol confirmed that these drugs could interact with

the stimulus properties of amphetamine. Cocaine resulted in

an overall increase in drug lever responding relative to

vehicle control tests and an elevation of the amphetamine

stimulus generalization function. Thus, it appears that 63 cocaine produced stimuli capable of summating with the stimulus properties of amphetamine. In contrast, haloperidol appeared to interfere with the amphetamine stimuli as indicated by the overall decrease in drug lever responses and lowering of the stimulus generalization functions. The effects of apomorphine depended on the dose employed. The low dose of apomorphine (0.05 mg/kg) appeared to interfere with the cueing effects of amphetamine as the generalization function was lowered relative to the vehicle control curve.

The high dose (0.2 mg/kg) of apomorphine appeared to generalize with the stimulus properties of amphetamine as the rats responded consistently on the drug lever irrespective of the amphetamine dose. The intermediate dose

(0.15 mg/kg) of apomorphine produced both non-significant decreases in drug-lever responding elicited by the higher doses (0.5 and 1.0 mg/kg) of amphetamine, and also a substantial amount of drug lever responding when administered alone (i.e., with 0.0 mg/kg amphetamine). This latter pattern of effects could be interpreted as a disruption of stimulus control. However, given the results obtained with the high and low doses of apomorphine it is reasonable to conclude that the drug effects reflected the capacity for the intermediate dose to produce both partial generalization and partial interference with the stimulus properties of amphetamine.

These effects of cocaine, apomorphine, and haloperidol on the amphetamine stimulus generalization functions are 64 consistent with the known actions of these drugs on dopaminergic neurotransmission. Cocaine enhances dopamine neurotransmission by blocking the reuptake and subsequent degradation of this neurotransmitter (Ritz et al., 1987).

High doses of apomorphine mimic the effects of dopamine at postsynaptic receptor sites (Anden et al., 1967) whereas low doses of this drug decrease dopamine neurotransmission by acting at presynaptic autoreceptors to inhibit impulse dependent release (Gonon & Buda, 1985; Grace & Bunney, 1985;

Lane & Blaha, 1986; Zetterstrom & Ungerstedt, 1984).

Haloperidol antagonizes dopamine neurotransmission by blocking postsynaptic dopamine receptors (Anden et al.,

1970). In view of the parallels between the effects of these drugs on dopamine neurotransmission and on amphetamine stimulus generalization functions, it is reasonable to conclude that the changes in the generalization functions were related to the dopaminergic actions of the drugs.

Specifically, the drugs may have modified the cueing efficacy of amphetamine by acting on dopaminergic substrates

that mediate the stimulus properties of this drug.

The changes in the amphetamine stimulus generalization

functions also were consistent with the known hedonic actions of cocaine, apomorphine, and haloperidol. Cocaine and apomorphine (at doses affecting post-synaptic receptors)

can produce rewarding effects in laboratory animals, whereas

haloperidol has been shown to attenuate the rewarding

effects of various drugs, including amphetamine (for reviews 65 see Carr et al., in press; Yokel, 1988). Accordingly, the elevations of the generalization functions by cocaine and high doses of apomorphine may have reflected the summation of the hedonic properties of these drugs with those of amphetamine, and the lowering of the functions by haloperidol may have been due to the capacity for this drug to interfere with the hedonic actions of amphetamine. This interpretation would not necessarily supersede an explanation of the results in terms of the dopaminergic actions of the test drugs. In fact, the hedonic actions of cocaine, apomorphine and haloperidol may result from their influences on mesocortical dopamine systems (Carr et al., in press; Ritz et al., 1987; Roberts & Vickers, 1984; 1987;

Roberts, Corcoran, & Fibiger, 1977; Zito, Vickers, &

Roberts, 1985). Thus, the separate interpretations of the present results in terms of the dopaminergic or the hedonic actions of the test drugs might simply represent different functional and neurochemical descriptions of the same neuropharmacological events.

In the present experiment, the effects of apomorphine and haloperidol on amphetamine stimulus generalization functions were measured even at doses that strongly inhibited lever-pressing for food, and at a dose of apomorphine (0.2 mg/kg) that slightly reduced the consistency with which rats responded on the selected lever.

Thus, it appeared that performance factors did not interfere with the ability of these drugs to selectively alter the 66 dependent measure of the amphetamine stimuli. This resistence of the discrimination measure to disruptive effects of drugs contrasts with other behavioral paradigms that have been employed to assess the dopaminergic and/or hedonic actions of pharmacological compounds. In many of these latter paradigms the performance disrupting effects of high drug dosages often confound the dependent variable being measured, and selective effects may only be obtained within a limited dose range (Fibiger, 1978; Wise, 1982). In contrast, the effects of a wide range of drug dosages on amphetamine stimulus generalization functions may be measured without appreciable interference of performance

factors with the dependent variable. 67

EXPERIMENT 2

Effects of Nicotine on Amphetamine Stimulus

Generalization Functions

Nicotine is a general CNS stimulant that is self- administered by both laboratory animals and humans

(Henningfield & Goldberg, 1983). Recent studies have

indicated that this drug increases both the firing rate of mesocortical dopamine neurons and the extracellular concentrations of dopamine in the nucleus accumbens

(Imperato et al., 1986; Mereu et al., 1987). Thus, it appears that nicotine is capable of enhancing dopamine neurotransmission at synapses within the nucleus accumbens by increasing impulse flow in the mesoaccumbens neurons.

This pharmacological action may be the basis for nicotine's rewarding effects, as 6-OHDA lesions of the nucleus accumbens block the self-administration of this drug by rats

(Singer, Wallace, & Hall, 1982).

If the pharmacological enhancement of dopamine neurotransmission or the production of rewarding effects represent sufficient conditions for stimulus generalization with amphetamine, then such generalization should be obtained after nicotine injections. However, previous attempts to demonstrate such generalization have reported

inconsistent results. For example, Schecter and Rosecrans

(1973) reported that rats trained to discriminate 4.0 mg/kg amphetamine from saline emitted saline-appropriate responses when tested for generalization to nicotine. In a separate 68 study (Ho & Huang, 1975), rats trained with a lower dose of amphetamine (0.8 mg/kg) appeared to show partial generalization to a nicotine stimulus. However, as these rats emitted only 41% of their responses on the drug lever, it was not possible to establish whether these results reflected true generalization or a general impairment in discriminative performance. Recently, Reavill and Stolerman

(1988) confirmed that nicotine can produce a limited amount of amphetamine-appropriate responding when given alone, and can increase the amount of drug-appropriate responding elicited by low amphetamine doses. This result suggested that nicotine indeed may have produced a stimulus that could induce both partial generalization and summation with the stimulus properties of amphetamine.

The present experiment attempted to confirm that nicotine can produce an amphetamine-like stimulus which would elevate stimulus generalization functions. This result would verify both predictions under investigation in the present thesis: 1) that stimulus summation will occur between amphetamine and other drugs that can enhance mesoaccumbens dopamine neurotransmission; and 2) that stimulus summation will occur between amphetamine and drugs that can produce rewarding effects.

The effects of nicotine on locomotor activity also were assessed. As indicated in the introduction (p. 38), changes in locomotor activity levels often reflect parallel changes in mesoaccumbens dopamine neurotransmission. In fact, the 69 locomotor stimulant properties of nicotine may be blocked by

6-OHDA lesions of the nucleus accumbens (Ksir & Kline,

1987). Within the present context, the locomotor activity tests provided independent behavioral confirmation of nicotine's facilitatory effects on dopamine neurotransmiss ion.

Methods

Twelve experimentally naive male hooded rats were used as subjects for this experiment. These rats were trained to discriminate 1.0 mg/kg amphetamine from saline using the procedures outlined in the general methods section. Rats that acquired the discrimination task subsequently were given generalization tests with the same doses of amphetamine that were employed in Experiment 1 (0.0, 0.25,

0.50 and 1.0 mg/kg) after receiving injections of 1-nicotine bitartrate (0.2 and 0.4 mg/kg) or saline. The injections of nicotine or its vehicle were given SC, 20 min before the start of each session.

Upon completion of the discrimination experiment, all twelve rats were tested for the effects of nicotine on locomotor activity. Each rat was given three 30 min activity tests 20 min after SC injections of nicotine (0.2 and 0.4 mg/kg) or saline.

Results

Eleven of the rats used for this experiment learned to discriminate amphetamine from saline within 30 trials.

During subsequent generalization tests, pre-reinforcement 70 responses on the drug-appropriate lever were found to increase as a direct function of the increasing amphetamine dose. Analysis of the effect of amphetamine dosage on drug- lever responding (collapsed across the nicotine dosage factor) confirmed the statistical significance of this trend

(F[3,30] = 20.33; p < .0001).

As may be seen in Figure 3, the amphetamine dose- response gradients obtained from generalization tests with nicotine were elevated relative to the vehicle control curve. Analyses of the nicotine dosage effect on discriminated responses revealed significant differences between the drug conditions (F[2,20] = 7.10; p < .005), which post hoc analyses indictated were due to significant increases in drug-lever responding at both doses of nicotine

(0.2 and 0.4 mg/kg) relative to tests with the nicotine vehicle. Although these increases were evident primarily at the lower doses of amphetamine (0.0 and 0.25 mg/kg), the interaction effect of the nicotine and amphetamine dosages did not reach statistical significance (F[6,601 = 2.08; p =

.07) .

Analyses of the selected-lever response percentages

(Table 4) revealed significant effects of the nicotine dosage (F[2,20) = 4.11; p < .05) and the nicotine and amphetamine dosage interactions (F[6,59J = 2.23; p = .05), but not of the amphetamine dosage (F[3,30] = 1.46; p > .05).

The nicotine dosage effect reflected a significant increase in the consistency of selected lever responses at the lower 71

Figure 3: Effects of nicotine on amphetamine stimulus generalization functions. Both closes of nicotine (0.20 and

0.40 mg/kg) increased drug-lever responses significantly and elevated the amphetamine stimulus generalization functions relative to the control curve. 72

AMPHETAMINE DOSE (mg/kg) 73

TABLE 4

Percentages of responses on the initially selected lever after nicotine.

AMPHETAMINE DOSE (ma/kg)

0.0 0.25 0.5 1.0

NICOTINE DOSE: vehicle 98 88 96 94

0.2 mg/kg 99 99 100 99

0.4 mg/kg 91 97 100 100 74 dose of nicotine (0.2 mg/kg) relative to tests with saline.

Post hoc analyses of the drug-dosage interactions revealed that selected-lever response percentages were higher when nicotine was injected in combination with certain doses of amphetamine relative to tests with 0.25 mg/kg amphetamine plus the nicotine vehicle. These latter interaction effects might best be attributed to random error rather than a specific action of nicotine on response accuracy, as the effects do not follow any logical order.

Analyses of the number of responses during the last 15 min of the generalization sessions revealed significant effects of nicotine dosage (F[2,20] = 4.63; p < .025), amphetamine dosage (F[3,30] = 3.02; p < .05) and the interaction of nicotine and amphetamine dosages (Ft 6,60] =

2.46; p < .05). Post hoc analyses of the nicotine dosage effect indicated that the rats emitted more responses following injections of the lower dose of nicotine (0.2 mg/kg) than during tests with either the high dose of nicotine (0.4 mg/kg) or the nicotine vehicle (see Table 5).

However, post hoc tests of the interaction effects indicated that this increase in responding was significant only when

0.2 mg/kg nicotine was injected alone (i.e., with 0.0 mg/kg amphetamine). This increase after injection of 0.2 mg/kg nicotine alone also may account for the significant amphetamine dosage effect, which was due to increases in responding during tests with 0.0 mg/kg amphetamine relative to tests with 1.0 mg/kg amphetamine. TABLE 5

Total number of responses after nicotine.

AMPHETAMINE DOSE (mg/kg)

0.0 0.25 0.5 1.0

NICOTINE DOSE: vehicle 1199 1079 1200 1101

0.2 mg/kg 1642 1223 1255 1171

0.4 mg/kg 1357 1320 1029 1028 76

The effects of nicotine on locomotor activity are shown in Figure 4. Analyses of the activity scores obtained from these tests revealed significant effects of the nicotine dosage (F[2,22] = 10.86; p < .001), the time period (F[5,55]

= 65.77; p < .0001) and the interaction of nicotine dose with the time period (F[10,110] = 3.33; p < .001). Post hoc tests indicated that the time period effect was due to a greater amount of activity earlier in the session relative to the later time periods, regardless of the drug condition.

Analyses of the nicotine dosage effects revealed that activity scores were increased significantly after injections of both doses of nicotine (0.2 and 0.4 mg/kg) relative to tests with the drug vehicle. The significant interaction between nicotine dosage and time period effects reflected differences in the time course of the separate nicotine doses. The lower dose of nicotine exerted its strongest stimulant actions earlier in the sessions, whereas the higher dose produced greater effects during the later periods in the session. Importantly, the stimulant actions of both doses were present 15 to 20 min into the session (35 to 40 min after injection); the time interval when the rats would usually be making their first discriminated responses during generalization tests with nicotine.

Discussion

In the present experiment rats trained to discriminate

1.0 mg/kg amphetamine from saline were given tests with a range of amphetamine doses after receiving injections of 77

Figure 4: Effects of nicotine on locomotor activity. The data represent the total activity counts measured during each 5-minute block over a 30-minute test period following injections of vehicle, 0.20 or 0.40 mg/kg nicotine. Both doses of nicotine significantly elevated the activity scores relative to tests with the vehicle. 78 79 either nicotine or its vehicle solution. During vehicle control tests the amount of responding on the drug- appropriate lever was found to increase as a direct function of the increasing amphetamine dose, reflecting an orderly stimulus generalization function. Pretreatments with nicotine resulted in functions that were elevated relative to the control curve. This finding suggests that the stimulus properties of nicotine may have summated with those of amphetamine and augmented the cueing efficacy of this psychomotor stimulant drug.

The summation between nicotine and amphetamine stimuli may reflect a common dopaminergic substrate for the stimulus properties of these drugs. Indeed, the increases in locomotor activity in the present experiment suggested that the doses of nicotine employed were capable of augmenting dopaminergic function within the nucleus accumbens.

