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Investigations of the neurobiological and behavioural actions of cocaine Brown, Erin E. 1993

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INVESTIGATIONS OF THE NEUROBIOLOGICAL AND BEHAVIOURAL ACTIONSOF COCAINEByERIN EARL BROWNB.Sc. (Honours), University of Alberta, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDivision of Neurological SciencesWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1993© Erin Earl BrownIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of ^NJ E `-'424::, L—Orc I C AL SC( €-4  ce- sThe University of British ColumbiaVancouver, CanadaDate  5.---er- 2_4 ) igq DE-6 (2/88)iiAbstractAlthough once considered a benign recreational stimulant, cocaine is now recognizedto possess substantial abuse potential with considerable medical and social consequences.Accordingly, these experiments examined the behavioural and neurobiological effects ofcocaine in the rat.The behavioural and neurochemical interactions between cocaine and buprenorphinewere examined using a conditioned place preference (CPP) procedure and in vivomicrodialysis. Cocaine and buprenorphine both elicited CPP; moreover, these drugsinteracted to produce significantly larger CPPs when given in combination. Both cocaineand buprenorphine increased interstitial concentrations of dopamine in the nucleusaccumbens; the effect of cocaine was potentiated by the coadministration of buprenorphine.Taken as a whole, these results indicate that buprenorphine can interact with cocaine in asynergistic manner.The ability of stimuli previously paired with cocaine to elicit similar neurochemicalchanges as cocaine was assessed by in vivo microdialysis. Although acutely administeredcocaine produced a significant increase in interstitial dopamine concentrations in the nucleusaccumbens, the presentation of a cocaine-paired environment did not. Despite the absenceof a conditional neurochemical effect, significant conditioned locomotion was observed.These data do not support the hypothesis that stimuli paired with cocaine produce theirbehavioural effects by eliciting similar neurochemical effects as cocaine.To understand better the neurobiology of cocaine-induced environment-specificconditioning, expression of c-Fos, a putative marker of neuronal activity, was examined inthe forebrain of rats exposed to an environment in which they had previously receivedcocaine. Compared to saline-treated controls, cocaine produced an increase in locomotorbehaviour that was accompanied by an increase in c-Fos expression within specific limbicregions, as well as the basal ganglia. Exposure of rats to the cocaine -paired environmentiiialso produced an increase in locomotion that was associated with an increase in c-Fosexpression within specific limbic regions, but not within the basal ganglia. These findingssuggest that specific limbic regions exhibit increased neuronal activation during thepresentation of cocaine-paired cues and may be involved in the formation of associationsbetween cocaine's stimulant actions and the environment in which the drug administrationoccurred.Given the large body of evidence implicating the amygdaloid complex in the learningof stimulus-reward associations, the effects of quinolinic acid lesions of the amygdala oncocaine-induced conditional locomotion and CPP were examined. Although destruction ofthe amygdala did not affect basal locomotion, cocaine-induced locomotion or cocaine-induced conditional locomotion, cocaine-induced CPP was completely blocked by theamygdaloid lesions. These data demonstrate that cocaine-induced stimulus-rewardconditioning can be differentially affected by lesions of the amygdala.These studies provide a further understanding of the neurobiology of cocaine'sbehavioural actions. The implications for the treatment of cocaine abuse are discussed.ivTable of ContentsPageAbstract ^  iiTable of Contents ^  ivList of Tables  viList of Figures ^  viiAcknowledgements  xI. Introduction ^  1II. Behavioural and Neurochemical Interactionsbetween Cocaine and Buprenorphine ^  12(A) Introduction ^  12(B) Materials and Methods ^  14(C) Results ^  19(D) Discussion  37(E)^Notes ^  45III.^Cocaine-Induced Conditioned Locomotion:Absence of Associated Increases in Dopamine Release ^ 46(A) Introduction ^  46(B) Materials and Methods ^  47(C) Results ^  52(D) Discussion  66VIV.^Evidence for Conditional Neuronal Activation Following Exposure to aCocaine-Paired Environment: Role of Forebrain Limbic Structures ^ 72(A) Introduction ^  72(B) Materials and Methods ^  73(C) Results ^  77(D) Discussion  92V.^Differential Effects of Excitotoxic Lesions of the Amygdala on Cocaine-InducedConditioned Locomotion and Conditioned Place Preference ^ 99(A) Introduction ^  99(B) Materials and Methods ^  100(C) Results ^  104(D) Discussion  117VI. General Discussion ^  121VII. References ^  132viList of TablesPageTable 1^Diagnostic criteria for psychoactive substance dependence ^ 3Table 2^Diagnostic criteria for psychoactive substance abuse  4vi iList of FiguresPageFigure 1^Schematic representation of the phases of thecocaine abstinence syndrome ^  7Figure 2^The effects of cocaine on the time spent in the drug-pairedcompartment before and after conditioning ^  21Figure 3^The effects of buprenorphine on the time spent in the drug-pairedcompartment before and after conditioning ^  23Figure 4^^The effects of low doses of cocaine and buprenorphine aloneand in combination on the time spent in the drug-pairedcompartment before and after conditioning ^  25Figure 5^The effects of moderate doses of cocaine and buprenorphine aloneand in combination on the time spent in the drug-pairedcompartment before and after conditioning ^  27Figure 6^The effects of cocaine on dialysate concentrations ofdopamine and metabolites from the nucleus accumbens ^ 30Figure 7^The effects of buprenorphine on dialysate concentrations ofdopamine and metabolites from the nucleus accumbens ^ 32Figure 8^The effects of cocaine + buprenorphine on dialysate concentrationsof dopamine and metabolites from the nucleus accumbens ^ 34Figure 9^Summary of the effects of saline, cocaine, buprenorphine andcocaine + buprenorphine on dialysate concentrationsof dopamine from the nucleus accumbens ^  36Figure 10^Photomicrograph of a coronal section through the nucleus accumbensof a rat implanted with a dialysis probe ^  54viiiFigure 11^The effects of saline and cocaine on the dialysate concentrationsof dopamine and metabolites from the nucleus accumbens ^ 56Figure 12^Locomotor counts of control, pseudoconditioned and conditionedsubjects when exposed to the conditioned environment ^ 58Figure 13^Dialysate concentrations of dopamine and its metabolitesfrom the nucleus accumbens prior to and during exposureto an environment paired with cocaine or saline ^  61Figure 14^Dialysate concentrations of dopamine and its metabolitesfrom the nucleus accumbens prior to and during exposureto an environment paired with cocaine or saline ^  63Figure 15^Locomotor activity of conditioned and pseudoconditionedsubjects when exposed to the conditioned environment ^ 65Figure 16^Camera lucida drawings of representative sections used forthe counting of Fos-positive nuclei ^  79Figure 17 Locomotor counts for control, pseudoconditioned and conditionedsubjects when exposed to the conditioned environment, as well asthe locomotor counts of saline- and cocaine-treated subjects   81Figure 18^Number of Fos-positive nuclei within the cingulate cortex, claustrum,piriform cortex and nucleus accumbens ^  83Figure 19^Number of Fos-positive nuclei within the striatum, lateral septum,paraventricular nucleus of the thalamus and amygdala ^ 85Figure 20^Photomicrographs of Fos immunoreactivity in the cingulate cortex ^ 88Figure 21^Photomicrographs of Fos immunoreactivity in the lateral habenula ^ 90Figure 22^Photomicrograph of the amygdaloid region following infusionsof quinolinic acid ^  106Figure 23^Schematic of a representative bilateral lesion of the amygdalafollowing infusions of quinolinic acid ^  108ixFigure 24^Locomotor counts for control, pseudoconditioned and conditionedsubjects during 10 days of conditioning with cocaine ^ 112Figure 25^Locomotor counts for control, pseudoconditioned and conditionedsubjects when exposed to the conditioned environment ^ 114Figure 26^^The