Furthermore, previous studies have suggested a role for dopamine in mediating some of nicotine's stimulus properties. Rats trained to discriminate nicotine from saline were found to generalize partially to both amphetamine and to the direct Dl dopamine receptor agonist

SKF 38393 (Chance, Murfin, Krynock, & Rosecrans, 1977;

Reavill & Stolerman, 1988). In contrast, the cueing effects of nicotine were attenuated by the dopaminergic antagonists haloperidol, pimozide and Sch 23390 (Reavill & Stolerman,

1988). Although the specific dopaminergic pathways mediating the stimulus properties of nicotine have not been 80 determined, it is possible that they may include the same dopaminergic processes that mediate the stimulus properties of amphetamine.

The results of the present experiment are consistent with the prediction that stimulus summation will occur between amphetamine and other drugs with facilitatory actions on dopaminergic neurotransmission. In addition, the results verify the prediction that summation may occur with drugs that are capable of producing rewarding effects.

Nicotine is readily self-administered by both humans and laboratory animals (Henningfield & Goldberg, 1983).

Injections of this drug also may produce place preference conditioning (Fudala & Iwamoto, 1986; Fudala, Teoh, &

Iwamoto, 1985) and can increase response-rates for LH and

VTA brain-stimulation rewards (Clarke & Kumar, 1983a;

Druhan, Fibiger, & Phillips, in press; Newman, 1972; Olds &

Domino, 1969; Schaefer & Michael, 1987). Recent evidence has indicated that these rewarding effects of nicotine may involve actions on mesoaccumbens dopamine neurons, as 6-OHDA lesions of the nucleus accumbens can block the self- administration of this drug (Singer et al., 1982). Thus, the successful prediction of nicotine's effects on amphetamine stimulus generalization functions both from its rewarding effects and from its dopaminergic actions may reflect a common neuronal basis for these two characteristics of nicotine. 81

EXPERIMENT 3

Effects of Morphine on Amphetamine Stimulus

Generalization Functions

Morphine is an opiate compound that is readily self- administered by laboratory animals and humans (Jasinski,

1977; Martin & Jasinski, 1977). Like nicotine, morphine can

increase the firing rate of mesoaccumbens dopamine neurons and extracellular concentrations of dopamine within the nucleus accumbens (Di Chiara & Imperato, 1986; Gysling &

Wang, 1983). These actions of morphine on dopamine neurotransmission may be partly responsible for the hedonic effects of this drug. Rats will self-administer morphine directly into the VTA and develop preferences for environments that have been paired with opiate injections

into this region (Bozarth, 1987; Bozarth & Wise, 1981;

Phillips & LePiane, 1980, 1982; Phillips et al., 1983).

In view of the abovementioned dopaminergic actions and rewarding effects of morphine, this drug might be expected to augment the stimulus properties of amphetamine. Morphine previously has been shown to potentiate the threshold

lowering effects of amphetamine on lateral hypothalamic ICSS

(Hubner, Bain, & Kornetsky, 1987) and to enhance the level

of euphoria produced by amphetamine in human subjects

(Jasinski & Preston, 1986). Nevertheless, attempts to demonstrate generalization between morphine and amphetamine

have yeilded conflicting or ambiguous results. In one study

(Jarbe, 1982), pigeons trained to discriminate amphetamine 82

(1.6 mg/kg) from saline failed to generalize to morphine

(1.5 and 3.0 mg/kg). In contrast, morphine (5.0 mg/kg) produced 50% drug-lever responding when administered to rats trained to discriminate 0.75 mg/kg amphetamine from saline

(Hernandez, Holohean, & Appel, 1978). However, morphine also decreased drug-lever responding to a level of 63% when co- injected with the training dose of amphetamine. It is unclear whether these results reflected generalization between the stimulus properties of morphine and amphetamine or merely a disruption of discriminative performance.

The present experiment assessed the ability of morphine to summate with amphetamine stimuli. Rats trained to discriminate 1.0 mg/kg amphetamine from saline were tested for stimulus generalization to lower doses of amphetamine following pretreatment with either saline or low doses of morphine (1.0 and 2.0 mg/kg). If morphine could produce amphetamine-like stimulus properties, then this drug might augment the cueing effects of amphetamine and elevate the stimulus generalization functions relative to a curve obtained in the absence of the drug. In contrast, any disruptive actions of morphine might be reflected as concomitant increases and decreases in drug-lever responding at low and high doses of amphetamine, respectively. As in the previous experiment, the stimulant actions of morphine were assessed independently during locomotor activity tests conducted after completion of all generalization sessions. 83

Methods

Twelve experimentally naive male hooded rats were used as subjects for this experiment. These rats were trained to discriminate 1.0 mg/kg amphetamine from saline using the procedures outlined in the general methods section. All of the rats acquired the discrimination task and were given generalization tests with the standard range of amphetamine doses injected 60 min after pretreatment with morphine sulphate (1.0 and 2.0 mg/kg) or saline. Subsequently, these twelve rats were given three 30 min activity tests 60 min after SC injections of morphine (1.0 and 2.0 mg/kg) or saline.

Results

All 12 rats utilized in this experiment learned the discrimination task within 20 to 30 trials. During subsequent generalization tests, these rats increased their responses on the drug lever as a function of increasing amphetamine doses. Analysis of the effect of amphetamine dosage on drug-lever responding confirmed the statistical significance of this trend (F[3,33 = 28.43; p < .0001).

The effects of morphine on amphetamine discriminated response gradients are shown in Figure 5. Analyses of the effects of morphine on amphetamine-lever responses indicated a significant dosage effect (F[2,22] = 5.06; p < .025), which was due to decreases in drug lever-responding at the higher dose of morphine (2.0 mg/kg). These decreases resulted in a lowering of the discriminated response 84

Figure 5: Effects of morphine on amphetamine stimulus generalization functions. Responses on the drug lever were significantly reduced at the high dose of morphine (2.0 mg/kg), causing the stimulus generalization function to be lowered relative to the curves obtained following pretreatment with either the drug vehicle or the low dose of morphine. 85 86 gradient relative to the curves obtained after vehicle or the low dose of morphine (1.0 mg/kg). Analysis of the drug- dosage interactions did not reveal significant differences in the effects of morphine at the separate amphetamine dosages (F[6,61] = 1.28; p > .05).

The ANOVA's performed on the percentage of selected lever responses (Table 6) did not reveal significant effects of either morphine dosage (F[2,22] = 1.75; p > .05) or amphetamine dosage (F[3,33J =0.26; p > .05). However, there was a significant interaction effect (F[6,61] = 2.44; p <

.05). Post hoc analysis revealed that this interaction effect was due to slightly more consistant selected-lever responding after injections of 1.0 mg/kg amphetamine plus

1.0 mg/kg morphine relative to tests with 0.25 mg/kg amphetamine alone. These differences likely reflect random variation rather than a specific facilitatory action of morphine.

Morphine produced significant effects on the amount of responding exhibited during the last 15 min of the generalization sessions (F[2,22] = 19.80; p < .0001; Table

7). Post hoc analyses indicated that responding was significantly lower at both doses of morphine relative to tests with the morphine vehicle, and that the higher dose of morphine (2.0 mg/kg) produced significantly greater decrements than the lower dose (1.0 mg/kg). Significant effects of the amphetamine dosage also were observed

(F[3,33] = 14.05; p < .0001), which were due to the lower 87

TABLE 6

Percentages of responses on the initially selected lever after morphine.

AMPHETAMINE DOSE (mo/kg)

0.0 0.25 0.5 1.0

MORPHINE DOSE: vehicle 92 81 87 85

1.0 mg/kg 91 95 87 96

2.0 mg/kg 91 88 90 82 88

TABLE 7

Total number of responses after morphine.

AMPHETAMINE DOSE (mg/kg)

0.0 0.25 0.5 1.0

MORPHINE DOSE: vehicle 1370 1351 1362 821

1.0 mg/kg 1180 1204 1157 659

2.0 mg/kg 997 982 983 356 89 levels of responding at the high dose of amphetamine (1.0 mg/kg) relative to tests with the lower doses of this drug

(0.0, 0.25 and 0.5 mg/kg). Analyses of the drug-dosage interactions did not reveal significant differences in the effects of morphine at the different amphetamine doses

(F[6,66] = 0.14; p > .05).

As may be seen in Figure 6, morphine increased activity levels during locomotion tests. An ANOVA confirmed the significance of the morphine dosage effect (F[2,221 = 19.22; p < .0001). Post hoc tests indicated that activity scores were elevated significantly after both doses of morphine relative to tests with the morphine vehicle, and that activity levels were greater at the high dose (2.0 mg/kg) than at the lower dose (1.0 mg/kg) of this drug.

Significant interaction effects also were revealed

(F[10,110] = 2.28; p < .025). Post hoc analyses indicated that the magnitude of the activity increases varied throughout the session, being non-significant during the first 5 min and highly significant during later periods in the sessions. Analysis of the effects of time period on activity scores revealed significant differences (F[5,55] =

76.10; p < .0001), which reflected the progressive decline in activity levels observed over the course of each session.

Discussion

The present experiment assessed the effects of morphine on amphetamine stimulus generalization functions in rats trained to discriminate 1.0 mg/kg amphetamine from saline. 90

Figure 6: Effects of morphine on locomotor activity. Both doses of morphine (1.0 and 2.0 mg/kg) increased activity scores significantly relative to those obtained following vehicle injections. 91 92

The doses of morphine employed in this experiment previously have been shown to increase neuronal firing rates and extracellular concentrations of dopamine (Di Chiara &

Imperato, 1986; Glysing & Wang, 1983), support place preference conditioning (Mackey & Van der Kooy, 1985; Mucha,

Van der Kooy, 0'Shaughnessy, & Bucenieks, 1982) and enhance self-stimulation behavior (Hubner et al., 1987; Kornetsky &

Esposito, 1979). These individual doses also have served as rewards for intravenous self-administration responding, and rats commonly respond at a rate that maintains an average dosage level of 2.0 to 3.0 mg/kg/hour (Smith, Guerin, Co,

Barr, & Lane, 1985; Smith, Shultz, Co, Goeders, & Dworkin,

1987). Nevertheless, these doses failed to augment the stimulus properties of amphetamine during generalization tests. Instead, the higher dose of morphine (2.0 mg/kg) appeared to interfere with the cueing effects of amphetamine as indicated by the lowering of the generalization gradient relative to the curves obtained from tests with the vehicle or low dose of morphine.

Although morphine also reduced operant response levels during the generalization tests, the interference with the amphetamine stimuli did not appear to reflect a general loss of stimulus control over discriminated responding. Analyses of the selected-lever response percentages did not reveal differences between tests with morphine or its vehicle solution, indicating that the influence of the reinforcing stimulus on discriminated responses was maintained after 93 morphine injections. The effects of morphine on the amphetamine stimuli also did not appear to reflect a general depressant action of the drug, as both doses of morphine exerted strong stimulant actions during subsequent activity tests. Rather, it appears that the high dose of morphine may have attenuated the cueing efficacy of amphetamine by interfering with the perception of its stimulus properties.

The absence of additive interactions between the stimulus properties of morphine and amphetamine would appear to suggest that morphine did not exert facilitatory actions on neural processes that give rise to amphetamine stimuli.

However, this interpretation would not be consistent with the evidence that morphine can enhance mesoaccumbens dopamine neurotransmission (Di Chiara & Imperato, 1986;

Gysling & Wang, 1983). A more viable explanation might be that the detection of such facilitatory interactions may have been obscured by stronger, antagonistic actions of morphine on the stimulus properties of amphetamine. These latter inhibitory effects may have involved selective actions on processes post-synaptic to the mesoaccumbens dopamine neurons. Alternatively, other mesocortical dopamine projections may play a role in mediating the amphetamine stimuli, and morphine's inhibitory effects may have resulted from actions on processes that are post-synapt ic to these neurons. Finally, the inhibition may have resulted from a non-specific masking of the amphetamine cues by non- dopaminergic stimulus properties of morphine. 94

EXPERIMENT 4

Effects of Midazolam on Amphetamine Stimulus

Generalization Functions

Midazolam is a benzodiazepine compound that is readily self-administered by laboratory animals (Ator & Griffiths,

1987; Falk & Tang, 1985; Griffiths, Lukas, Bradford, Brady,

& Snell, 1981; Szostak et al., 1987). However, unlike the compounds employed in Experiments 1 through 3, midazolam has been reported to reduce both the spontaneous firing rate of mesocortical dopamine neurons (cf. Haefley et al., 1981) and extracellular dopamine concentrations in the nucleus accumbens of unanesthetized rats (Finlay et al., 1987).

These studies suggest that midazolam may inhibit mesoaccumbens dopamine neurotransmission.

The abovementioned hedonic and dopaminergic actions of midazolam offer conflicting predictions with respect to the drug's interactions with the stimulus properties of amphetamine. If such interactions were determined by the rewarding efficacy of drugs, then the cueing effects of midazolam and amphetamine might summate and elevate the stimulus generalization functions relative to a control curve. In contrast, if interactions with amphetamine stimuli were related to drug actions on dopamine neurotransmission, then midazolam might reduce the cueing effects of amphetamine and lower the generalization functions. The present experiment assessed the relative importance of the rewarding effects or the dopaminergic actions of midazolam 95 as predictors of generalization with amphetamine, by determining the influence of this drug on amphetamine stimulus generalization functions. As in the previous two experiments, the doses employed for the discrimination tests also were administered prior to locomotor activity tests to obtain an independent assessment of the possible dopaminergic actions of midazolam.

Methods

Twelve experimentally naive male hooded rats were used as subjects for this experiment. These rats were trained to discriminate 1.0 mg/kg amphetamine from saline using the procedures outlined in the general methods section. Ten of these rats acquired the discrimination and were given further test sessions with the usual doses of amphetamine, 5 min after SC injections of midazolam maleate (0.1 and 0.2 mg/kg) or distilled water. All twelve rats were subsequently given three 30 min locomotor activity tests 5 min after SC injections of midazolam (0.2 and 0.4 mg/kg) or distilled water.

Results

Ten of the 12 rats employed for this experiment learned the discrimination task within 20 to 30 trials. During subsequent generalization tests, these rats emitted more responses on the drug-lever at high doses of amphetamine relative to tests with lower doses. Analysis of the amphetamine dosage effect for the experiment confirmed the significance of this trend (F[3,27] = 10.15; p < .0001). 96

The results of the generalization tests with midazolam are shown in Figure 7. ANOVA's performed on the percentage of drug-lever responses during these tests revealed a significant effect of midazolam dosage (F[2,18] = 5.76; p <

.025), but no significant interaction between the effects of midazolam and amphetamine dosages (Ft 6,54] = 0.78; p >

.05). Post hoc tests indicated that the midazolam dosage effect was due to significant reductions in drug-lever responding at both doses of this drug (0.1 and 0.2 mg/kg) relative to tests with saline. These decreases resulted in the amphetamine dose-response gradients being lowered relative to curves obtained from tests with the midazolam vehicle.