effects of cocaine on the time spent in the drug-pairedcompartment before and after conditioning for non-lesionedand lesioned subjects ^  116xAcknowledgmentsI would like to acknowledge the invaluable assistance of Campbell Clark, LilliCollu, Geert Damsma, Jamie Day, Janet Finlay, Mark Kimmins, George Nomikos, GeorgeRobertson, Sandra Sturgeon, Chui-Si Tham, Danielle Wenkstern and Catriona Wilson. Theguidance, encouragement and friendship of the aforementioned people made this anenjoyable endeavor. The supervision and guidance of Chris Fibiger, The Big Guy", notonly aided in the completion of this thesis, but also helped mature and refine myunderstanding and appreciation of science. Finally, I would like to acknowledge the personwho saw me through both the good times and bad, my wife, Kim Capri. She tolerated yearsof being a "science widow", yet continued to provide me with encouragement and support.1I. INTRODUCTIONShortly after cocaine became commercially available in Europe and the United Statesin the 1880's it began to receive endorsements and praise for its action as a tonic for thebody and mind (Musto, 1992). At this time the stimulant properties of cocaine wereemphasized, while its addictive potential was generally dismissed. Within thirty years,however, the adverse effects of cocaine had become apparent and severe restrictions on itsuse were enacted. In the United States the recognition of the abuse potential of cocaineculminated in the passage of the Harrison Act of 1914. Cocaine use diminished during the1920's as the adverse consequences associated with its use became more widelyacknowledged (Musto, 1992). As cocaine use began to increase rapidly in the 1970's andearly 1980's (Anthony, 1992), it was again suggested by medical professionals that cocainewas a safe, nonaddicting stimulant (Grinspoon and Bakalar, 1980; Van Dyke and Byck,1982). These assertions were partly the result of an absence of strong empirical evidenceregarding the actions of cocaine. At present, clinical and epidemiological research hasclearly documented numerous severe consequences of cocaine use (Anthony, 1992; Benowitz,1992) and it is recognized that cocaine possesses substantial abuse potential. Given thatcocaine abuse remains a serious medical and social issue, considerable interest remains inelucidating the actions and effects of this powerful stimulant.Clinical Characteristics of Cocaine AddictionAcute administration of cocaine produces a sense of alertness, euphoria and well-being (Jaffe, 1989; Johanson and Fischman, 1989). There are also decreases in hunger andneed for sleep. In addition to these behavioural changes, a number of physiological signsoccur following cocaine, such as tachycardia, pupillary dilation and elevated blood pressure.Cocaine can also induce paranoia, suspiciousness and overt psychosis, especially following aprolonged "binge" (Gawin and Ellinnwood, 1988; Jaffe, 1989; Satel et al., 1991).2Of those individuals who try intranasal cocaine ("snorting") the National Institute ofDrug Abuse estimates that 10 to 15% become abusers (Gawin, 1991). Most cocaine usersseeking treatment report that their initial use was intermittent. Eventually, however,episodes of high-dosage use become more frequent until "runs" or binges occur (DSM-III-R;Gawin and Ellinwood, 1988; Jaffe, 1989). Binges, which can last several days, arecharacterized by the user compulsively readministering cocaine every 10 to 30 minutes.During the binge, the user experiences periods of extreme euphoria, the memories of whichwill later be contrasted with the mood of the undrugged state and can produce cravings forthe drug (Gawin and Ellinwood, 1988; Gawin and Kleber, 1986). Binges tend to beinterrupted only when drug toxicity occurs, the user collapses from physical exhaustion orthe cocaine supplies are depleted. The termination of the binge is generally followed by anintense and unpleasant "crash".Clinical findings appear to suggest that cocaine smoking or intravenousadministration tend to produce a more rapid progression from infrequent use to cocaineabuse or dependence than intranasal use of cocaine (DSM-III-R; Jaffe, 1989; Gawin andEllinwood, 1988). This result may reflect the fact that these routes of administrationproduce a more rapid rise in blood and brain concentrations of cocaine and an intense"rush", followed by a rapid decline in blood and brain concentrations (Jones, 1990).Psychoactive substance disorders, as defined in the DSM-III-R, are divided intopsychoactive substance dependence, and a residual category, psychoactive substance abuse,which is reserved for individuals who exhibit pathological drug use, but fail to meet thecriteria for substance dependence. The criteria for psychoactive substance dependence andabuse are presented in Tables 1 and 2, respectively. Most individuals who exhibit high-dose, binge use of cocaine would meet the criteria for cocaine dependence. Unfortunately,not all authors use these terms with reference to the DSM-III-R diagnostic criteria, or theyuse other terms, such as cocaine addiction, making it difficult to make comparisons betweenthe results of different investigators.3Table 1.Diagnostic Criteria for Psychoactive Substance Dependence A.^At least three of the following:1). substance often taken in larger amounts or over a longer period than theperson intended.2). persistent desire or one or more unsuccessful efforts to cut down or controlsubstance use.3). a great deal of time spent in activities necessary to get the substance (e.g.theft), taking the substance, or recovering from its effects.4). frequent intoxication or withdrawal symptoms when expected to fulfill majorrole obligations at work, school, or home (e.g. does not go to work becausehung over, goes to school or work "high", intoxicated while taking care of hisor her children), or when substance use is physically hazardous (e.g. drivingwhile intoxicated).5). important social, occupational, or recreational activities given up or reducedbecause of substance use.6). continued substance use despite knowledge of having a persistent or recurrentsocial, psychological, or physical problem that is caused or exacerbated by theuse of the substance.Note: The following items may not apply to cannabis, hallucinogens or phencyclidine8). characteristic withdrawal symptoms (e.g. fatigue, insomnia or hypersomnia,and/or psychomotor agitation for cocaine withdrawal).9). substance often taken to relieve or avoid withdrawal symptoms.B.^Some symptoms of the disturbance have persisted for at least 1 month, or haveoccurred repeatedly over a longer period of time.Adapted from DSM-III-R Diagnostic and Statistical Manual of Mental Disorders, ed 3,revised. American Psychiatric Association, Washington, DC, 1987.4Table 2.Diagnostic Criteria for Psychoactive Substance Abuse A.^A maladaptive pattern of psychoactive substance use indicated by at least one of thefollowing:1). continued use despite knowledge of having a persistent or recurrent social,occupational, psychological, or physical problem that is caused or exacerbatedby the use of the psychoactive substance.2). recurrent use in situations in which use is physically hazardous (e.g. drivingwhile intoxicated).B.^Some symptoms of the disturbance have persisted for at least 1 month, or haveoccurred repeatedly over a longer period of time.C.^Never met the criteria for psychoactive substance dependence for this substance.Adapted from DSM-III-R Diagnostic and Statistical Manual of Mental Disorders, ed 3,revised. American Psychiatric Association, Washington, DC, 1987.5A triphasic syndrome associated with the abstinence from cocaine has been describedby Gawin and Kleber (1986). This syndrome is represented schematically in Figure 1. Thefirst phase, the crash, immediately follows the cessation of a cocaine binge, and ischaracterized by symptoms that vary throughout the crash phase. During the initial periodof phase 2, withdrawal, the individual experiences improved mood, normalized sleep andlow levels of cocaine craving; however, as this phase proceeds anhedonia, anergia, anxietyand increased cocaine craving, especially in response to stimuli previous associated withcocaine use, become apparent. The third phase, extinction, appears to represent a period ofextended vulnerability to relapse, although mood and withdrawal anhedonia havenormalized.A clear understanding of the clinical characteristics of cocaine abuse or dependenceis useful for a number of reasons. First, the informed clinician will have greater predictivepower regarding the probable course of cocaine abuse for individuals seeking treatment, andhence be capable of formulating more rational treatment programs that specifically addressproblems associated with the abstinence from cocaine. Second, the relative dirth ofempirical clinical data regarding cocaine's abuse potential that contributed to the belief thatcocaine was a harmless stimulant will be avoided in the future. Finally, an awareness of thesalient features of cocaine abuse will allow basic researchers investigating this topic toconsider their findings in a broader context.Neurobiology of Cocaine-induced RewardThe investigation of the neurobiology of cocaine abuse has proven to be aparticularly fruitful endeavor, as a number of animal models have been developed toinvestigate this topic. For example, the self-administration paradigm (Weeks, 1962) hasbeen used by numerous investigators to examine the neurochemical and neuroanatomicalsubstrates that mediate cocaine's ability to function as a positive reinforcer. The results of alarge number of studies using various pharmacological challenges and specific brain lesionsFigure 1. Schematic representation of the phases of the cocaine abstinence syndrome.Duration and intensity of symptoms vary on the basis of binge characteristics andpatient history and diagnosis. Binges range from under four hours to six or moredays. [Modified from Gawin and Kleber, 1986]6^Phase 1^ Phase 2^ Phase 3Crash Withdraw! Extinction9 hours to 4^days^1 to 10 weeks^Indefinite...111110.Cocaine BingeRelapseEarlyAgitationDepressionAnorexiaHigh Cocaine CravingMiddleFatigueDepressionNo Cocaine CravingInsomnia WithIncreasing Desirefor SleepLateExhaustionHypersomnolenceHyperphagiaNo Cocaine CravingEarlySleep NormalizedEuthymic MoodLow Cocaine CravingLow AnxietyMiddle and LateAnhedoniaAnergiaAnxietyHigh Cocaine CravingConditioned CuesExacerbateCravingNormal HedonicResponseEuthymic MoodEpisodic CravingConditioned CuesTrigger CravingAbstinence8strongly implicate the mesolimbic dopaminergic pathway in the rewarding effects of cocaine(Fibiger et al., 1992; Fibiger and Phillips, 1987; Johanson and Fischman, 1989). Forexample, Ritz and colleagues (1987) have reported that although cocaine blocks the reuptakeof noradrenaline, dopamine (DA) and serotonin, its action at the DA uptake site appearsmost directly related to its rewarding effects. This conclusion is in agreement with studiesthat have demonstrated that low doses of specific DA receptor antagonists increase the rateof responding for intravenous cocaine (De Wit and Wise, 1977; Ettenberg et al., 1982;Roberts and Vickers, 1984; Koob et a/., 1987a), while neither a- or /3-adrenergic receptorantagonists affect cocaine self-administration (De Wit and Wise, 1977; Wilson and Schuster,1974; Woolverton, 1987). These results have been interpreted by some investigators toindicate that low doses of DA receptor antagonists produce a partial DA receptor blockadethat results in an increase in the rate of responding to maintain the previous level ofpostsynaptic activation. However, it has also been proposed that "dopamine (receptor)antagonists exert their effect by antagonizing effects of cocaine unrelated to itsreinforcement" (Johanson and Fischman, 1989). Recent findings, however, lend additionalsupport for the hypothesis that DA receptor antagonists can affect the self-administration ofcocaine in a reward-related manner. Roberts et al. (1989) found that a low dose ofhaloperidol, which had previously been shown to produce an increase in responding forcocaine on a continuous reinforcement schedule (Roberts and Vickers, 1984), produced asignificant reduction in the break point for cocaine in the progressive ratio self-administration paradigm, a measure which varies directly with the strength of the rewardingeffects of the self-administered drug. Moreover, Bergman and colleagues (1990) havereported that the dose-response curve for cocaine self-administration is shifted to the rightby selective D1 and D2 receptor antagonists. These data, while consistent with thehypothesis that DA receptor antagonists attenuate the rewarding properties of cocaine, arenot readily accounted for by the simple rate-reduction antagonism hypothesis (Johanson andFischman, 1989). It should be noted, however, that rate of responding has been shown to be9affected by factors independent of the rewarding effects of cocaine, and that conclusionsbased on changes in the simple rate of responding must be interpreted with caution(Johanson and Fischman, 1989).The previously discussed results are further supported by studies that haveestablished that 6-hydroxydopamine (6-OHDA) lesions of the mesolimbic dopaminergicpathway, which produce extensive depletions of DA in the nucleus accumbens, producedramatic suppression in the rate of lever pressing for cocaine (Pettit et a/., 1984; Roberts etal., 1977, 1980). This finding does not appear to be the result of a generalized motor deficitor other nonspecific effects, as Dworkin and Smith (1988) have demonstrated that 6-OHDAlesions of the nucleus accumbens suppress the ascending limb of the cocaine self-administration dose-effect curve, while responding for food and water was unaffected.Furthermore, Koob et al. (1987b) have reported that the break point for cocaine in theprogressive ratio self-administration paradigm was significantly reduced by 6-OHDA lesionsof the nucleus accumbens.Although the aforementioned results suggest that the dopaminergic projection to thenucleus accumbens plays a critical role in the reinforcing effects of cocaine, otherobservations suggest that additional factors are involved in the self-administration of thispsychomotor stimulant. For example, Roberts and Koob (1982) reported that while 6-OHDA lesions of the ventral tegmental area, the origin of the dopaminergic projections tothe nucleus accumbens and other limbic structures, produced significant reductions in therate of cocaine self-administration, there was no significant correlation between themagnitude of the DA depletion in the nucleus accumbens and the change in respondingfrom prelesion rates. Moreover, several rats with considerable depletions of accumbens DAresponded at near normal pre-lesion rates for cocaine. The authors speculated that thedopaminergic innervation of other structures, such as the olfactory bulb, frontal cortex oramygdala, or a combination of structures may be critical in the self-administration ofcocaine. To this end, Goeders and Smith (1983) reported that rats will self-administer10cocaine directly into the medial prefrontal cortex, but not into the nucleus accumbens. Toexamine the role of this dopaminergic projection area in the role of systemicallyadministered cocaine, Martin-Iverson et al. (1986) investigated the effect of 6-OHDAlesions of the medial prefrontal cortex on the rate and pattern of cocaine self-administration. Although 6-OHDA lesions produced approximately a 95% depletion in DAin the medial prefrontal cortex, no significant effect of these lesions on either the rate orpattern of cocaine self-administration was observed. These results strongly suggest that thedopaminergic innervation of the medial prefrontal cortex is not necessary for the reinforcingeffects of intravenously administered cocaine. Given the previously discussed shortcomingsof using rate of responding as a measure of the reinforcing properties of cocaine,reexamination of the importance of the medial prefrontal cortex and other DA projectionareas is warranted.The rewarding properties of cocaine have also been extensively investigated using theconditioned place preference (CPP) paradigm. This paradigm is based on the principle thatif rewarding or appetitive stimuli are reliably associated with a given environment thenthere will be an increased tendency to approach or maintain contact with that environment(Carr et al., 1989). Although the findings of self-administration and CPP studies aregenerally in agreement, it should be noted that these paradigms have divergent theoreticalbackgrounds and behavioural demands and cannot be assumed necessarily to examineidentical