The downward shifts of the amphetamine discriminated response gradients were not associated with concomitant changes in either the consistency of selected-lever responses (Table 8) or the number of responses emitted during tests with midazolam (Table 9). Analyses of the percentages of responses emitted on the selected lever did not reveal any significant effects related to the midazolam dosage (F[2,18] = 0.14; p > .05), the amphetamine dosage

(F[3,27] = 1.39; p > .05) or the interaction of the dosage variables (F[6,52] = 1.84; p > .05). Similarly, analyses of the response levels also did not reveal significant effects of the midazolam dosage (F[2,18) = 0.06; p > .05), the amphetamine dosage (Ft 3,27] = 0.76; p > .05) or the interaction of these variables (F[6,54] = 1.17; p > .05). 97

Figure 7: Effects of midazolam on amphetamine stimulus generalization functions. Drug lever responses were reduced significantly by both doses of midazolam (0.10 and 0.20 mg/kg) causing the amphetamine stimulus generalization functions to be lowered relative to the vehicle control curve. 98

0.0 0.25 0.50 10 AMPHETAMINE DOSE (mg/kg) 99

TABLE 8

Percentages of responses on the initially selected lever after midazolam.

AMPHETAMINE DOSE (mq/kq)

0.0 0.25 0.5 1.0

MIDAZOLAM DOSE: vehicle 98 92 99 99

0.1 mg/kg 100 96 98 96

0.2 mg/kg 98 98 97 97 100

TABLE 9

Total number of responses after midazolam.

AMPHETAMINE DOSE (mg/kg)

0.0 0.25 0.5 1.0

MIDAZOLAM DOSE: vehicle 1926 1861 1633 1639

0.1 mg/kg 1890 1799 1669 1715

0.2 mg/kg 1832 1798 1879 1475 101

As may be seen in Figure 8, midazolam reduced activity scores obtained during subsequent locomotion tests. Analyses of the midazolam dosage effect confirmed the significance of these reductions (F[10,110] = 6.13; p < .01), and post hoc tests indicated that both doses of the drug (0.1 and 0.2 mg/kg) were effective in decreasing activity relative to scores obtained after vehicle injections. As in previous experiments, the analysis of the time-period effect revealed significant differences in activity levels over the course of the session (F[5,55] = 38.14; p < .0001), with scores being highest at the start of each test. Analysis of the interaction of the midazolam dosage and time-period variables did not reveal significant differences in the effects of midazolam as a function of time (F[10,55] = 1.20; p > .05) .

Discussion

In the present experiment, midazolam was found to attenuate the cueing effects of amphetamine so that stimulus generalization functions obtained from tests with this drug were lower than the vehicle control curve. This result was contrary to the effects predicted from the rewarding effects of midazolam. If interactions with amphetamine stimuli were determined by the hedonic actions of drugs, then the stimulus properties of midazolam should have summated with those of amphetamine and elevated the stimulus generalization functions. The absence of an elevation in the present experiment suggested that the capacity to produce 102

Figure 8: Effects of midazolam on locomotor activity. Both doses of midazolam (0.10 and 0.20 mg/kg) significantly reduced locomotor activity counts relative to tests with vehicle injections. 103

500 -i

• Vehicle

450 ° 01 mg/kg • 0.2 mg/kg

400

350 H

£ 300 H

250

200

l I i I I 10 15 20 25 30 TIME (minutes) 104 rewarding effects was not a sufficient condition for inducing stimulus summation with amphetamine.

The effects of midazolam on amphetamine stimulus generalization functions corresponded with the known actions of this drug on dopamine neurotransmission. Previous studies have found that midazolam can inhibit the firing rate of mesocortical dopamine neurons (cf. Haefley et al., 1981) and reduce extracellular dopamine concentrations in the nucleus accumbens of rats (Finlay et al., 1987). The efficacy of the doses employed in the present experiment for producing such effects was suggested by the findings that midazolam decreased locomotor activity without otherwise affecting discriminated response levels. However, midazolam's inhibitory effects on locomotion and amphetamine generalization also could have resulted from actions on

GABA-ergic processes post-synaptic to the dopamine projections. This possibility requires further investigation. Notwithstanding, the present results concur with the prediction that the interactions of a drug with the stimulus properties of amphetamine would reflect the dopaminergic actions of the compound. 105

EXPERIMENT 5

Effects of Ethanol on Amphetamine Stimulus

Generalization Functions

The self-administration of ethanol by humans has been well documented (see Mello, 1981; Mello & Mendelson, 1977).

In laboratory animals the rewarding effects of ethanol have been less consistent, but numerous studies have demonstrated that self-administration behaviors may be established when animals are given extensive prior exposure to the drug

(Numan, 1981), when schedule induction procedures are employed (concomitent fixed interval schedule of food presentation; Falk, Samson, & Winger, 1972) or when operant responding is first established with a highly rewarding drug

(e.g., cocaine or pentobarbital; cf. Mello & Mendelson,

1977). With respect to its neurochemical actions, many studies have reported that ethanol does not affect levels of either dopamine or its metabolite, dihydroxyphenylacetic acid within the nucleus accumbens (Ellingboe and Mendelson,

1982; Hellevuo & Kiianmaa, 1988; Kalant, 1975; Nutt & Glue,

1986). However, some reports have indicated that ethanol may

increase the firing rate of VTA dopamine neurons (Gessa,

Muntoni, Collu, Vargiu, & Mereu, 1985) and the release of this neurotransmitter within the nucleus accumbens (Di

Chiara & Imperato, 1986; Imperato & Di Chiara, 1986).

The present experiment determined the effects of ethanol on amphetamine stimulus generalization functions. If

interactions with the stimulus properties of amphetamine 106 were determined by a drug's capacity to produce rewarding effects, then ethanol might augment amphetamine stimuli and elevate generalization functions relative to a vehicle control curve. In contrast, ethanol might not affect these functions if facilitatory actions on dopamine neurotransmission were a prerequisite for summation with amphetamine. As indicated above, most studies of ethanol's neurochemical effects have indicated that this substance has no effects on dopamine neurotransmission.

Previous neurochemical studies into ethanol's effects on dopamine neurotransmission have been inconsistent enough to merit an independent assessment of ethanol's effects on a dopaminergically mediated behavior. Therefore, the present experiment also evaluated the effects of ethanol on locomotor activity. As indicated above (pp. 38 & 60), changes in mesoaccumbens dopamine neurotransmission often result in parallel alterations of activity levels.

Accordingly, locomotor activity may provide a behavioral index of the possible effects of ethanol on dopaminergic function. Comparisons of the results of discrimination and locomotor activity tests might clarify the relationship between ethanol's effects on amphetamine stimulus generalization functions and its actions on neurotransmission at dopaminergic synapses.

Methods

Twelve experimentally naive male hooded rats were used as subjects for this experiment. These rats were trained to 107 discriminate 1.0 mg/kg amphetamine from saline using the procedures outlined in the general methods section. Eleven of these rats acquired the discrimination task and were given generalization tests with the usual range of amphetamine doses, 15 min after IP injections of ethanol

(0.5 and 1.0 g/kg) or saline. Upon completion of this phase of the experiment, 11 of the original 12 rats were tested for the effects of ethanol on locomotor activity. Each rat was given three 30 min activity tests 15 min after IP injections of ethanol (0.5 and 1.0 g/kg) or saline.

Results

Eleven of the 12 rats employed for this experiment learned the discrimination task within 20 to 30 trials.

During subsequent generalization tests, these rats showed an orderly increase in drug-lever responding as a direct function of amphetamine dose. Analysis of the amphetamine dosage effect for the experiment confirmed the significance of this trend (F[3,30] = 39.22; p < .0001).

The effects of ethanol on amphetamine discriminated response functions are shown in Figure 9. Although the response gradients obtained following ethanol injections appear to be elevated relative to the control curve, analyses of the ethanol dosage effects on drug-lever responses revealed that the observed differences fell short of statistical significance (F[2,20] = 2.87; p = .08).

Analysis of the interactions between the ethanol and amphetamine dosage variables also did not reveal significant 108

Figure 9: Effects of ethanol on amphetamine stimulus generalization functions. Although ethanol produced large increases in drug lever responding at intermediate amphetamine doses, the elevation of the generalization function failed to reach statistical significance. 109

AMPHETAMINE DOSE (mg/kg) 110 differences in drug lever responses (F[6,54] = 1.38; p >

.05) .

Analyses of the percentage of responses emitted on the initially selected lever (Table 10) revealed significant effects of ethanol dosage (F[2,20] = 3.57; p < .05) and the interaction of ethanol and amphetamine dosage variables

(F[6,51] = 3.70; p < .005), but no significant main effect of amphetamine dosage (F[3,30] = 0.55; p > .05). Post hoc analyses of the ethanol dosage effect indicated that rats responded more consistently on the selected lever at the high dose of ethanol (1.0 g/kg) relative to tests with the ethanol vehicle. Analyses of the interaction effect revealed that selected-lever responses were significantly less consistent when 0.5 mg/kg amphetamine was injected alone relative to all other test conditions. This latter effect was likely due to random variation rather than a loss of stimulus control at this dose of amphetamine.

Analyses of the amount of responding during the last 15 min of each generalization session (Table 11) revealed significant effects of ethanol dosage (F[2,20] = 12.81; p <

.0005) and amphetamine dosage (F[3,30] = 3.48; p < .05), but no significant interaction effects (F[6,60] = 1.46; p >

.05). Post hoc analyses of the main effects indicated that responses were reduced significantly at the high dose of ethanol (1.0 g/kg) relative to tests with the low dose (0.5 g/kg) or the ethanol vehicle, and responses also were reduced at the high dose of amphetamine relative to tests 111

TABLE 10

Percentages of responses on the initially selected lever after ethanol.

AMPHETAMINE DOSE (mg/kg)

0.0 0.25 0.5 1.0

ETHANOL DOSE: vehicle 99 96 88 97

0.5 g/kg 99 94 100 95

1.0 g/kg 99 99 100 100 112

TABLE 11

Total number of responses after ethanol.

AMPHETAMINE DOSE (ma/kg)

0.0 0.25 0.5 1.0

ETHANOL DOSE: vehicle 1203 1188 1282 826

0.5 g/kg 1015 1089 1208 872

1.0 g/kg 862 819 788 671 113 with 0.5 mg/kg of this drug.

Ethanol did not alter activity scores during subsequent locomotion tests, as indicated by that lack of significant effects of either the ethanol dosage (Figure 10; F[2,20] =

0.47; p > .05) or the interaction of the dosage and time- period variables (F[10,100] = 0.66; p > .05). However, as in previous experiments there was a significant effect of the time period (F[5,50] = 49.37; p < .0001), which was due to the steady decline in activity levels over the course of the test sessions.

Discussion

In the present experiment, rats were trained to discriminate 1.0 mg/kg amphetamine from saline and then given tests with a range of amphetamine doses injected in combination with either ethanol (0.5 or 1.0 g/kg) or saline.

The results of these tests were somewhat ambiguous with respect to ethanol's effects on discriminated responding.

Ethanol appeared to produce large increases in drug-lever responses at the 0.25 and 0.50 mg/kg amphetamine doses relative to tests with vehicle injections. However, ethanol did not result in drug lever responding when administered alone (ethanol plus 0.0 mg/kg amphetamine), and the overall differences between the amphetamine stimulus generalization functions obtained with ethanol or vehicle injections failed to reach statistical significance.

The absence of amphetamine-appropriate responding when ethanol was administered alone suggests that the stimulus 114

Figure 10: Effects of ethanol on locomotor activity. Ethanol had no significant effects on locomotor activity scores. 115

500 -i

• Vehicle

o 450 H 0.5 g/kg ° 1.0 g/kg

400

350 H

b- 300 H

250

200 H

10 15 20 25 30 TIME (minutes) 116 properties of this drug did not generalize with those of amphetamine. This conclusion would be consistent with previous reports that the stimulus properties of ethanol do not involve dopaminergic components that could generalize with an amphetamine cue (Signs & Schecter, 1988). Although the apparant increases in drug-lever responding at intermediate doses may have indicated stimulus summation between the cueing properties of ethanol and amphetamine, this result also could have resulted from pharmacokinetic interactions between the two drugs. Ethanol can increase blood levels of amphetamine by interfering with its metabolic degradation (Ellinwood, Eibergen, & Kilbey, 1976), and such an effect might have accounted for the pattern of results obtained in the present experiment.

The lack of an effect of ethanol on locomotor activity suggests that ethanol may not have altered dopamine neurotransmission in the nucleus accumbens at the doses employed here. If this was the case, then the absence of generalization between amphetamine and ethanol would be consistent with the predicted relation between such generalization and the dopaminergic actions of drugs. In contrast, the results of this experiment refute the prediction that amphetamine will generalize to drugs that produce rewarding effects. Although ethanol may be self- administered in humans and laboratory animals (Mello, 1981;

Mello & Mendelson, 1977), this rewarding capacity was not sufficient to produce generalization with amphetamine. 117

EXPERIMENT 6

Effects of Electrical Stimulation of the VTA on

Amphetamine Stimulus Generalization Functions

If either the induction of positive affect or the enhancement of mesoaccumbens dopamine neurotransmission were sufficient conditions for stimulus summation with amphetamine to occur, then such summation might be obtained with non-pharmacological stimuli that possess these properties. For example, the stimulus properties of amphetamine might summate with those produced by electrical stimulation of the VTA. Stimulation of this region can serve as a potent reward for operant responding in rats (Fibiger et al., 1987), and recent ex-vivo chromatographic and in- vivo electrochemical measures have suggested that dopamine may be released in the ipsilateral nucleus accumbens of rats responding for VTA stimulation (Blaha et al., 1988; Fibiger et al., 1987).