phenomena (Wise, 1989). Cocaine has been shown to produce a CPP by numerousinvestigators (Bardo et al., 1984; Mackey and van der Kooy, 1985; Morency and Beninger,1986; Nomikos and Spyraki, 1988; Spyraki et al., 1982a, 1987). However, it appears thatmultiple mechanisms are potentially involved in cocaine-induced CPP, as this effect hasbeen reported to be dependent on dopaminergic transmission under some conditions(Morency and Beninger, 1986; Spyraki et al., 1987), while independent of dopaminergictransmission in other studies (Mackey and van der Kooy, 1985; Morency and Beninger,1986; Spyraki et al., 1982a). Although these data suggest that DA is not necessary for11cocaine-induced CPP following i.p. or s.c. administration, they cannot be assumed toindicate that dopaminergic transmission is not normally involved in this behaviour. Clearly,an understanding of the apparent DA-independent actions of cocaine in the CPP paradigmwill assist in further addressing these discrepancies.The advent of in vivo microdialysis has dramatically increased current understandingof the neurochemical actions of cocaine. The ability of behaviourally relevant doses ofcocaine to increase interstitial concentrations of DA in the rat nucleus accumbens has beenwell established (Bradberry and Roth, 1989; Di Chiara and Imperato, 1988a; Moghaddamand Bunney, 1989a; Pettit and Justice, 1989, 1991). Moreover, Pettit and Justice (1989,1991) have demonstrated that cocaine self-administration produces significant dose-relatedincreases in interstitial DA in the nucleus accumbens. Interestingly, cocaine has beenreported to preferentially increase interstitial DA in the nucleus accumbens, as compared toits effect in the striatum (Di Chiara and Imperato, 1988a) and the medial prefrontal cortex(Moghaddam and Bunney, 1989a). When considered together with the previously discussedbehavioural studies, recent microdialysis findings provide further support for the importanceof the mesolimbic dopaminergic projection to the nucleus accumbens in the rewardingproperties of cocaine.SummaryCocaine is presently recognized as a powerful stimulant possessing significant abusepotential, as well as a specific pattern of abuse and withdrawal (DSM-III-R; Gawin, 1991;Jaffe, 1989). A large body of evidence indicates that the mesolimbic DA system plays afundamental role in the reinforcing properties of cocaine (Di Chiara and Imperato, 1988;Fibiger et al., 1992; Fibiger and Phillips, 1987; Johanson and Fischman, 1989; Roberts et al.,1977; 1989). Despite the growth of both the clinical understanding of cocaine abuse and theneurobiological actions of cocaine, few attempts have been made to integrate the findings ofthese fields of investigation in a meaningful way. The following studies are a modestattempt to approach this objective.12II. BEHAVIOURAL AND NEUROCHEMICAL INTERACTIONS BETWEEN COCAINEAND BUPRENORPHINE(A) IntroductionBuprenorphine (BUP) is a synthetic opioid with the pharmacological profile of amixed agonist-antagonist. It has potent antinociceptive actions (Cowan et al., 1977; Dumand Herz, 1981) with a limited capacity for producing physical dependence (Cowan et al.,1977; Jasinski et al., 1978; Mello and Mendelson, 1980; Yanagita et al., 1981). BUP can alsoattenuate opiate self-administration in humans (Mello and Mendelson, 1980; Mello et al.,1982) and in non-human primates (Mello et al., 1983).Recently, it has been reported that BUP suppresses cocaine self-administration byrhesus monkeys (Mello et al., 1989). Based on these data, it was suggested that BUP mightbe useful in the pharmacotherapy of cocaine abuse. This proposal is supported by apreliminary report by Kosten and colleagues (1989) that indicated that heroin addicts treatedwith BUP had a significant reduction in cocaine-positive urines, compared to those subjectstreated with methadone. However, BUP is a potential drug of abuse, as indicated byepidemiological (Chowdhury and Chowdhury, 1990; Lewis, 1985; O'Connor et al., 1988;Strang, 1985) and primate self-administration studies (Lukas et al., 1986; Mello et al., 1981;Woods, 1977; Young et al., 1984). Moreover, as is the case for a variety of drugs of abuse,BUP decreases the threshold for intracranial self-stimulation (Hubner and Kornetsky, 1988).Cocaine or morphine pretreatment also suppresses responding for cocaine by non-humanprimates (Balster et al., 1992; Herling et al., 1979; Stretch, 1977), indicating that decreases inthe rate of responding in self-administration paradigms can occur for reasons other thandecreases in the rewarding properties of the reinforcer. These observations raise thepossibility that the BUP-induced decrease in cocaine self-administration reported by Melloet al. (1989) was due to a summation of the rewarding properties of these agents. In view13of these considerations, the conditioned place preference (CPP) paradigm was utilized toaddress two specific questions. First, can BUP produce CPPs? Second, is cocaine-inducedCPP affected by BUP?Although cocaine blocks the reuptake of a variety of biogenic amines, its action atthe DA uptake site appears to be most directly related to its rewarding effects (Ritz et al.,1987). This conclusion is consistent with self-administration studies that utilized specificneurotoxic lesions to illustrate that the integrity of the mesolimbic DA pathway to thenucleus accumbens is necessary for cocaine self-administration (Pettit et al., 1984; Roberts etal., 1977; Roberts et al., 1980). Moreover, low doses of specific DA receptor antagonistsproduce predictable alterations in self-administration of cocaine (De Wit & Wise, 1977;Ettenberg et al., 1982; Roberts & Vickers, 1984; Roberts et al., 1989), and local infusions ofthe DA receptor antagonist spiroperidol into the nucleus accumbens disrupts cocaine self-administration (Phillips & Broekkamp, 1980). In vivo microdialysis studies further suggestthat the rewarding properties of cocaine are mediated by increases in interstitial DA in thenucleus accumbens (Di Chiara & Imperato, 1988a; Moghaddam & Bunney, 1989a; Pettit &Justice, 1989, 1991).Although the association between the rewarding properties of cocaine and the releaseof DA in the nucleus accumbens is well established, the importance of mesolimbic DA inthe rewarding properties of opioids is unclear. Ettenberg et al. (1982) reported thatpretreatment with the neuroleptic a-flupenthixol produced a compensatory increase incocaine self-administration, but had no effect on heroin self-administration. In agreementwith this finding, 6-OHDA lesions, which reduced cocaine self-administration, had nolasting effect on heroin self-administration (Pettit et al., 1984). However, other findingssuggest that the DA projections from the ventral tegmental area (VTA) to the nucleusaccumbens may play a role in the rewarding effects of opioids. First, it has been reportedthat infusions of opioids directly into the VTA are rewarding, as assessed by self-administration (Bozarth and Wise, 1981a; Welzl et al., 1989) and CPP (Bals-Kubik et al.,141993; Phillips and LePiane, 1980, 1982). Second, heroin-induced CPPs are attenuated byneuroleptics (Bozarth & Wise, 1981b; Spyraki et al., 1983). Third, infusions of opioidreceptor antagonists directly into the VTA decrease the rewarding properties of systemicheroin (Britt and Wise, 1983). Finally, results from in vivo microdialysis studies demonstratethat p receptor agonists, such as morphine, methadone, fentanyl and ED-Ala2 , N-methyl-Phe4 , G1y5 -oli-enkephalin (DAMGO), increase interstitial DA concentrations in the nucleusaccumbens (Di Chiara and Imperato, 1988b; Spanagel et al., 1990). Because BUP increasesDA turnover at behaviourally relevant doses (Cowan et al., 1976), it is possible that theeffect of BUP on cocaine self-administration (Mello et al., 1989) may be the result ofinteractions between these agents on DA release in the nucleus accumbens. To determine ifBUP alters the effects of cocaine on interstitial DA concentrations, in vivo microdialysisstudies were also undertaken.