In a previous study, D'Mello (1981) failed to demonstrate stimulus generalization between amphetamine and

VTA stimulation. However, recent in-vivo electrochemical experiments have indicated that the square-wave stimulation parameters employed by D'Mello may have been inappropriate

for activating dopamine neurons. In these studies, dopamine was released in forebrain regions only when the stimulation

involved trains of either square-wave pulses with long durations (i.e., > 0.5 msec; Millar, Stamford, Kruk, &

Wightman, 1985) or 60 Hz sine-waves (Blaha et al., 1988; 118

Fibiger et al., 1987). Accordingly, the present experiment reassessed the effects of VTA stimulation on discriminated responses to a range of amphetamine doses using sine-wave currents that could both increase the release of dopamine in the nucleus accumbens and produce rewarding effects. If the dopaminergic or hedonic actions of this non-pharmacological stimulus were sufficient to produce amphetamine-like cueing effects, then the VTA stimulation should summate with the stimulus properties of amphetamine and elevate generalization functions relative to a control curve.

Methods

Fifteen male hooded rats were employed as subjects for this experiment. One week after electrode implantation, the rats were given five 30 min ICSS sessions in which they could lever-press for 200 msec trains of 20 uA, 60 Hz sine wave stimulation on a CRF schedule. This initial self- stimulation screening ensured that the brain-stimulation was capable of supporting ICSS. Subsequently, the rats were trained to discriminate 1.0 mg/kg amphetamine from saline using the procedure described in the General Methods section.

Following acquisition, the rats were given generalization tests with a range of amphetamine doses (0.0,

0.125, 0.25, 0.5 and 1.0 mg/kg) either alone or in combination with intermittent VTA stimulation. At the start of the brain-stimulation trials, the rats were injected with one of the solutions and then immediately attached to 119 electrode leads and placed in the testing chambers. The intermittent delivery of electrical stimulation then began three minutes later and continued throughout the test session. The stimulation was maintained at a constant intensity within each test (15 or 20 uA), and presented every 10 sec. Each presentation consisted of four 200 msec trains of 60 Hz sine-wave stimulation delivered 200 msec apart. During control tests with amphetamine alone, the rats were attached to the electrode leads but no stimulation was delivered. The rats were reinforced (FR-32 schedule) during these tests for continuing to press the lever on which the first FR-32 response requirement was completed.

Results

The electrode placements for the 15 rats employed in the present experiment are shown in Figure 11. These rats acquired the discrimination task at a rate comparable to that observed for rats in the previous experiments. During subsequent generalization tests, the rats were found to emit more drug-lever responses at the high amphetamine doses relative to when lower doses were injected. An ANOVA performed on the amphetamine dosage effects for the experiment confirmed the significance of this trend (F[4,56]

= 25.93; p < .0001).

Statistical analyses of the influence of VTA stimulation on discriminated responses to amphetamine revealed significant effects of both the current intensity (F[2,28] =

5.34; p < .025) and the interaction of the current intensity Figure 11: Electrode placements for the 15 rats employed in

Experiment 6. All electrodes were implanted in the left hemisphere. The filled triangles on the left side of the brain represent the electrode placements in rats for which the amphetamine-appropriate lever was contralateral to the stimulating electrode, whereas the triangles on the right represent placements in rats for which the drug-lever was ipsilateral to the electrode. The numbers to the right of the diagrams represent the plate numbers corresponding to the coronal sections from the brain atlas of Konig and

Klippel (1963).

122

and amphetamine dosage variables (Ft8/104] = 5.84; p <

.0001). Post hoc analyses of the effects of current intensity indicated that the rats emitted more responses on the drug lever during tests with both 15 and 20 uA relative to control sessions without stimulation (Figure 12a). Thus, the amphetamine dose-response functions were elevated in the presence of the VTA stimulation relative to the control curve. The significant interaction reflected the selective increases in drug-lever responses when each stimulation intensity was delivered in combination with 0.0 and 0.125 mg/kg amphetamine and when 15 uA was given with 0.25 mg/kg, relative to tests with these doses in the absence of VTA stimulation.

The increased bias towards responding on the drug lever in the present experiment could have been related to a general sensorimotor asymmetry associated with unilateral stimulation of the VTA. The influence of such sensorimotor effects was examined by analysing separately the effects of the stimulation on discriminated responses when the drug- lever was either ipsilateral or contralateral to the stimulating electrode. These analyses indicated that for the ipsilateral group (Figure 12b), there was a significant interaction between the current intensity and amphetamine dosage variables (F[8,43] = 4.74; p < .0005) but no significant main effect of the current intensity (F[2,12] =

1.43; p > .05). Post hoc tests revealed that the interaction was due to significant increases in drug-lever responding 123

Figure 12: Effects of electrical stimulation of the VTA on amphetamine stimulus generalization functions. Rats were tested for generalization to amphetamine in the absence of

VTA stimulation (0), with the delivery of VTA stimulation at a constant intensity of 15 uA (O)/ or with VTA stimulation delivered at a constant intensity of 20 uA (Q).

A) Generalization functions averaged across all rats. B) generalization functions obtained from rats for which the drug-lever was ipsilateral to the stimulating electrode. C)

Generalization functions obtained from rats for which the drug-lever was contralateral to the stimulating electrode.

Significant increases in drug-lever responding were observed under all three conditions, causing the generalization functions to be elevated relative to the control curves.

125 when each stimulation intensity was delivered in combination with 0.0 and 0.125 mg/kg amphetamine and when 15 uA was given with 0.25 mg/kg, relative to tests with these doses in the absence of VTA stimulation. Analyses of the data for the contralateral group (Figure 12c) revealed significant effects of both the current intensity (F[2,28] = 5.34; p <

.025) and the interaction of current intensity with amphetamine dose (F[8,53] = 2.81; p < .01). Post hoc analyses indicated that the current intensity effect was due to a significant increase in drug-lever responding at the 20 uA intensity relative to tests without VTA stimulation. The interaction effect reflected significant increases in drug- lever responding when each intensity was presented in combination with saline injections (0.0 mg/kg amphetamine).

These results confirmed that VTA stimulation could produce increases in drug-lever responding regardless of whether the lever was ipsilateral or contralateral to the stimulating electrode.

Analyses of the influence of VTA stimulation on the consistency of selected-lever responses (Table 12) did not reveal any significant effects of either the current intensity (F[2,28] = 0.92; p > .05), the amphetamine dosage

(F[4,56] = 2.44; p > .05) or the interaction of these

variables (F[8/104] = 1.11; p > .05). In contrast, ANOVAs •i performed on the response levels during the last 15 minutes of the generalization tests (Table 13) revealed significant effects of the current intensity (F[2,28] = 26.31; p < 126

TABLE 12

Percentages of responses on the initially selected lever during VTA stimulation.

AMPHETAMINE DOSE (mq/kq)

0.0 0.125 0.25 0.5 1.0

INTENSITY LEVEL: no stimulation 99 92 96 98 99

15 uA 98 94 92 91 97

20 uA 97 97 92 97 98 127

TABLE 13

Total number of responses during tests with VTA stimulation.

AMPHETAMINE DOSE (mg/kg)

0.0 0.125 0.25 0.5 1.0

INTENSITY LEVEL: no stimulation 1978 1660 1788 1978 1498

15 uA 1709 1406 1548 1318 859

20 uA 1257 1071 1235 939 643 128

.0001) and amphetamine dosage variables (F[4,56] = 4.62; p <

.005), but no significant interaction effect (F[8,108] =

1.61; p > .05). Post hoc analyses of the current intensity effect revealed that responses were reduced significantly at both stimulation intensities relative to tests without the stimulation, with significantly fewer responses being emitted at the higher intensity (20 uA) relative to tests with the lower intensity (15 uA) . Analyses of the amphetamine dosage effect indicated that responses were reduced at the high dose of amphetamine (1.0 mg/kg) relative to the lower doses of this drug.

Discuss ion

The present experiment examined the effects of VTA stimulation on amphetamine stimulus generalization functions in rats trained to discriminate 1.0 mg/kg amphetamine from saline. The results of this experiment confirmed that amphetamine generalization functions could be elevated by electrical stimulation of the VTA. Although unilateral stimulation of regions containing dopamine neurons can produce both contralateral circling behavior (Gratton &

Wise, 1985) and greater responsiveness to stimuli in the contralateral sensory field (Bandler & Flynn, 1971; Beagley

& Holley, 1977; Nakahara & Ikeda, 1984; Smith, 1972), such sensorimotor effects did not appear to be responsible for the observed biasing of responses toward the drug-lever. The rats increased their responses on the drug-lever during stimulation trials regardless of whether this lever was 129 ipsilateral or contralateral to the stimulating electrode.

Admittedly, the effects on amphetamine generalization functions appeared to be strongest when the drug-lever was contralateral to the electrode, thus the sensorimotor effects may have exaggerated the response biases of these rats. However, the significant elevation observed with the ipsilateral group suggests that the main effect of the stimulation was to produce stimulus properties which could interact in an additive manner with the cueing effects of amphetamine.

The summation observed between the stimulus properties of VTA stimulation and amphetamine in this experiment contrasts with earlier findings by D'Mello (1981). In this previous study, VTA stimulation appeared to have only disruptive effects on amphetamine discriminated response functions. These incongruent results may have been related to differences in the types of stimulation parameters employed by the two studies. The stimulation parameters employed by D'Mello consisted of trains with short-duration

(i.e., 0.2 msec) square-wave pulses. Recent in vivo electrochemical measurements have indicated that such parameters may be insufficient to activate mesotelencephalic dopamine neurons (Millar et al., 1985). In the present experiment, 60 Hz sine-wave currents were used to stimulate the VTA. This type of stimulation appears to be adequate for activating dopamine neurons,, as indicated by the increases in dopamine release and turnover within the nucleus 130 accumbens of rats following sine-wave stimulation of the VTA

(Blaha et al., 1988; Fibiger et al., 1987). Conceivably, this capacity to activate mesoaccumbens dopamine neurons could.have accounted for the summation between the stimulus properties of the brain-stimulation and those of amphetamine in the present experiment.

The results of the present experiment were consistent with the prediction that the stimulus properties of amphetamine might summate with those of any test stimulus with facilitatory actions on dopamine neurotransmission.

These findings also were consistent with predictions that summation may occur with rewarding stimuli. All of the rats employed for this experiment displayed ICSS during preliminary tests, thereby confirming the rewarding efficacy of the brain-stimulation. Accordingly, the shifts of the discriminated response functions observed in the present experiment could have reflected an additive interaction between stimuli associated with rewarding actions of amphetamine and the electrical brain-stimulation. However,

it is noteworthy that the VTA stimulation also was rewarding for many of the rats in the D'Mello (1981) study, yet the stimulation did not generalize or summate with the stimulus properties of amphetamine. Thus, amphetamine does not appear to generalize to all of the rewarding effects of VTA stimulation. Instead, such generalization may be related only to the hedonic effects produced by activation of the mesoaccumbens dopamine neurons. 131

EXPERIMENT 7

The Role of the Rewarding Effects of VTA Stimulation

in Determining Generalization with the

Stimulus Properties of Amphetamine

Experiment 6 revealed that the stimulus properties of amphetamine could be augmented by electrical stimulation of the VTA. In the discussion of that experiment it was suggested that the augmentation may have reflected a summation between the stimulus properties of amphetamine and stimuli associated with dopaminergically mediated hedonic properties of the brain-stimulation. The present experiment investigated this issue further, focussing specifically on the putative role of the hedonic properties of the brain- stimulation in determining the interactions with the stimulus properties of amphetamine.

In the initial phase of this experiment, rats trained to discriminate 1.0 mg/kg amphetamine from saline were given six drug-free generalization tests with different parameters of VTA stimulation (15 or 20 uA trains, presented 10, 5 or

2.5 sec apart). Subsequently, each rat was given a single test wherein ICSS rates were measured at the two current intensities employed during the discrimination phase of the experiment (15 and 20 uA) . The results of these tests were then compared to determine whether generalization between amphetamine and VTA stimulation might correlate with the rewarding effects of this stimulation. If rats that displayed the strongest stimulus generalization between the 132

VTA stimulation and amphetamine also were found to have the highest ICSS rates, then this might suggest that the generalization was related to the hedonic properties of the brain-stimulation.

Methods

Twenty male hooded rats were used as subjects for this experiment. Fifteen of these rats had been used as subjects

in Experiment 6, whereas the other five animals were experimentally naive. The procedures for ICSS screening and discrimination training employed for the 5 new rats were

identical to those described in Experiment 6.

For the tests with VTA stimulation, each rat was given a saline injection and placed in the chamber with the electrode leads attached. As in Experiment 6, the

intermittent presentations of VTA stimulation began 3 min after the start of the half-hour session and continued throughout. The brain-stimulation remained at a constant

intensity (15 or 20 uA) within a session and was presented either once every 10 sec (.1 Hz), once every 5 sec (.2 Hz)

or once every 2.5 sec (.4 Hz). Each presentation consisted

of four 200 msec trains of 60 Hz sine-wave stimulation, delivered 200 msec apart.

Two days after the final generalization test, each rat was placed in a separate self-stimulation chamber and given

10 free stimulations (200 msec trains of 60 hz sine-wave

spaced 1 sec apart) of 15 uA current intensity. The rats

could then lever-press during a 5 min period to receive a 133 single train of VTA stimulation upon each response (CRF reinforcement schedule). The total number of responses emitted during this period were recorded on mechanical counters. At the end of this initial 5 min, the current intensity was increased to 20 uA and responses were recorded for a further 5 min. Rats that were not already lever- pressing at the end of the first 5 min were given 10 free stimulations at the new intensity.

Results

The results of generalization tests with VTA stimulation are shown in Figure 13. As indicated by Figure 13a, the rats emitted an average of 48 to 64% of their responses on the drug-lever during tests with different parameters of VTA stimulation. ANOVAs performed on the drug-lever responses obtained from the stimulation tests did not reveal significant effects of either the current intensity manipulation (F[l,18] = 0.64; p > .05) or the different stimulus presentation rates (F[2,38] = 0.06; p > .05).

However, there was a significant interaction between the intensity and presentation rate variables (F[2,34] = 6.75; p

< .005), which post hoc tests indicated was due to significantly more drug-lever responses when 20 uA stimulation was delivered at a presentation rate of 0.4 Hz relative to tests with the 10 uA current intensity presented at this same rate.