(B) Materials and MethodsSubjectsSubjects were 268 male Long Evans rats (Charles River, Quebec), weighing 270-350g at the beginning of the experiment. The rats were group housed (four per cage), on a12:12 h light:dark cycle (lights on 07:00), with food and water available ad libitum. Allsubjects were handled daily for one week prior to initiation of the experiment. Allexperimental procedures were conducted at approximately the same time each day, duringthe animal's light phase.15ApparatusPlace preference conditioning was conducted in four identical shuttle boxes (78 x 25cm, 35 cm high). Each box was divided into two compartments (36 x 25 cm) joined by atunnel (6 x 8 x 8 cm) that could be closed at both ends by guillotine doors. The twocompartments differed in the appearance of the walls and the type of floor (solid brownwalls and a 1.2 cm wire mesh floor versus walls with black and white strips 1 cm wide anda floor of parallel bars spaced 1.2 cm apart). Translucent Plexiglas lids allowed a moderateamount of light from overhead incandescent lights to enter each compartment. Each shuttlebox was mounted on a fulcrum, allowing its position to be detected by microswitches. Thetime spent in each compartment and the number of crosses between compartments wasrecorded with dedicated electronic equipment.ProcedureThe procedure for CPP consisted of three phases: habituation, conditioning and test.During the three day habituation phase, rats were placed in one of the compartments of theshuttle box (hereafter referred to as the start side), following a counterbalanced design.Following placement in the start side, and with both guillotine doors raised, the rats weregiven access to both compartments during the 900 s trial. During each trial, the time spentin each compartment and the number of crosses between compartments were recorded. Theconditioning phase was conducted over the next eight days. On days 1, 3, 5 and 7, ratswere given drug injections and immediately confined to the non-start side for 30 min. Onalternate days, rats were injected with saline and confined to the start side for 30 min. Onthe test day, each rat was placed in the start-side and given access to both compartments;the time spent in each compartment and the number of crosses between compartments wererecorded.16Cocaine-Induced CPPThe first experiment was conducted to determine the dose-response relationship forcocaine in the CPP paradigm. Animals were randomly assigned to one of four groups (n=12per group), that received 0, 1.25, 2.5 or 5.0 mg/kg cocaine.BUP-Induced CPPThe dose response relationship for BUP in the CPP paradigm was determined in thisexperiment. Animals were randomly assigned to one of nine groups (n=12 per group) thatreceived 0, 0.005, 0.01, 0.03, 0.075, 0.15, 0.3, 0.6 or 0.9 mg/kg BUP.Cocaine & BUP, CPP ExperimentsBased on the dose-response data obtained in the first two experiments, two studieswere conducted to examine the effects of combinations of cocaine and BUP in the CPPparadigm. In the first of these studies, animals were randomly assigned to one of fourgroups (n=12 per group) that received either vehicle + vehicle, cocaine + vehicle, vehicle +BUP or cocaine + BUP. Cocaine and BUP were given in doses of 1.5 and 0.01 mg/kg,respectively. The second study of this experiment used the same design as the first, exceptthat cocaine and BUP were given in doses of 5.0 and 0.075 mg/kg, respectively.Cocaine & BUP, Dialysis ExperimentsRats were anaesthetized with sodium pentobarbital (50 mg/kg i.p.), mounted in astereotaxic instrument, and a vertical microdialysis probe was implanted into the nucleusaccumbens (AP: +3.6 mm; ML: -1.5 mm; DV: -8.2 mm from dura; relative to bregma;Pellegrino et al., 1979). The microdialysis probe was a variant of the concentric verticaldesign (outer diameter = 250 pm, molecular weight cut-off = 6000 Dalton; Spectra Pordialysis fibre). The active surface was 2 mm in length, and was 0.2 mm from the tip of theprobe. Recovery of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid17(HVA) and 5-hydroxyindoleacetic acid (5-HIAA) was measured in vitro at 37°C and at aflow rate of 5 pl/min. The in vitro recoveries (%) for this probe were (mean ± SD, n=6):3.5 ± 0.4 (DA), 4.0 ± 0.5 (DOPAC), 3.7 ± 0.4 (HVA) and 4.3 ± 0.5 (5-HIAA).Following surgery, the rats were individually housed for 48 h prior to testing. Onthe test day, after a stable baseline had been established (not more than 5% variation for 3consecutive samples), each rat was injected with either cocaine + saline, BUP + saline,cocaine + BUP or saline + saline. Microdialysis samples were taken for five hours post-injection. Histological verification of the probe placement was conducted following testing.Microdialysis experiments were conducted on-line (Damsma et al., 1990), such thatthe microdialysis probe was perfused at 5 p1/min (Harvard Apparatus perfusion pump); theresulting dialysate was directed to the sample loop (100 pl) of an injector (Valco) throughthe outlet tubing (PE-10, Clay Adams). Samples were automatically injected into a highperformance liquid chromatographic (HPLC) system with electrochemical detection every 20min. The perfusion fluid contained: NaCl (147 mM), KC1 (3.0 mM), CaCl2 (1.3 mM),MgC12 (1.0 mM), and sodium phosphate (1.5 mM, pH 7.3).The concentrations of DA, DOPAC, HVA and 5-HIAA in the dialysate weredetermined with HPLC in conjunction with electrochemical detection. Separation of DAand the acid metabolites was achieved by reversed-phase liquid chromatography (150 x 4.6mm, Nucleosil 5 C18, Chrompack). The flow rate of the mobile phase [0.1 M acetic acidadjusted to pH 4.1 with solid sodium acetate, 0.01 mM EDTA, 0.35 - 0.5 mM octanesulfonicacid (Kodak) and 10 % methanol] was 1.85 ml/min. Detection of the amines was achievedby the sequential oxidation and reduction of the samples by a coulometric detection system(coulometric electrode = +0.4 V; amperometric electrode = -0.2 V; ESA Inc.). The acidmetabolites were quantified by their oxidation at the coulometric electrode, while DA wasmeasured at the subsequent amperometric electrode. The detection limit of this assay wasapproximately 5 fmol/injection for DA and 20 fmol/injection for DOPAC, 5 - HIAA andHVA.18DrugsCocaine hydrochloride (Sigma) and buprenorphine hydrochloride (Schering) weredissolved in isotonic saline and were injected intraperitoneally (i.p.). In both the cocaineand BUP CPP experiments, drugs were injected in a volume of 3 ml/kg; in the combinationexperiments (cocaine and BUP), drugs were injected in a volume of 1 ml/kg. Doses ofcocaine are expressed as weight of salt, while those of buprenorphine are expressed as thebase.Statistical AnalysisTrend analysis was used to evaluate the results from the cocaine and BUP CPPexperiments. Data were weighted based on the dose of the drug. Within-group comparisonswere then conducted using the Bonferroni t statistic to assess which doses producedsignificant CPP. An initial analysis of variance illustrated that the preconditioning valuesfor the time spent on the non-start side did not differ between groups [F(20,231)=0.30,p=0.999], justifying the use of within-subject comparisons. The results from thecombination CPP experiments (cocaine and BUP) were also analyzed using the Bonferroni tstatistic. A Reverse Helmert analysis was conducted to further examine some of the CPPdata. The data from the dialysis experiments (% values), from time 0 to 300 min, wereevaluated using a two-way analysis (Treatment X Time) of variance with repeated measures(Geisser-Greenhouse adjustment of d.f.). Percentage values of dialysate concentrations werebased on an average of the three samples prior to the injection of drug. Comparisons ofdifferences between groups at specific time points were made using the Bonferroni tstatistic. All statistics were performed using SPSS:X version.3 software.