Although inspection of the group means from this experiment suggested only intermediate levels of drug-lever 134

Figure 13: Stimulus generalization between amphetamine and

VTA stimulation during drug-free substitution tests. The data are presented as the percent of responses emitted on the drug lever as a function of different rates of stimulus presentation: once every 10 sec (0.1 Hz), every 5 sec (0.2

Hz) or every 2.5 sec (0.4 Hz). The current intensity was kept at a constant level of either 15 uA (open symbols) or

20 uA (filled symbols). A): Average percentage of drug lever responses for all rats during tests at different current

intensities and presentation rates. B): Average percentages of drug lever responses for 3 sub-groups of rats that consistently showed either strong generalization (triangles

= > 67%; N=8), intermediate generalization (squares = 33 to

67%; N=6) or no generalization (circles = < 33%; N = 6) between amphetamine and the VTA stimulation.

136 responding during stimulation trials, closer examination of the data revealed that the stimulation could elicit strong biases towards responding on the drug lever in many of the rats. In fact, 8 of the rats responded primarily on the drug lever ( > 67% of pre-reinforcement responses) across all tests with VTA stimulation. Another six rats showed

intermediate levels of drug lever responding (34 to 67% of pre-reinforcement responses), whereas the remaining six responded predominantly on the saline lever ( < 33% of pre- reinf orcement responses). The average levels of drug-lever responding for these three subgroups of rats are shown in

Figure 13b. Separate analyses of the effects of the parameter manipulations for these sub-groups did not reveal any significant differences related to either the current

intensity, the stimulus presentation rate or the interaction of these variables either for: A) the rats that responded consistently on the drug lever (F[l,7] = 1.55; p > .05;

F[2,14] = 0.46 p > .05; and F[2,14] = 3.05; p > .05); B) the rats that showed intermediate amounts of drug-lever responding (F[l,4] = 0.52; p > .05; F[2,10] = 0.03; p > .05; and F[2,8] = 0.55; p > .05); or C) rats that emitted few responses on the drug-lever (F[l,5] = 2.35; p > .05; F[2,10]

= 1.70; p > .05; and F[2,8] = 1.48; p > .05).

Figure 14 indicates the electrode placements for the rats employed in the present experiment. Inspection of the

figure reveals that moderate to high levels of drug- appropriate responding could be obtained from stimulation of 137

Figure 14: Relationship between the electrode placements and stimulus generalization with amphetamine for the 20 rats employed in Experiment 7. All electrodes were implanted in the left hemisphere. The different symbols reflect the levels of drug-appropriate responding > 67%; fl = 33 to

67%; 0 = < 33%) when each electrode site was stimulated at a current intensity of either 15 uA (left sides of the sections) or 20 uA (right sides of the sections). The response percentages for each group of rats were obtained by averaging over the tests with different stimulus presentation rates. The numbers to the right of the diagrams represent the plate numbers corresponding to the coronal sections from the brain atlas of Konig and Klippel (1963).

139 regions surrounding the anterior portion of the interpeduncular nucleus (Plate #'s 47b and 48b) and ventromedial placements at a level just anterior to this nucleus (Plate # 46b). Placements that produced the least amount of drug-appropriate responding were located primarily in the dorsal part of the region just anterior to the interpeduncular nucleus (Plate #'s 45b and 46b). Two additional placements associated with low levels of drug- appropriate responding were situated at dorsal and ventral extremities in the posterior VTA (Plate # 49b).

Analyses of the percentages of selected-lever responses measured during the generalization tests (Table 14) did not reveal any significant effects of either the current intensity (F[l,18] = 0.87; p > .05), the stimulus presentation rate (F[2,38] = 1.91; p > .05) or the interaction of these variables (F[2,33] = 0.24; p > .05).

However, there were significant effects of the brain- stimulation on the number of responses emitted during these sessions (Table 15). Statistical analyses revealed significant effects of both the current intensity (Ftl,18] =

12.14; p < .005) and the stimulus presentation rate (F[2,38]

= 10.76; p < .0005), but there was no interaction between these variables (F[2,36] = 0.82; p > .05). Post hoc tests revealed that the number of responses were reduced during tests with the high current intensity (20 uA) relative to tests with the lower intensity (15 uA) . The number of responses also were reduced as a function of increasing the 140

TABLE 14

Percentages of responses on the initially selected lever during substitution tests with different parameters of VTA stimulation.

PRESENTATION RATE (Hz)

0.1 0.2 0.4

INTENSITY LEVEL;

15 uA 94 92 91

20 uA 93 87 88 141

TABLE 15

Total number of responses during substitution tests with different parameters of VTA stimulation.

PRESENTATION RATE (Hz)

0.1 0.2 0.4

INTENSITY LEVEL:

15 uA 1294 1059 962

20 uA 898 797 499 142 stimulus presentation rate, with response levels measured at each rate being significantly different from response levels observed for the other two rates.

During subsequent self-stimulation tests, all of the rats responded for the VTA stimulation. However, there were substantial differences in the ICSS rates obtained from the individual rats. Importantly, there appeared to be a relationship between the ability of the stimulation to elicit amphetamine-appropriate responses during stimulus generalization tests and its efficacy in producing rewarding effects (see Figure 15). In general, the rats that emitted a high percentage of their responses on the amphetamine- appropriate lever during generalization tests also responded at high rates during ICSS sessions. Rats that did not emit drug-appropriate responses during generalization tests tended to respond at low rates for the brain-stimulation reward. To determine the significance of this relationship, the individual percentages of drug-lever responses emitted at each intensity (averaged across all presentation frequencies) were correlated with the self-stimulation rates measured at these intensities using Pearson's correlation coefficient. These analyses revealed significant positive correlations between drug-lever responses and ICSS rates for tests with both the low intensity (15 uA, Figure 15a; r =

0.46; d.f. = 19; p < .05) and the higher intensity (20 uA,

Figure 15b; r = 0.71; d.f. = 18; p < .01).

Figure 16 again shows the electrode placements of the 143

Figure 15: Scattergrams showing the correlations between stimulus generalization with amphetamine and ICSS rates obtained with VTA stimulation. The data are expressed as the percent of responses emitted on the drug-lever during generalization tests (y-axis) relative to the response rates for the VTA stimulation (x-axis; average responses per min) obtained during ICSS tests at: A) 15 uA (Pearsons r = .46; p

< .05); and B) 20 uA (Pearsons r = 0.71; p < .01). The generalization data represent the averaged scores for the three tests at the different rates of stimulus presentation.

The solid lines through the scattergrams represent the lines of best fit obtained from the regression equations. 144

S3SNOdS3« U3A31 DfiyQ lN30d3d 145

Figure 16: Relationship between electrode placements and

ICSS rates for the 20 rats employed in Experiment 7. All electrodes were implanted in the left hemisphere. The different symbols reflect the levels of self-stimulation obtained > 80 presses/min; | = 40 to 80 presses/min;

40 presses/min) when each electrode site was stimulated at a current intensity of either 15 uA (left sides of the sections) or 20 uA (right sides of the sections). The numbers to the right of the diagrams represent the plate numbers corresponding to the coronal sections from the brain atlas of Konig and Klippel (1963). 146 147 rats in the present experiment, with the different symbols presented to indicate the level of self-stimulation obtained at each site. Inspection of the left side of the figure reveals that moderate to high self-stimulation rates were obtained from all but six sites when the current intensity was set at 15 uA. Four of these latter six sites were situated at various extremities of the VTA and correspond to the sites which also were associated with low levels of drug-appropriate responding during stimulus generalization tests (compare with Figure 14). The right side of the Figure

16 shows that all of the sites yielded moderate to high levels of ICSS when the current intensity was set at 20 uA.

Many of the sites that yielded high self-stimulation rates also were associated with high levels of amphetamine- appropriate responding during generalization tests, whereas sites that yielded only moderate ICSS rates tended to produce only moderate to low levels of drug-appropriate responding. There were also a few sites that yeilded high

ICSS rates but low levels of amphetamine-appropriate discriminated responding.

Discuss ion

The results of the present experiment confirmed that rats trained to discriminate 1.0 mg/kg amphetamine from saline would emit drug-appropriate responses when tested with electrical stimulation of the VTA. As a group, the rats emitted between 48 and 64% of their responses on the amphetamine-appropriate lever during stimulation trials. 148

This level of drug-appropriate responding was similar to that observed when the stimulation was delivered alone

(i.e., with 0.0 mg/kg amphetamine) in the previous experiment.

Given that the average percentage of responses emitted

on the drug-appropriate lever did not exceed a maximum of

64% for the group, and the discriminated responses did not vary systematically with changes in the stimulation parameters, the results of the stimulation tests could have reflected disruptive effects of the VTA stimulation on

operant performance. However, the results of Experiment 6 revealed that the stimulation could augment the cueing effects of low amphetamine doses and increase drug-lever responses to levels above that of chance performance. Such augmentation would not occur if the stimulation was exerting

only disruptive actions. Moreover, examination of the data

from individual animals in the present experiment revealed that eight of the rats responded predominantly on the amphetamine-appropriate lever ( > 67% of their pre-

reinforcement responses) during each of the tests with VTA stimulation. Six other rats exhibited intermediate amounts

of responding on the drug-appropriate lever (33 to 67%) during stimulation trials, whereas the remaining 6 animals responded consistently on the saline-appropriate lever.

Thus, the apparent partial biasing of responses toward the drug-lever indicated by the group mean was not a reflection

of partial biasing within each rat. In fact, many of the 149 rats responded primarily on the drug lever during stimulation trials, suggesting that VTA stimulation could generalize with the stimulus properties of amphetamine in certain individuals.

The ability of the brain-stimulation to substitute for the stimulus properties of amphetamine appeared to be related to the placement of the stimulating electrodes within the VTA. Examination of Figure 14 revealed that the stimulus generalization with amphetamine was strongest when the electrodes were located within the VTA either in the regions immediately adjacent the anterior interpeduncular nucleus or in a ventromedial location just anterior to this nucleus. These sites correspond with the location of cell bodies for the mesocortical dopamine neurons (Fallon &

Moore, 1978). Electrode sites that did not produce generalization tended to be located in regions lacking high densities of dopamine perikarya. This relation between stimulus generalization with amphetamine and anatomical proximity of the electrodes to dopamine neurons suggests that the generalization might have been determined by the efficacy of the stimulation for activating mesocortical dopamine projections.

During subsequent ICSS tests, all of the rats were found to self-stimulate when the brain-stimulation was made available as a reward. However, there were individual differences among the rats, with some exhibiting higher ICSS rates than others. As was the case during stimulus 150 generalization tests, the efficacy of the stimulation appeared to depend on the electrode placements. The rats with the highest ICSS rates generally were found to have electrodes located within regions containing high densities of dopamine cells (Fallon & Moore, 1978) whereas rats with lower ICSS rates tended to have electrodes outside of these regions.

Comparisons of the data obtained during generalization and ICSS tests for each rat revealed a significant positive correlation between the amount of drug-appropriate responding elicited by the stimulation and the rate of responding for the brain-stimulation reward. Thus, stimulus generalization with amphetamine was strongest when the VTA stimulation was highly rewarding, but was absent when the stimulation had relatively weak rewarding effects. This result suggested that the ability of the VTA stimulation to generalize with amphetamine may have been related to its capacity to produce rewarding effects. In particular, the generalization may have reflected the activation of dopaminergic substrates for the brain-stimulation reward, as the strongest effects were produced when electrodes were within regions containing high densities of dopamine perikarya. 151

EXPERIMENT 8

Effects of Amphetamine and Haloperidol on Discriminative

Stimuli Produced by Electrical Stimulation of the VTA

The results of Experiments 6 and 7 suggested that electrical stimulation of the VTA could produce stimulus properties similar to those of amphetamine. At a neuronal level, the similarity between stimuli may have reflected the ability of the VTA stimulation to activate dopaminergic processes that mediate the stimulus properties of amphetamine. Indeed, the generalization between amphetamine and VTA stimulation observed in Experiment 7 was strongest when the stimulating electrodes were located within regions containing high densities of mesocortical dopamine cells.

Furthermore, previous studies have indicated that the sine- wave currents employed for the generalization tests were capable of increasing the turnover and release of dopamine within various forebrain regions, including the nucleus accumbens (Blaha et al., 1988; Fibiger et al., 1987).

However, direct attempts to demonstrate dopaminergic substrates for brain-stimulation cues have yielded conflicting results. Several reports have indicated that the discriminative stimulus properties of LH or VTA brain- stimulation were unaffected by injections of amphetamine, cocaine or haloperidol (Druhan, 1985; Druhan, Martin-

Iverson, Wilkie, Fibiger, & Phillips, 1987a; Kornetsky &

Esposito, 1981; Schaefer & Michael, 1985). Instead, the stimulus properties of VTA stimulation appeared to be 152 modulated by cholinergic drugs (Druhan, 1985; Druhan,

Martin-Iverson, Wilkie, Fibiger, & Phillips, 1987b; Druhan,

Fibiger & Phillips, in press). On the other hand, Colpaert,

Niemegeers, & Janssen (1977) found that LH brain-stimulation

cues could be blocked by haloperidol, and in a subsequent

study Colpaert (1977a) demonstrated that detection

thresholds for such stimuli could be increased by

haloperidol and decreased by cocaine. These latter effects

were consistent with the capacity of haloperidol and cocaine

to respectively interfere with and enhance neurotransmission

at dopaminergic synapses.

A plausible explanation for these inconsistent drug

effects is that LH and VTA stimulation may produce several

neurochemically distinct stimuli, each which may be measured

under appropriate circumstances. For example, studies

suggesting the involvement of non-dopaminergic substrates

for the brain-stimulation cues employed massed-trial

procedures wherein rats were required to make discrete

discriminated responses to brief presentations of brain-

stimulation. In contrast, the studies implicating

dopaminergic substrates used procedures that were similar to

the methods commonly employed in drug-discrimination

experiments. Thus, rats received single daily trials in

which intermittent trains of brain-stimulation were

delivered for 5 min prior to access to the response levers.

The rats then could respond on the appropriate lever for a

further 15 min (on an FR-10 schedule of reinforcement) in 153 the continued presence of the brain-stimulation cue.

In the present experiment, a discrimination procedure was developed which incorporated certain characteristics of this latter method, and this new procedure was employed to investigate the possible involvement of dopamine neurons in mediating the stimulus properties of VTA stimulation. Rats were trained with a massed-trial procedure to discriminate between high and low intensities of VTA stimulation, but with each intensity being delivered intermittently over a 2- minute period prior to a choice-trial. These rats were then given generalization tests with intermediate current intensities after pretreatment with amphetamine, haloperidol or the drug vehicles. If dopamine neurons mediated the brain-stimulation cues produced with this new procedure, then these cues would be enhanced by amphetamine and attenuated by haloperidol.