(C) ResultsCocaine-induced CPPThe results of this experiment indicate that the time spent in the drug-paired (non-start) side was directly related to the dose of cocaine (Figure 2). Specifically, a significantlinear dose-response function was observed [F(1,44)=7.26, p<0.02]. Within-groupcomparisons illustrated that only the 5.0 mg/kg group spent significantly more time on thedrug-paired side of the apparatus [t(11)=2.6, p<0.05].BUP-induced CPPBUP elicited a dose-related CPP (Figure 3). The data were best fit by a cubic dose-response function [F(1,99)=13.88, p<0.001], although a linear dose-response function was alsoobserved [F(1,99)=4.83, p<0.05]. Within-group comparisons indicated that at 0.03 mg/kg,0.075 mg/kg, 0.15 mg/kg and 0.3 mg/kg BUP produced significant CPP [t(11)=5.28, 4.54,5.78, 3.61, respectively, p<0.001].Cocaine & BUP, CPP ExperimentsConsistent with the data from the previous experiments, low doses of BUP (0.01mg/kg) or cocaine (1.5 mg/kg) did not by themselves produce CPP (Figure 4). However, awithin-group comparison indicated that animals pretreated with a combination of BUP andcocaine did exhibit CPP [t(11)=2.63, p<0.05]. This finding was replicated using anothergroup of animals [t(I1)=2.55, p<0.05].CPP was produced by moderate doses of cocaine (5.0 mg/kg) [z(11)=2.89, p<0.01] andBUP (0.075 mg/kg) [t(11)=4.09, p<0.001], as well as by a combination of these drugs[t(11)=6.25, p<0.001] (Figure 5). A Reverse Helmert analysis of the three treatmentconditions indicated that, while the cocaine and BUP treatment groups did not differ1920Figure 2. The effects of cocaine on the time spent in the drug-paired (non-start)compartment before (^) and after (u) conditioning (n = 12/group). Values representthe mean ± SEM. * indicates a significant within-group difference (p<0.05) of pre-versus post-conditioning scores.21EI Pre—conditioningI. Post—conditioning*7;a) 600 —cawa 550 -(75cw 500 -cc7,7cCL(91 450 -=cca 400 -zzI— 350 -wCL00.0^1.25^2.5COCAINE (mg/kg)MIw 300 -MI7-5.022Figure 3. The effects of buprenorphine on the time spent in the drug-paired (non-start)compartment before (0) and after (•) conditioning (n = 12/group). Values representthe mean ± SEM. * indicates a significant within-group difference (p<0.05) of pre-versus post-conditioning scores.2324Figure 4. The effects of saline, cocaine (1.5 mg/kg), BUP (0.01 mg/kg), and cocaine (1.5mg/kg) + BUP (0.01 mg/kg) on the time spent in the drug-paired (non-start)compartment before (^) and after (■) conditioning (n = 12/group). Values representthe mean ± SEM. * indicates a significant within-group difference (p<0.05) of pre-versus post-conditioning scores.257;650-Nw0 600-E50w 550-cc47(^-a.01^500-ncca 450-z}-- 400-Zwo_coO Pre—conditioning• Post—conditioning*0.0+Coc 1.5+Coc 0.0 +C oc 1.5+Coc0.0 Bup 0.0 Bup 0.01 Bup 0.01 Bup26Figure 5. The effects of saline, cocaine (5.0 mg/kg), BUP (0.75 mg/kg), and cocaine (5.0mg/kg) + BUP (0.75 mg/kg) on the time spent in the drug-paired (non-start)compartment before (0) and after (■) conditioning (n = 12/group). Values representthe mean ± SEM. * indicates a significant within-group difference (p<0.05) of pre-versus post-conditioning scores. f indicates a significant difference (p<0.05) betweenthe combination group (cocaine + BUP) and the cocaine and the BUP treatmentgroups, as determined by a reverse Helmert analysis.270.0+Coc 5.0+ Coc 0.0 Coc+5.0+Coc0.0 Bup 0.0 Bup 0.075 Bup 0.075 Bup1U• 700-co.......w0 650-(7)0 600-wcc•Tia. 550-1^-0ccD 500-0z 450-I—z 400-wa_(/)350-wMI=^ Pre—conditioningIN Post—conditioning**significantly from each other [F(1,33)=0.75], the combined treatment was significantlydifferent from the individual treatments [F(1,33)=5.29, p<0.05].Cocaine & BUP, Dialysis ExperimentsThe average baseline output of DA (± SEM, n=16) was 1.84 fmol/min ± 0.48, anddid not differ significantly between groups [F(3,12)=0.68]. The average basal outputs (±SEM, n=16) for DOPAC, HVA and 5-HIAA were 533 ± 65, 297 ± 42, and 245 ± 54fmol/min, respectively. Basal values of DOPAC [F(3,12)=1.53], HVA [F(3,12)=0.18] and 5-HIAA [F(3,12)=0.69] did not differ significantly between the groups.Cocaine produced a rapid increase in interstitial concentrations of DA[F(3.29,19.74)=11.76, p<0.001] that returned to baseline within 120 to 160 min (Figure 6).Cocaine also significantly decreased DOPAC [F(2.70,16.23)=4.85, p<0.05] and HVA[F(2.77,16.65)=4.68, p<0.05] without affecting the serotonin metabolite, 5-HIAA[F(2.41,12.05)=1.45]. All statistical comparisons are made against saline injected controls(data not shown).Interstitial DA was gradually increased by BUP [F(3.99,23.92)=9.80, p<0.001],reaching approximately 200% of baseline values five hours post injection (Figure 7).Interstitial concentrations of DOPAC [F(3.17,19.04)=5.21, p<0.01] and HVA[F(3.32,19.92)=15.73, p<0.001] were also increased by BUP, although the effect on DOPACwas not sustained over the five hour test period. Interstitial 5-HIAA was unaffected byBUP [F(2.60, 15.62)=1.63].Co-administration of cocaine and BUP produced a rapid increase in the interstitialconcentration of DA that returned to baseline within 120 to 160 min [F(2.45,14.73)=8.80,p<0.005] (Figure 8). DA concentrations showed a further modest increase after 180 min.DOPAC [F(3.12,18.72)=12.18, p<0.001] and HVA [F(2.96,17.76)=14.89, p<0.001] were alsosignificantly altered by the drug combination, as compared to saline injected controls. TheDA metabolites showed a transient decrease, followed by a gradual increase over the2829Figure 6. The effects of cocaine (5.0 mg/kg) + saline on microdialysis output of DA (o),DOPAC (^), HVA (,n,) and 5-HIAA (0). Cocaine was administered at time 0. Valuesrepresent the group mean (n = 4) + SEM. Percentage values of dialysateconcentrations were based on an average of the three samples prior to the injectionof drug.200 -13180 -cliiw 160 -<vCO^-'--140 -0ae---120 -I—^-0_  100 -I—^_D0 80-60 -Cocaine (5.0 mg/kg)o — DopamineO — DOPACA — HVAO — 5—HIAA301 I^.^I 1—60^0^60^120^180^240^300TIME (min)31Figure 7. The effects of BUP (0.01 mg/kg) + saline on microdialysis output of DA (o),DOPAC (0), HVA (6.) and 5-HIAA (o). BUP was administered at time 0. Valuesrepresent the group mean (n = 4) + SEM. Percentage values of dialysateconcentrations were based on an average of three samples prior to the injection ofdrug.Buprenorphine (0.01 mg/kg) 1o - Dopamine^ - DOPACA - HVAO - 5-HIAA200 -a). 0 180 -7) ^ -NCO(0 160 -"a 140 -120 -I—ci- 100 -i—0 80 -3260 -I^.^I^I^.^1^..^1^..^1^.^i-60^0^60^120^180^240^300TIME (min)33Figure 8. The effects of the combination of cocaine (5.0 mg/kg) and BUP (0.01 mg/kg) onmicrodialysis output of DA (o), DOPAC (^), HVA (A) and 5-HIAA (0). The drugswere administered at time 0. Values represent the group mean (n = 4) ± SEM.Percentage values of dialysate concentrations were based on an average of threesamples prior to the injection of drug.o — Dopamineo — DOPACA — HVAo — 5—HIAAI^I^I^I^1^I^I^I1 1.^I300 -0 260 -c1r)0COvo 220 -"a180 -? AI- - 140-0I-D0 100 -60 -Cocaine (5.0 mg/kg) +Buprenorphine (0.01 mg/kg)34—60^0^60^120^180^240^300TIME (min)35Figure 9. A summary of the effects of saline + saline (0), cocaine (5.0 mg/kg) + saline (o),BUP (0.01 mg/kg) + saline (A) and cocaine (5.0 mg/kg) + BUP (0.01 mg/kg) (o) onmicrodialysis output of DA. Drugs were administered at time 0. Values representthe group mean (n = 4) + SEM. Percentage values of dialysate concentrations werebased on an average of three samples prior to the injection of drug.300 -a).E 260 -a)(,)racO220 -IS!I 180 -liiz---< 140 -a.00 100-o — Cocaine + Buprenorphine^ — Cocaine + SalineA - Buprenorphine + SalineO — Saline + SalineI^I^I—60^0^60^120^180^240^300TIME (min)361^1 1^137remainder of the session. This profile encompasses the individual effects that werepreviously observed for both cocaine and BUP alone. 5-HIAA was unaffected by theadministration of the drug combination [F(3.07,18.39)=1.21].The combination of cocaine and BUP resulted in a significantly larger peak effect(20 min) on interstitial DA than was observed after cocaine [46)=2.99, p<0.05] (Figure 9).Although the cocaine-BUP group continued to have larger average concentrations of DAthan the cocaine-saline group over the subsequent 40 min (two samples), these differencesfailed to reach significance. The combination of cocaine and BUP also produced effects oninterstitial concentrations of DOPAC and HVA that were different from the effect ofcocaine alone. Analyses of individual time points indicated that DOPAC was significantlyhigher in the combination group than in the cocaine group from 160 min [46)=3.92, p<0.01]to the end of the session [t(6)=5.83, p<0.005]. HVA showed a similar profile, but thedifference between the groups did not become significant until 180 min [t(6)=3.38, p<0.05]and then remained significant until the end of the session [t(6)=4.68, p<0.005]. DAconcentrations also appeared to differ between the cocaine-BUP and the cocaine-salinegroups after three hours; however, these differences failed to reach statistical significance.(D) DiscussionThe first experiment confirmed that cocaine can produce a CPP (Morency &Beninger, 1987; Mucha et al., 1982; Spyraki et al., 1987). Moreover, cocaine-induced CPPwas directly related to the dose administered.The CPP elicited by BUP was also dose-dependent, with maximal effects occurringbetween 0.03 and 0.15 mg/kg. In so far as the CPP is predictive of abuse liability (Carr etal., 1989), these results are consistent with previous reports which have indicated that BUPmay have abuse potential (Hubner and Kornetsky, 1988; Lukas et al., 1986; Mello, et al.,381981; Young et al., 1984). BUP produces complete generalization in rats trained todiscriminate u-agonists (Colpaert, 1978), suggesting that BUP possesses p-like discriminativeeffects. Given that other ih-agonists reliably produce CPP (Finlay et al., 1988; Mucha &Herz, 1985; Shippenberg et al., 1993; Spyraki et al., 1983), the finding that BUP inducesCPP is not unexpected. The inverted U-shaped function obtained for BUP-induced CPPmay reflect the agonist-antagonist properties of this drug. The analgesic actions of BUPshow a similar profile of action, with maximal effects occurring at 0.5 mg/kg (Dum & Herz,1981; Sadêe et al., 1982). Although the data from the present CPP experiment may be dueto the agonist-antagonist actions of BUP, other explanations exist. Due to its unusually slowdissociation from opioid receptors and its high degree of lipophilicity, BUP has a longduration of action (Hambrook & Rance, 1976; Lewis, 1985; Schulz & Herz, 1976). At thehigher doses of BUP, behaviourally relevant amounts of the drug may have remained in theanimals for extended periods. Specifically, the animals may have been unable to make thediscrimination between the BUP-paired and the saline-paired compartments if BUP waspresent in behaviourally relevant amounts 24 h later, during the next day's (i.e. salinetraining day's) pairing. This hypothesis may be supported by the finding that rhesusmonkeys responding for intravenous infusions of BUP and saline on alternate days failed torespond preferentially for BUP (Mello et al., 1981). However, if three days of saline wereinterposed between the BUP sessions, the monkeys took significantly more BUP than saline.These previous self-administration results illustrate that the potentially prolongedbehavioural effects of BUP should be considered when interpreting results obtained withthis long acting opioid.The results from the drug combination experiments indicate that cocaine and BUPcan interact synergistically to produce a CPP. Thus, subthreshold doses of BUP and cocaine,themselves incapable of eliciting CPP, produced a significant CPP when given incombination. In addition, moderate doses of cocaine and BUP that were individuallycapable of eliciting CPP interacted to produce a significantly larger CPP. Taken together,39these data suggest that BUP can increase the rewarding properties of cocaine. Although thisfinding is contrary to the recent interpretations offered by Mello et al. (1989), other resultssupport this conclusion (Hubner and Kornetsky, 1988). Previous self-stimulation studieshave shown a clear synergistic interaction between psychomotor stimulants and opiates(Hubner et al., 1987; Izenwasser & Kornetsky, 1989), which appears to be related to a DA-opioid interaction (Izenwasser & Kornetsky, 1989). Subjects in a clinical study also reporteda greater degree of euphoria following the administration of d-amphetamine and morphine,than when receiving either drug alone (Jasinski & Nutt, 1972). Given the results of thepresent study, and previous studies that indicate BUP has rewarding properties, it isreasonable to expect that, like other opiates, BUP can augment the rewarding effects ofpsychomotor stimulants.Following the completion of this study a number of investigators have published theresults of both preclinical and clinical investigations regarding cocaine-BUP interactions. Insupport of the present findings, it has been demonstrated that although BUP does not itselfpossess cocaine-like discriminative-stimulus properties, it can potentiate the discriminative-stimulus effects of cocaine (Dykstra et al., 1992; Kamien and Spealman, 1991). Dykstra etal. (1992) reported that BUP increased cocaine-appropriate responding by rats following alow dose of cocaine in a discrimination paradigm, while Kamien and Spealman (1991)observed that BUP shifts the full cocaine dose-effect curve to the left in monkeysdiscriminating between cocaine and saline. Taken as a whole, these data suggest that BUPcan potentiate the discriminative-stimulus properties of cocaine. The neurochemicalinteractions between cocaine and BUP observed in the present study may provide themechanism for this behavioural effect.Mello and colleagues (1990, 1992, 1993) have replicated their finding that BUPsuppresses cocaine self-administration. These authors report that this effect is relativelyspecific for cocaine because food reinforced responding, although significantly reduced byBUP, is not consistently reduced in all subjects and because many subjects become tolerant40to BUP's effect on food-maintained responding. Despite the reproducible nature of theBUP-induced suppression of cocaine self-administration, previously discussed issuesregarding the interpretation of these findings have not been adequately addressed in thesesubsequent studies. Specifically, a decrease in responding cannot be assumed to reflect adecrease in the rewarding effects of cocaine, as rate of responding is not determined solelyby these effects of the drug (Johanson and Fischman, 1989). This point is exemplified bythe fact that cocaine, amphetamine or morphine pretreatment suppress responding forcocaine by non-human primates (Balster et al., 1992; Herling et al., 1979; Stretch, 1977).Other investigators have also documented that BUP suppresses responding for intravenous orinhaled cocaine (Carroll and Lac, 1992; Carroll et al., 1992; Winger et al., 1992). However,the results of these studies illustrated that BUP suppresses behaviour maintained by severaldrug reinforcers, as well as non-drug reinforcers. Moreover, Winger et al. (1992)demonstrated that BUP did not shift the cocaine dose-response curve to the right, as wouldbe expected if BUP were antagonizing the effects of cocaine. Rather, BUP produced ageneralized suppression of responding across the cocaine dose-response curve in thisparadigm, a response similar to that observed for heroin. Although the mechanismresponsible for the BUP-induced suppression of responding is unknown, it is apparent thatthe decrease in responding for intravenous cocaine cannot be assumed to necessarily reflect adecrease in the rewarding effects of cocaine (Johanson and Fischman, 1989)Two recent studies have re-examined the ability of BUP to affect cocaine-inducedCPP. Suzuki et al. (1992) reported that the acute administration of 0.5 mg/kg BUPsuppressed cocaine-induced CPP, while Kosten et al. (1991) observed that the chronicadministration of 0.5 mg/kg of BUP twice a day also reduced cocaine-induced CPP. Asdiscussed previously, following the administration of large doses of BUP, behaviourallyrelevant amounts of the drug may have remained in the animals for extended periods oftime (Hambrook & Rance, 1976; Lewis, 1985; Schulz & Herz, 1976). Specifically, theanimals may have been unable to make the discrimination between the cocaine-paired and41the saline-paired compartments if BUP was present in behaviourally relevant amountsthroughout testing.In addition to the large number of preclinical studies examining cocaine-BUPinteractions, clinical trials have directly investigated the effect of BUP on the subjectiveresponses of human subjects to cocaine challenge. In an initial study, Mendelson et al.