Methods

Nine male hooded rats were employed as subjects for this experiment. One week after implantation of the electrodes, the rats were given five 30 min ICSS sessions in which they could lever-press for 200 msec trains of 20 uA, 60 Hz sine wave stimulation on a CRF schedule. Subsequently, the rats received eight ICSS sessions in which their response rates were measured over a range of current intensities (6 to 26 uA) . For these tests, the current intensity was set initially at 6 uA and subsequently increased by 2 uA every 5 min until 26 uA was reached. The same range of currents (26 154

to 6 uA) was then delivered in a descending order of

presentation. Each change in the current intensity was

signalled by the free delivery of 10 stimulation trains (2 trains/sec) at the new intensity.

The ascending and descending rate-intensity functions measured over the final 4 days of this phase were averaged to obtain a single function for each rat relating response rate to current intensity. From this function, one low current intensity (10 or 12 uA) and one high current

intensity (10 uA higher than the low intensity) were chosen

for use as discriminative stimuli. The low intensity supported threshold ICSS rates (10 to 20 presses per min) and the high intensity supported near assymptotic ICSS rates

(approximately 250 presses per min).

Discrimination Training

After ICSS testing was complete, the rats were trained during two 30 min sessions to lever-press for 45 mg Noyes

food pellets on a CRF schedule. Subsequently, each rat was given daily discrimination sessions with 12 trials spaced

120 to 180 sec apart (variable interval - 150 sec). The beginning of each trial was signalled by a brief (0.05 sec)

flash of the houselight, followed 1 sec later by the delivery of the first of six presentations of either high

(20 or 22 uA) or low (10 or 12 uA) intensity VTA stimulation. Each stimulus presentation consisted of four

200 msec trains of 60 Hz sine-wave stimulation delivered 200 msec apart. The stimulation was maintained at a constant 155 high or low intensity throughout a given cueing period and was delivered at 20 sec intervals. A final 20 sec interval followed the sixth stimulus presentation, after which the houselight was turned on to signal the availability of food.

The houselight remained on for 30 sec during which time the rat could respond to obtain food on the lever appropriate for the stimulus intensity presented during the cueing period. Responses on the incorrect lever had no programmed consequence. The lever appropriate for each current intensity was counterbalanced between rats.

During initial training, each response on the correct lever resulted in the delivery of one food pellet.

Subsequently, the response requirement was increased so that a food pellet was delivered after every sixth response on the correct lever (FR-6), regardless of whether intervening responses were made on the other lever. The accuracy of the discrimination was assessed by recording the lever on which a rat first completed six responses.

Stimulus Generalization After Amphetamine and Haloperidol

Rats that acquired the discrimination task (8 of the 9) were given generalization tests 10 min after receiving intraperitoneal injections of either d-amphetamine sulphate

(0.5 and 1.0 mg/kg) or saline. During these generalization tests, rats were presented with four equally spaced intermediate intensities (2 uA apart) delivered randomly along with the usual training currents. Each intermediate intensity was delivered once and each training current was 156 presented four times within a single generalization test.

During trials with intermediate intensities, the rats were reinforced for continuing to respond on the lever on which the first FR-6 response requirement had been completed.

Upon completion of the tests with amphetamine, further generalization tests were given 45 min after intraperitoneal

injections of either haloperidol (0.10 and 0.125 mg/kg) or distilled water. The order of dose administration was counterbalanced across rats during both the amphetamine and haloperidol test phases. At least two regular training sessions were interposed between each generalization test.

The data obtained from each generalization session were expressed in terms of the percentage of responses emitted prior to the first reinforcement that occurred on the HS

lever after each current intensity. The percentages obtained

from the amphetamine and haloperidol tests were analysed separately using two-way repeated measures ANOVA's with the amphetamine or haloperidol dose as one factor and the stimulation intensity as the second factor. As in previous experiments, post hoc analyses were conducted using Newman-

Keuls test (p < 0.05) when the ANOVA's revealed differences significant beyond a probability of 0.05.

Results

The eight rats that learned the discrimination task reached an accuracy level of over 80% correct choices per session. The electrode placements for these rats are shown

in Figure 17. During generalization tests, the rats 157

increased their responses on the lever appropriate for high

intensity stimulation (HS lever) as a function of the

increasing current intensities. This trend was reflected in

the significant current intensity effects obtained during

the experiments with amphetamine (F[5,35] = 11.75; p <

.0001) and haloperidol (F[5,35] = 13.33; p < .0001).

The results of the tests with amphetamine and haloperidol

are shown in Figure 18. Analyses of the amphetamine

experiment (Figure 18a) revealed significant effects of the

amphetamine dosage (F[2,12] = 9.89; p < .005), which were

due to increases in HS-lever responding at both dosages of

amphetamine relative to tests with saline injections. These

effects of amphetamine resulted in elevations of the

discriminated response gradients for the range of current

intensities. Analysis of the haloperidol experiment (Figure

18b) revealed a significant effect of haloperidol dosage

(F[2,12] = 4.28, p < .05), which was due to decreases in HS-

lever responding at the high dosage of this drug (0.125

mg/kg) relative to tests with the vehicle solution. This decrease in HS-lever reponding resulted in a lowering of the discriminated response function at the high dose. Analyses

of the interactions between drug dosage and current

intensity variables did not reveal significant effects for

experiments involving either amphetamine (F[10,60] = 1.46; p

> .05) or haloperidol (F[10,51] = 1.56; p > .05). 158

Figure 17: Electrode placements for the 8 rats that acquired the discrimination task in Experiment 8. All electrodes were

implanted in the left hemisphere. The numbers to the right of the diagrams represent the plate numbers corresponding to the coronal sections from the brain atlas of Konig and

Klippel (1963). 159 160

Figure 18: Effects of amphetamine and haloperidol on stimulus generalization to a range of current intensities in rats trained to discriminate between high and low

intensities of VTA stimulation. The data are expressed as the mean percentage of responses emitted on the lever appropriate for high-intensity stimulation (HS) as a function of the average current intensity delivered. A)

Amphetamine (0.5 and 1.0 mg/kg) resulted in significant

increases in HS lever responding, causing the generalization

functions to be elevated relative to those obtained with vehicle injections. B) Haloperidol (0.125 mg/kg) resulted in significant decreases in HS lever responses, so that the generalization functions were lowered relative to the vehicle control curve. A AMPHETAMINE B. HALOPERIDOL 162

Discussion

In the present experiment, rats were trained to discriminate between high and low intensities of VTA

stimulation and then given generalization tests with a range

of current intensities after pretreatment with amphetamine,

haloperidol or the respective vehicle solutions. Amphetamine

appeared to enhance the cueing effects of the brain-

stimulation, as indicated by elevations of the stimulus

generalization functions relative to the vehicle control

curve after pretreatment with this drug. In contrast,

haloperidol appeared to interfere with the stimulus

properties of the brain-stimulation as the generalization

function was lowered at the high dose of this drug. These

drug effects are consistent with the known dopamine agonist

properties of amphetamine (Chiueh & Moore, 1975; Ferris,

Tang, & Maxwell, 1972) and the selective dopamine receptor

antagonist actions of haloperidol (Anden et al., 1970).

Accordingly, the results of the present experiment might be

taken as evidence of a dopaminergic substrate for the

stimulus properties of VTA brain stimulation.

The results of the present experiment contrast with

those of earlier pharmacological studies in which the

stimulus properties of VTA stimulation were enhanced by the

acetylcholinesterase inhibiter physostigmine and the direct

muscarinic acetylcholine receptor agonists pilocarpine and

RS-86, but not by amphetamine, haloperidol or nicotine

(Druhan, 1985; Druhan et al., 1987a; Druhan et al., in 163 press). These reports suggested that cues produced by VTA stimulation might be mediated by non-dopaminergic processes which involve muscarinic cholinergic receptor mechanisms.

However, these studies employed training procedures that differed in many respects from those used in the present experiment. For the earlier studies, rats were trained to discriminate brief presentations of VTA stimulation and emit discrete responses on the appropriate one of two levers to receive food reinforcement. In the present experiment rats were trained to discriminate VTA stimulation that was presented intermittently over a 2-minute period prior to the choice trial. The rats could then respond for 30 sec on the appropriate lever to receive food on an FR-10 schedule of reinforcement. It is conceivable that these different training procedures may have resulted in the measurement of neurochemically distinct cue properties of VTA stimulation.

As indicated above, procedures involving brief presentations of VTA stimulation yielded cues that appeared to involve cholinergic rather than dopaminergic processes. In contrast, the brain-stimulation cues measured with the present procedure appeared to be mediated by independent dopaminergic substrates. The independence of these latter cues from those measured with brief presentations of stimulation has been substantiated by a recent study in which the cue properties of VTA stimulation measured with the present procedure were not affected by physostigmine

(Druhan et al., 1987b). 164

In Experiments 6 and 7 it was suggested that the amphetamine-like cueing effects of sine-wave VTA stimulation might have resulted from the production of dopaminergically mediated stimuli (possibly of a hedonic nature). This suggestion was consistent with previous evidence that sine- wave stimulation of the VTA could increase the release and turnover of dopamine in various forebrain regions (Blaha et al., 1988; Fibiger et al., 1987). Also, Experiment 7 revealed that the generalization between amphetamine and VTA stimulation was strongest when the stimulating electrodes were within regions of the VTA that contained high densities of dopamine cell bodies. The present experiment provided further evidence in support of a dopaminergic basis for the amphetamine-like cueing actions of the brain-stimulation, in demonstrating that some of the stimuli produced by electrical stimulation of the VTA may be mediated by dopaminergic substrates. 165

GENERAL DISCUSSION

The studies reviewed in the Introduction indicated that the discriminative stimulus properties of amphetamine may result from the facilitatory actions of this drug on mesoaccumbens dopamine neurotransmission. As such actions also give rise to amphetamine's rewarding effects (Aulisi &

Hoebel, 1983; Carr & White, 1983, 1986; Lyness et al., 1979;

Monaco et al., 1980; Spyraki et al., 1982), it is conceivable that amphetamine stimuli may reflect the hedonic actions of this drug. The present series of experiments determined whether amphetamine stimulus generalization functions could be affected by psychoactive drugs from diverse pharmacological classes and VTA stimulation in a manner that was consistent either with the actions of the test stimuli on dopamine neurotransmission or with their hedonic effects.

The first experiment assessed the effects of the psychomotor stimulant cocaine, the dopamine receptor agonist apomorphine and the dopamine receptor antagonist haloperidol on amphetamine stimulus generalization functions. Cocaine elevated amphetamine stimulus generalization functions relative to the vehicle control curve in a dose dependent manner, suggesting that this drug may have augmented the stimulus properties of amphetamine. In contrast, haloperidol lowered the generalization functions in a manner suggestive of an antagonistic interaction with the stimulus properties of amphetamine. The effects of apomorphine varied according 166

to the particular dosage employed. At the lowest dose (0.05

mg/kg), apomorphine appeared to antagonize the stimulus

properties of amphetamine as the generalization function was

lowered relative to the control curve. At the highest dose

(0.2 mg/kg), these inhibitory actions were superseded by the drug's capacity to generalize with the stimulus properties

of amphetamine, and the generalization functions were

elevated relative to the control curve. The intermediate

dose of apomorphine (0.15 mg/kg) appeared both to substitute

partially for the stimulus properties of amphetamine and to

inhibit the stimulus properties of high amphetamine doses.

However, these latter effects were not statistically

significant and the overall generalization function at this

dose did not differ from the curve obtained under control

conditions.

Experiments 2 through 5 examined whether amphetamine

stimulus generalization functions could be influenced by

drugs other than psychomotor stimulants or dopamine receptor

agonists and antagonists. These compounds included the

general CNS stimulant nicotine, the opiate narcotic

morphine, the benzodiazepine midazolam and the sedative

hypnotic, ethanol. Nicotine was observed to elevate

amphetamine stimulus generalization functions relative to

the control curve, in a manner suggestive of an additive

interaction with the amphetamine stimuli. Morphine and

midazolam lowered the generalization functions, suggesting

that these drugs may have attenuated the amphetamine 167 stimuli. Ethanol increased the amount of drug-appropriate responding emitted at the intermediate doses of amphetamine, but it did not elicit drug-appropriate responses when injected alone (i.e. with 0.0 mg/kg amphetamine). This pattern of results may have been due to an interference with the metabolic degradation of amphetamine by ethanol

(Ellinwood et al., 1976), rather than a summation of the cueing effects of the two drugs.

Experiments 2 through 5 also examined the effects of nicotine, morphine, midazolam and ethanol on locomotor activity. To the extent that changes in activity levels often reflect parallel changes in dopaminergic function, these tests could provide independent behavioral evaluations of the dopaminergic actions of each drug. The results of these tests appeared to confirm previous neurochemical and physiological reports concerning the effects of the drugs on dopamine neurotransmission. Locomotor activity was increased by nicotine and morphine, decreased by midazolam and unaffected by ethanol.

Experiment 6 determined whether the stimulus properties of amphetamine could be augmented by a non-pharmacological stimulus produced by electrical stimulation of the ventral tegmental area. The VTA contains the cell bodies for the mesocortical dopamine projection (Fallon & Moore, 1978), and stimulation of this site can both increase dopamine release and turnover in forebrain regions and produce rewarding effects (Blaha et al., 1988; Fibiger et al., 1987). On the 168 basis of each of these actions, it was predicted that VTA stimulation might summate with the stimulus properties of amphetamine. Indeed, this expectation was confirmed in

Experiment 6, wherein the VTA stimulation was found to

elevate amphetamine stimulus generalization functions

relative to curves obtained in the absence of the

stimulation.

Experiment 7 investigated the possible role of hedonic

factors in determining the capacity of VTA stimulation to

produce amphetamine-like cueing effects. The rats employed

in Experiment 6 were given additional drug-free tests for

generalization to VTA stimulation delivered at two different

current intensities and at three separate rates of stimulus

presentation. Subsequently, each rat was given a single ICSS

test to determine the rewarding efficacy of the current

intensities employed during the generalization tests. The

results of these tests revealed that there were consistent

individual differences in the degree of generalization

produced by the stimulation, and these differences

correlated positively with the ICSS rates measured in these

rats. These individual differences also appeared to reflect

the proximity of the stimulating electrodes to regions of

the VTA that contain high densities of dopamine cell bodies

(Fallon & Moore, 1978). This pattern of results suggested

that the VTA stimulation might have produced its

amphetamine-like cueing effects by activating the same

dopaminergically-mediated affective processes that commonly 169 give rise to the stimulus properties of amphetamine.

Experiment 8 investigated whether the stimulus properties of VTA stimulation could be mediated by dopaminergic substrates. Rats were trained to discriminate between high and low intensities of VTA stimulation and then tested for generalization to a range of current intensities after injections of amphetamine, haloperidol or the respective vehicle solutions. These tests revealed that amphetamine could augment the stimulus properties of VTA stimulation, as indicated by elevations of the stimulus generalization functions relative to the vehicle control curve. In contrast, haloperidol appeared to attenuate the brain-stimulation cues as the stimulus generalization functions were lowered by this drug. Given the known capabilities of amphetamine (Chiueh & Moore, 1975; Ferris et al., 1972) to enhance dopamine neurotransmission and of haloperidol to block dopamine receptors selectively at the doses employed here (Anden et al., 1970), these effects on stimulus generalization gradients might reasonably be attributed to alterations in the activity of a dopaminergic substrate for the stimulus properties of VTA stimulation.

This dopaminergic substrate could conceivably be the neural mechanism by which the VTA stimulation produces its amphetamine-like cueing effects.

The pattern of results obtained in the present series of experiments allows several conclusions to be made about the stimulus properties of amphetamine. On the most basic level, 170 the findings of Experiment 1 confirmed that the amphetamine stimulus generalization paradigm employed in this thesis could produce results similar to those obtained with simple substitution and antagonism tests. For example, the elevations of the amphetamine stimulus generalization functions after cocaine and a high dose of apomorphine (0.2 mg/kg) were consistent with previous reports that these latter compounds can generalize with the stimulus properties of amphetamine (Colpaert et al., 1978; Huang & Ho, 1974a;

Huang & Wilson, 1986; Schecter, 1977; Schecter & Cook,

1975). Similarly, the lowering of the generalization function after haloperidol was consistent with evidence that this drug can attenuate the cueing effects of amphetamine

(Colpaert et al., 1978; Nielsen & Jepsen, 1985; Schecter &

Cook, 1975). Experiment 1 also revealed a previously unreported finding; that low doses of apomorphine (0.05 mg/kg) could attenuate the stimulus properties of amphetamine and lower stimulus generalization functions.

This effect was strongest at the two intermediate doses of amphetamine (0.25 and 0.50 mg/kg), whereas the inhibitory effects exerted at the training dose of amphetamine were relatively weak. Indeed, the inhibitory effects of apomorphine might not have been detected had the present generalization paradigm not been used. This result indicated that, in addition to providing results comparable to those obtained with simple substitution and antagonism paradigms, the amphetamine stimulus generalization paradigm employed in 171

the present thesis might have the advantage of being able to detect subtle interactions with the stimulus properties of

amphetamine that might otherwise go unnoticed.

The effects of cocaine, apomorphine, and haloperidol on

amphetamine stimulus generalization functions in Experiment

1 were consistent with the hypothesis that the stimulus

properties of amphetamine may be mediated by a dopaminergic

substrate. The selective dopamine receptor agonist actions

of the high dose of apomorphine (Anden et al., 1967) were

sufficient to produce amphetamine-like cueing effects.

Similar effects were produced by cocaine, which can increase

synaptic concentrations of dopamine by blocking the reuptake

and metabolic degradation of this transmitter (Ritz et al.,

1987). In contrast, the findings that amphetamine stimulus

generalization functions were lowered both by a low dose of

apomorphine and by haloperidol suggests that dopamine

release and receptor activation are necessary for the cueing

effects of amphetamine. Low doses of apomorphine can

decrease dopamine neurotransmission by blocking impulse-

dependent release of this transmitter (Gonon & Buda, 1985;

Grace & Bunney, 1985; Lane & Blaha, 1986; Zetterstrom &

Ungerstedt, 1984), and haloperidol has been shown to act as

a selective dopamine receptor antagonist at the doses

employed here (Anden et al., 1970).

The present thesis also provided some support for the

hypothesis that the stimulus properties of amphetamine

might, at a functional level, reflect the hedonic actions of 172 this compound. In Experiment 7, the capacity of VTA stimulation to generalize with the stimulus properties of amphetamine was positively correlated with the ability of

the stimulation to produce rewarding effects. Rats that displayed strong generalization between the stimulus

properties of amphetamine and VTA stimulation generally were

observed to respond at high rates for the stimulation when

it was made available as a reward. Conversely, rats that

showed intermediate or low levels of generalization tended

to respond at lower rates for the stimulation. In view of

this correlation, it is conceivable that the rats that

generalized between the stimulus properties of amphetamine and VTA stimulation may have done so on the basis of common

hedonic elements of the two stimuli.

A central issue in this thesis was whether amphetamine

stimulus generalization functions could be affected by test

stimuli other than psychomotor stimulants or dopamine

receptor agonists/antagonists in a manner consistent with

either the dopaminergic actions or hedonic effects of the

stimuli. This issue was addressed in Experiments 2 through

6, wherein the effects of nicotine, morphine, midazolam,

ethanol, and VTA stimulation on amphetamine stimulus

generalization functions were determined. The results of

these experiments revealed that the generalization functions

could indeed be influenced by these test stimuli in a manner that generally was consistent with the known effects

of the stimuli on mesoaccumbens dopamine neurotransmission. 173

For example, the functions were elevated relative to vehicle control curves by nicotine and VTA stimulation, both of which can increase indices of dopamine release and utilization within the nucleus accumbens (Blaha et al.,

1988; Fibiger et al., 1987; Imperato et al., 1986; Mereu et al., 1987). In contrast, the functions were lowered by midazolam, a compound that has been shown to decrease the activity of dopamine neurons and reduce accumbens dopamine concentrations (Finlay et al., 1987; Haefley et al., 1981).

Although ethanol appeared to increase drug-appropriate responding elicited at intermediate amphetamine doses, there was no evidence for stimulus generalization with amphetamine when ethanol was administered alone (i.e., with 0.0 mg/kg amphetamine). In interpreting this latter result, it is important to note that ethanol has been found to increase blood levels of amphetamine by directly interfering with its metabolic degradation (Ellinwood et al., 1976). This pharmacokinetic action may have been responsible for the increases in drug-appropriate responding observed when ethanol was injected with intermediate amphetamine doses. In contrast, the absence of generalization observed when ethanol was injected alone indicates that this substance may have lacked amphetamine-like stimulus properties.

Interpreted as such, the effects of ethanol would be consistent with evidence that ethanol does not affect mesoaccumbens dopamine neurotransmission (Ellingboe &

Mendelson, 1982; Kalant, 1975; Nutt & Glue, 1986). 174

Although the interactions of these test stimuli with amphetamine stimulus generalization functions generally appeared to reflect their known actions on dopamine neurotransmission, additional evidence is needed to confirm a dopaminergic basis for the effects. Some of this evidence may be obtained by determining whether animals trained to discriminate the individual test stimuli respond to cues associated with their dopaminergic actions. For example, previous studies have shown that the stimulus properties of nicotine may be mediated in part by dopaminergic substrates, as nicotine stimuli can both generalize partially to the Dl dopamine receptor agonist SKF 38393 and they can be attenuated partially by the dopamine receptor antagonists haloperidol, pimozide, and Sch 23390 (Reavill & Stolerman,

1988). Likewise, the results of Experiment 8 revealed that the stimulus properties of VTA stimulation may be mediated in part by dopaminergic substrates, as the cueing effects of the stimulation were augmented by amphetamine and attenuated by haloperidol. In view of these findings, it is conceivable that the dopaminergic components of the nicotine and brain- stimulation cues might have provided the basis for the summation observed between these test stimuli and the stimulus properties of amphetamine. Indeed, Experiment 7 revealed that the amphetamine-like cueing effects of VTA stimulation were most pronounced when the stimulating electrodes were placed in regions containing high densities of dopamine cell bodies. To date, only a limited number of 175 studies have investigated the stimulus properties o£ midazolam, and as yet there have been no attempts to determine whether the dopaminergic actions of this drug give rise to its stimulus properties.

Only one of the test stimuli employed in the present thesis was found to exert an effect on amphetamine stimulus generalization functions that was inconsistent with its actions on dopamine neurotransmission. Although morphine appears to enhance mesoaccumbens dopamine neurotransmission

(Di Chiara & Imperato, 1986; Glysing & Wang, 1983; Moleman et al., 1984), the amphetamine stimulus generalization function obtained after injections of this drug was lower than the vehicle control curve. This unexpected outcome may have resulted from a non-dopaminergic action of morphine that superseded the drug's facilitatory actions on dopamine function and attenuated the stimulus properties of amphetamine. The possible mechanisms that may have been responsible for this effect will be discussed further below

(p. 180). The important conclusion to be made here is that amphetamine stimulus generalization functions may be influenced by non-dopaminergic actions of drugs, even to the extent that pharmacological effects on dopamine neurotransmission may be obscurred.

A second major issue addressed in the present thesis was whether the stimulus properties of amphetamine might summate with those of any test stimulus that could exert positive hedonic actions, or whether such summation might occur only 176 with a limited range of hedonic stimuli. The results of

Experiments 2 through 6 provided a clear answer to this question. Of the wide range of test stimuli examined in these experiments, only nicotine and VTA stimulation were observed to elevate amphetamine stimulus generalization functions in a manner suggestive of an additive interaction with the amphetamine stimuli. Morphine and midazolam appeared to interfere with the stimulus properties of amphetamine, whereas ethanol produced a pattern of results which likely reflected pharmacokinetic effects on amphetamine metabolism rather than an interaction between the drug stimuli. These results indicated that the stimulus properties of amphetamine could summate with only a limited range of positive hedonic test stimuli.

The test stimuli that showed summation with the stimulus properties of amphetamine were those whose hedonic actions are mediated by mesoaccumbens dopamine neurons. The rewarding effects of cocaine, apomorphine, nicotine, and VTA stimulation all appear to involve agonistic actions on mesoaccumbens dopamine neurotransmission (Fibiger et al.,

1987; Roberts & Vickers, 1987; 1984; Roberts, Corcoran, &

Fibiger, 1977; Singer et al., 1982; Zito, Vickers, &

Roberts, 1985). In contrast, two of the drugs that did not show stimulus summation with amphetamine (midazolam and ethanol) previously have been suggested to have non- dopaminergic substrates for their rewarding effects (Amit &

Brown, 1982; Finlay et al., 1987). The third drug that 177 failed to show such summation was morphine. Although the rewarding effects of morphine may result in part from facilitatory actions on dopamine neurons in the VTA

(Phillips & Lepiane, 1980; Phillips et al., 1983; Stewart,

1984; Wise & Bozarth, 1982; 1984) there is some evidence that these effects also may be produced by morphine's actions in the periaqueductal gray, the lateral hypothalamus and on non-dopaminergic processes within the nucleus accumbens (Koob et al., 1987; Olds, 1979; 1982; van der

Kooy, Mucha, 0'Shaughnessy, & Bucenieks, 1982). These latter actions could have interfered with the perception of amphetamine-like stimuli (including those of amphetamine itself) and prevented the measurement of additive interactions between the stimulus properties of amphetamine and morphine. Whereas the effects of morphine will require further investigation, the pattern of results obtained with the other test stimuli would appear to suggest that generalization or summation with the stimulus properties of amphetamine might be limited to drugs that produce their positive hedonic properties by exerting facilitatory actions on dopamine neurotransmission.

Implications of the Present Findings for a Theory of the

Stimulus Properties of Amphetamine

As discussed above, the results of the present thesis provided support for the general hypotheses that the stimulus properties of amphetamine involve dopaminergic substrates, and that they reflect the drug's hedonic 178 effects. In fact, the present data allow specific statements to be made about the precise neurochemical mechanisms responsible for the transduction of amphetamine's pharmacological actions into a discriminative stimulus, and the specificity of the affective state that constitutes this stimulus at the perceptual level. With respect to the transduction mechanisms, it is important that the effects of the various test stimuli on amphetamine generalization functions were observed regardless of the specific means through which the drugs exerted their dopaminergic actions.

Although amphetamine's primary action is to stimulate the non-voltage dependent release of dopamine (Chiueh & Moore,

1975), amphetamine-like cueing activity also could be produced by a dopamine receptor agonist (the high dose of apomorphine; Anden et al., 1967), by a dopamine reuptake blocker (cocaine; Ritz et al., 1987), and by pharmacological and non-pharmacological agents that can increase the impulse-dependent release of dopamine (nicotine and VTA stimulation; Mereau et al, 1987; Powell, Carr, & Garner,

1987; Fibiger et al., 1987). Alternatively, the stimulus properties of amphetamine were attenuated by a dopamine receptor antagonist (haloperidol; Anden et al., 1970) and by compounds that can inhibit the activity of dopamine neurons

(the low dose of apomorphine and midazolam; Grace & Bunney,

1985; Haefley et al., 1981). This capacity of the amphetamine stimuli to be influenced by stimuli with diverse actions on dopamine neurotransmission suggests that the 179 common transducer of amphetamine-like cueing effects may be independent from the specific physiological or pharmacological activities of the individual test stimuli.

These results, and those of previous studies, might best be accounted for by a transduction theory that describes the stimulus properties of amphetamine in terms of the drug's consequences for postsynaptic dopamine receptor activity.

Specifically, the cueing actions of amphetamine may result from the capacity of this drug to increase synaptic concentrations of dopamine and thereby increase the amount of agonistic activity at postsynaptic receptors. This emphasis on the postsynaptic receptor activity would account for the results of generalization and summation tests wherein the cueing actions of amphetamine were mimicked both by dopamine agonists that acted directly on the postsynaptic receptors, and by test stimuli that increased the synaptic concentrations of dopamine by facilitating its release or by blocking its reuptake and metabolic degradation. This theory also would account for evidence that the stimulus properties of amphetamine can be attenuated by dopamine receptor antagonists and by compounds that decrease synaptic dopamine concentrations by interfering with transmitter synthesis, storage or release (see Silverman & Ho, 1977).

It is now well established that there are two separate subtypes of dopamine receptor that exist in the CNS. These receptor subtypes have been referred to as DI and D2 dopamine receptors (Creese, Sibley, Hamblin, & Leff, 1983). 180

Recently, Smith and Lyness (1988) demonstrated that the stimulus properties of amphetamine generalized to the D2 receptor agonist quinpirole but not the Dl receptor agonist

SKF 38393. However, both drugs were capable of increasing drug-appropriate responses elicited by a low amphetamine dose, and the Dl receptor antagonist SCH 23390 antagonized the cueing effects of amphetamine. Similarly complex patterns of results have been observed in other behavioral studies of dopamine receptor subtypes (Waddington, 1986), suggesting that Dl and D2 dopamine receptors may have an interactive influence in the control of dopaminergically mediated behaviors. The results of Smith and Lyness (1988) suggest a similar interactive involvement of the two receptor subtypes in mediating the stimulus properties of amphetamine.

As indicated in previous sections of this thesis, the stimulus properties of amphetamine appear to result from the drug's facilitatory actions at dopaminergic synapses within the nucleus accumbens. By increasing the availability of dopamine at postsynaptic receptors within this structure, amphetamine may initiate a chain of physiological events which ultimately results in a positive change in the affective state of the animal. It is this positive affective state that appears to be discriminated by animals during operant conditioning trials. The stimulus properties of amphetamine do not appear to represent a general state of positive affect, as they summated with only a limited range 181 of hedonically positive test stimuli in the present thesis.

In fact, such summation only was observed with test stimuli

(apomorphine, cocaine, nicotine, and VTA stimulation) that previously have been shown to produce their rewarding effects by facilitating dopamine neurotransmission. When rats were tested with drugs that appear to exert their rewarding effects through non-dopaminergic mechanisms

(midazolam and ethanol), there was no evidence for summation with the stimulus properties of amphetamine. These results suggested that rats may be able to discriminate between the positive hedonic consequences of enhanced mesoaccumbens dopamine neurotransmission and the positive affective states that result from the activation of non-dopaminergic processes.

One anomalous result that may have important implications for the theory outlined above was the apparent inhibitory actions of morphine on the stimulus properties of amphetamine. As indicated in previous sections of this thesis, morphine has been shown to increase both the firing rate of mesoaccumbens dopamine neurons and extracellular dopamine concentrations within the nucleus accumbens. In behavioral studies, morphine has been found to enhance both the euphoria-producing effects of amphetamine in humans

(Jasinski & Preston, 1986) and threshold lowering effects of amphetamine on lateral hypothalamic ICSS in rats (Hubner et al., 1987). Accordingly, it was anticipated that morphine also would summate with the stimulus properties of 182 amphetamine and elevate the stimulus generalization functions relative to the vehicle control curve. The failure of morphine to produce these effects (and its actual interference with the amphetamine stimuli) suggested that amphetamine stimulus generalization functions might be influenced by factors unaccounted for in the present theory.

The precise mechanisms responsible for morphine's effects on amphetamine stimulus generalization functions could not be determined from the experiments in the present thesis. However, a number of plausible explanations may be offered to account for the results. For example, the non- dopaminergic stimulus properties of morphine might have acted to mask stimuli associated with the combined dopaminergic actions of morphine and amphetamine. These masking stimuli could conceivably be related to other hedonic properties of morphine that result from the actions of this drug in the nucleus accumbens and the periaqueductal gray (Olds, 1982; van der Kooy et al., 1982). Indeed, masking stimuli are most effective when they are from the same sensory modality as the discriminative stimuli

(Kahneman & Treisman, 1984; Treisman, 1969).

A second explanation for morphine's effects takes into account the possibility that the stimulus properties of amphetamine may represent a stimulus complex composed of at least two independent subjective phenomena. Amphetamine can produce a variety of subjective effects in humans that potentially could control discriminated responses in 183 laboratory animals. The inhibitory effects of morphine on amphetamine stimuli might have resulted from antagonistic actions on processes (postsynaptic to dopamine projections) that mediate these non-hedonic stimulus properties of amphetamine, such that the overall intensity of the amphetamine stimuli were reduced. Although there is presently no evidence from animal studies to confirm or dispute this hypothesis, morphine has been reported to attenuate the anxiogenic properties of amphetamine in human drug users (Cox et al., 1983).

One final explanation for morphine's inhibitory effects deserves mention here. Although morphine frequently has been suggested to exert facilitatory actions on dopamine neurotransmission, there have been some reports that morphine actually may act as a dopamine receptor antagonist.

At a behavioral level, morphine can produce effects similar to those of dopamine antagonists such as the induction of catalepsy and the reduction of stereotyped behaviors produced by dopamine agonists (Lai, 1975). At a biochemical level, morphine has been found to inhibit the ability of dopamine to activate adenylate cyclase systems that are necessary for the functioning of DI dopamine receptors

(Neff, Parenti, Gentleman, & Olianas, 1981). Neff et al.

(1981) have suggested that morphine can modulate the functioning of dopamine receptors and attenuate their responsiveness to dopamine and dopaminergic agonists. This hypothesis would not conflict with evidence that morphine 184 can increase the firing rate of dopamine neurons and synaptic dopamine concentrations. These effects on dopamine function are commonly observed after administration of dopamine receptor antagonists (Bunney, Chiodo, Grace, &

Schenk, 1985; Blaha & Lane, 1984), presumably as a consequence of their ability to act at presynaptic dopamine receptors and interfere with the feedback inhibition of dopamine release. However, this hypothesis would not explain the contradiction between morphine's inhibitory effects on amphetamine stimuli and its enhancing effects on the hedonic properties of amphetamine (Jasinski & Preston, 1986; Hubner et al., 1987).

The Utility of Amphetamine Stimulus Generalization Paradigms as Screening Procedures for Assessing the Dopaminergic and

Hedonic Properties of Drugs

A major reason for conducting the present series of experiments was to determine whether an amphetamine stimulus generalization paradigm might be useful as a screening procedure to assess either the dopaminergic or hedonic actions of various test stimuli. With the exception of the tests with morphine, the results of these experiments indicated that such a paradigm indeed might be useful for assessing the dopaminergic actions of stimuli. In general, amphetamine stimulus generalization functions were elevated by test stimuli that could enhance dopamine neurotransmission and lowered by test stimuli that could interfere with dopamine neurotransmission. Ethanol, which 185 does not exert consistent actions on dopamine neurotransmission (Ellingboe and Mendelson, 1982; Kalant,

1975; Nutt & Glue, 1986), did not have an effect on the generalization functions indicative of a stimulus interaction. Importantly, these effects were obtained with drugs from diverse pharmacological classes and with a non- pharmacological stimulus produced by electrical stimulation of the VTA. Thus, the range of stimuli that could interact with the stimulus properties of amphetamine was not limited necessarily by pharmacological class boundries. However, the results of the tests with morphine indicated that the generalization paradigm sometimes might be limited by factors that have yet to be identified.

The results of the present experiments clearly indicated that amphetamine stimulus generalization paradigms would not be useful as a general screening procedure for assessing the hedonic properties of stimuli. Amphetamine stimulus generalization functions were elevated only by test stimuli which produce their rewarding effects by facilitating dopamine neurotransmission. Such elevations were not observed following injections of drugs whose rewarding effects appear to be mediated by non-dopaminergic mechanisms

(i.e., midazolam and ethanol), nor were they obtained after pretreatment with morphine, a drug that may produce only some of its rewarding effects by facilitating dopamine neurotransmission (Phillips et al., 1983; Stewart et al.,

1984; Wise and Bozarth, 1982). 186

Although the present amphetamine stimulus generalization procedure does not detect the hedonic actions of all test stimuli, the paradigm might be useful for a more limited purpose. Specifically, tests for summation with the stimulus properties of amphetamine may indicate whether the hedonic properties of a test stimulus are similar to those of amphetamine. Evidence that a test stimulus could elevate amphetamine stimulus generalization functions might suggest a common neural and perceptual basis for the hedonic properties of the amphetamine and test stimuli. The absence of effects or a lowering of the generalization functions would be less informative. These latter results could indicate either that the test stimulus was devoid of hedonic actions, that it produced its hedonic actions through non- dopaminergic mechanisms (as with midazolam and ethanol) or that it blocked the hedonic properties of amphetamine (as with haloperidol). Furthermore, the results with morphine indicated that certain drugs may lower amphetamine generalization functions even when some of their hedonic properties involve dopaminergic substrates.

Although the present stimulus generalization paradigm may be useful for making qualitative comparisons between amphetamine and test drug states, one should not attempt to use results from amphetamine stimulus generalization experiments to make quantitative statements regarding the relative abuse potential of different drugs. The magnitude of summation between the stimulus properties of amphetamine 187 and those of another drug may be influenced by a variety of factors other than the intensity of the affective state produced by the test drug. For example, it appeared that the ability of morphine to summate with the stimulus properties of amphetamine may have been limited by some non- dopaminergic property of the narcotic. Conceivably, other drugs might possess similar properties that would limit the extent to which they elevated amphetamine stimulus generalization functions. Another factor to consider is the extent to which tolerance or sensitization effects may influence the degree of summation observed between amphetamine and other drug stimuli. Repeated injections of amphetamine (which are necessary for discrimination training) commonly result in either increases or decreases in the efficacy of the drug in exerting various behavioral effects (Demellweek & Goudie, 1983; Stewart & Vezina, in press). Such chronic amphetamine treatments also may alter the magnitude of psychomotor stimulant-like effects produced by other drugs. For example, repeated amphetamine injections may enhance the locomotor stimulant actions of cocaine and morphine (Kalivas & Weber, 1987; Stewart & Vezina, 1987). At present, there are no data available to indicate the extent to which cross-tolerance and cross-sensitization might influence the magnitude of stimulus summation observed.

However, the possibility of these effects occurring should be considered when interpreting the changes in amphetamine stimulus generalization functions produced by various test 188 stimuli.

Implications of the Present Findings for Theories of Drug

Abuse

As indicated in the Introduction, recent theories of drug addiction have emphasized that the positive affective consequences of drug intake may represent a primary determinant of substance abuse. The role of positive affective properties in maintaining drug-taking behavior has been established most convincingly for the case of psychomotor stimulant compounds such as amphetamine and cocaine. These drugs appear to produce their hedonic effects by acting directly on dopaminergic processes that ordinarily determine affective and behavioral responses to exteroceptive appetitive motivational stimuli (Blackburn,

Phillips, & Fibiger, 1987; Blackburn, Phillips, Jakubovic, &

Fibiger, 1986; Stewart et al., 1984).

Recently, it has been suggested that addictive drugs from a variety of pharmacological classes may produce their hedonic effects by acting on the same neural systems that mediate the hedonic properties of psychomotor stimulants

(Wise & Bozarth, 1987; Wise, 1987). Thus, the abuse potential of different drugs may result from the capacity of each compound either to facilitate dopaminergic neurotransmission or to influence processes afferent or efferent to the dopamine neurons. Indeed, there is now evidence to suggest that dopamine neurons may play an important role in mediating the hedonic actions of opiates 189

(Bozarth, 1987; Bozarth & Wise, 1981; Phillips & LePiane,

1980; Smith et al., 1985; Stewart, 1984; Stewart et al.,

1984) and nicotine (Singer, Wallace, & Hall, 1982). However, few studies have investigated the neural basis for the hedonic properties of addictive drugs from other pharmacological classes.

The results of the present thesis may have important implications for the theory of drug addiction proposed by

Wise and Bozarth (1987) and Wise (1987). If all drugs of abuse produced hedonic effects similar to those of psychomotor stimulants, then each addictive drug employed in the present thesis might have possessed amphetamine-like stimulus properties. However, as noted above, only cocaine, apomorphine and nicotine were found to be capable of exerting amphetamine-like cueing effects. The three other hedonically positive compounds (morphine, midazolam, and ethanol) all failed to elevate amphetamine stimulus generalization functions in a manner suggestive of an additive interaction with the stimulus properties of amphetamine.

In the case of morphine, the absence of summation with the stimulus properties of amphetamine was contrary to expectations based on previous findings that at least some of the rewarding effects of opiates involve dopaminergic substrates (Phillips et al., 1983; Stewart et al., 1984;

Wise & Bozarth, 1984). Thus, it was concluded that the amphetamine-like stimulus properties of this drug may have 190 been obscurred by non-dopaminergic actions that remain to be identified. In contrast, the absence of summation between the stimulus properties of amphetamine and those of midazolam or ethanol could reasonably be attributed to an inability of these test drugs to exert amphetamine-like cueing actions. Unlike the results obtained with morphine, the absence of summation with midazolam and ethanol was consistent with previous reports that these drugs do not exert facilitatory actions on dopamine neurotransmission

(Ellingboe & Mendelson, 1982; Finlay et al., 1987; Kalant,

1975; Nutt & Glue, 1986). Furthermore, these drugs also failed to exert stimulant actions during locomotor activity tests with the same animals. Wise and Bozarth (1987) have suggested that any drug which produces its rewarding effects by facilitating mesoaccumbens dopamine function (or the afferents or efferents) should stimulate locomotor activity as a consequence of activating processes that normally regulate appetitive motivational responses to environmental stimuli. Indeed, locomotor stimulant actions have been reported for compounds such as psychomotor stimulants, opiates and nicotine (Clarke & Kumar, 1983b; Swerdlow,

Vaccarino, Amalric, & Koob, 1986; Vezina & Stewart, 1984).

The failure of midazolam and ethanol to produce either amphetamine-like stimulus properties or locomotor stimulant actions suggests that these drugs may not influence the same appetitive motivational processes that appear to be responsible for generating the positive affective properties of amphetamine.

Although the results of this thesis imply the existence of independent neural substrates for the positive affective properties of certain drugs, it is also true that many addictive drugs appear to produce their hedonic effects by acting on the same dopaminergic processes that mediate the hedonic actions of amphetamine. Accordingly, studies of the neural processes underlying amphetamine's affective properties may lead to a general understanding of the mechanisms responsible for the abuse potential of a wide variety of drugs. The drug discrimination paradigm employed in this thesis may offer a unique means of investigating these mechanisms. Whereas most procedures for studying the affective properties of drugs assess the changes in overt appetitive motivational responses associated with a drug's actions, measurements of the stimulus properties of amphetamine may provide a means of indexing changes in the more covert affective reactions to the drug after specific experimental manipulations. By evaluating the cueing efficacy of amphetamine after various pharmacological challenges, CNS lesions or intracerebral drug injections, we may come to understand the neural processes through which certain drugs of abuse give rise to subjective sensations of pleasure and euphoria. 192

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