(1991) reported that BUP maintenance (4 or 8 mg/day, sublingually) completely blocked thesubjective effects of 10 mg of intravenous morphine, as well as significantly reducingsubjects' reported craving for heroin. In contrast, the effect of BUP on cocaine craving andthe subjective response to a cocaine challenge were variable. Some subjects reported thatBUP diminished the intensity and quality of the cocaine challenge dose, while other subjectsreported that the BUP maintenance enhanced the subjective effects of the cocaine. In asubsequent study, it was reported that subjective responses to cocaine were diminshedfollowing maintenance on 4 mg/day BUP, while subjects treated with 8 mg/day of BUPreported that cocaine challenges were more intense and prolonged, as well as being of higherquality (Teoh et al., 1992). Mendelson and colleagues (1992) recently found that BUP (4mg/kg, sublingually) suppressed the acute cocaine-induced stimulation of bothadrenocorticotropin hormone (ACTH) and euphoria. Taken as a whole, these preliminaryclinical studies suggest that the subjective responses to cocaine may be differentiallyeffected by BUP treatment in a dose related manner. It is noteworthy that these humanstudies compared the same subjects across time (i.e. before and after BUP treatment) anddid not utilize control subjects.A number of clinical studies have also directly examined the effect of BUP oncocaine abuse. Two pilot studies provided evidence that BUP was highly efficacious inreducing cocaine abuse in heroin abusers, as evidenced by a decrease in cocaine positiveurines (Gastfriend et al., 1992; Kosten et al., 1989). Despite these promising initial results,two double-blind, controlled clinical trials of BUP for the treatment of opioid and cocainedependence have demonstrated that BUP is no more effective than methadone in reducing42cocaine abuse (Johnson et al., 1992; Kosten et al., 1992). Given that methadonemaintenance is not an effective treatment for cocaine abuse (Chambers et al., 1972; Kostenet al., 1986, 1987a, 1987b), the use of BUP in the pharmacotherapy of cocaine abuseremains questionable.The in vivo microdialysis experiments were designed to provide neurochemical datathat could potentially explain the positive interaction observed between cocaine and BUP inthe CPP drug-combination experiments. Specifically, these experiments providedinformation regarding the actions of cocaine, BUP, and a combination of these drugs oninterstitial concentrations of DA and its metabolites in the nucleus accumbens. Cocaineproduced an 82% mean peak increase in interstitial concentrations of DA that returned tobaseline within two to three hours. The DA metabolites, DOPAC and HVA, weresignificantly decreased by cocaine. This profile is similar to that reported in previousdialysis experiments (Kalivas & Duffy, 1990; Maissonneuve et al., 1990). Other dialysisexperiments examining the effects of systemic cocaine have reported minimal effects onDOPAC and HVA (Di Chiara & Imperato, 1988a; Hurd et al., 1989); these discrepanciesmay be the result of factors such as the amount of time between probe implantation andtesting, and differences in the Ca2+ concentration in the dialysis perfusion fluid. Both tissueand dialysate concentrations of the DA metabolites are elevated 24 hr post-implantation(Reiriz et al., 1989; Zis et al., 1991), raising the possibility that the results from the formerstudies which were conducted 24 hr post-implantation contained an implantation artifact.Moreover, the earlier experiments were performed using perfusion fluids that containedhigh, non-physiological Ca 2+ concentrations (2.3 - 3.5 mM), which have been shown toinfluence both the basal amounts of DA recovered as well as the effects of pharmacologicalmanipulations (Moghaddam & Bunney, 1989b; Westerink et al., 1988).BUP produced a considerably different neurochemical profile than cocaine.Interstitial DA concentrations increased gradually over the test period, and reached 200% ofbasal values 5 hours post-injection. DOPAC and HVA were also increased by BUP, but the43effect on DOPAC was shorter lasting. The gradual onset of action of BUP may be due tothe slow association kinetics of this drug (Schulz & Herz, 1976). In a study of the humanpharmacology of BUP, it was reported that the peak miotic effect occurred six hours post-injection (Jasinski et al., 1978).It is important to note that BUP has high affinity for both p and K opiate receptors(Lewis, 1985), and that p- and ic-agonists have opposing actions on DA release in thenucleus accumbens (Di Chiara & Imperato, 1988b). The present study suggests that lowdoses of BUP act predominantly at p opiate receptors to increase the release of DA in thisstructure. Electrophysiological studies have shown that morphine increases the frequency offiring of VTA-DA neurons (Nowycky et al., 1978; Glysing & Wang, 1983); therefore, theincrease in interstitial DA following BUP administration is probably due to an increase inthe firing rate of the DA neurons in the VTA. The increase in DOPAC and HVA afterBUP-treatment also suggests an increase in activity in these neurons.The combination of cocaine and BUP produced a 163% increase in interstitial DAconcentrations in the first sample after drug administration. This effect was approximatelydouble that seen for cocaine + saline (82%), providing neurochemical evidence that BUP canenhance cocaine-induced increases in interstitial DA in the nucleus accumbens. Thisenhancement is not readily accounted for by simple additivity, as BUP + saline produced anincrease in interstitial DA (20%) that was not significantly different from saline + saline inthe first sample after drug administration. These neurochemical data provide a potentialexplanation for the synergism observed in the CPP studies. Cocaine increases interstitial DAby blocking reuptake into presynaptic terminals (Nomikos et al., 1990; Richelson &Pfenning, 1984). BUP, like other opiates, probably increases the firing rate of dopaminergicneurons (Nowycky et al., 1978; Glysing & Wang, 1983), thereby resulting in increased DArelease. BUP had only a minor effect (20%) on interstitial concentrations of DA shortlyafter injection, probably due to a small increase in the firing rate of VTA dopaminergicneurons. An increase in the firing rate would, however, be expected to have a larger effect44on interstitial concentrations of DA if reuptake was compromised, as occurs followingcocaine administration. An additional finding of potential interest is the apparentdiminution of the long-term effects of BUP on interstitial DA concentrations in the drugcombination group, as evident in Figure 8. Although cocaine may affect the long-termBUP-induced release of DA, the large standard error present in the combination group atthese later time points caution against this speculation without further study.It is unlikely that the larger effect on DA seen in the combination group was due tosimple metabolic interactions between BUP and cocaine. First, cocaine is metabolized byliver and plasma cholinesterases (Vitti & Boni, 1985), while BUP is largely inactivated byconjugation with glucuronic acid (Rance & Shillingford, 1976). Second, if BUP decreasedthe metabolism of cocaine then it would be expected that cocaine concentration woulddecrease more gradually, resulting in a more prolonged effect on DA. An examination ofthe time course of the DA concentrations after these two treatments shows that only the firstfew time points differed (Figure 9).The present data do not support the view that BUP may be useful in thepharmacotherapy of cocaine abuse because it antagonizes the reinforcing properties of thisstimulant (Mello et al. 1989, 1990, 1992). Differences in the doses of BUP used in thepresent study and those of Mello and colleagues may explain these different conclusions.Previous research indicates that BUP has dose-related agonist-antagonist properties (Cowanet a/., 1977; Dum & Herz, 1981; Yanagita, 1981); therefore, different doses of this drugcould result in different, even opposite effects. The present experiment concentratedprimarily on low to moderate doses, while Mello et a/. (1989, 1990, 1992) examined onlyhigh doses of BUP. Although it is clear that the doses used in the combination experimentsof the present investigation were in the agonist range, it is uncertain where the doses usedby Mello et al. (1989, 1990, 1992) fall on the dose-response curve for BUP. Nevertheless, ifthe doses of BLJP used by Mello et al. (1989, 1990, 1992) were in the agonist portion of thedose-response curve, then an alternative explanation for their results can be proposed.45Specifically, a summation of the rewarding properties of cocaine and BUP could equallywell account for the findings of Mello et al. (1989, 1990, 1992), and such an interpretationis clearly supported by the present results, as well as the finding that BUP can potentiate thediscriminative-stimulus effects of cocaine (Kamien and Spealman, 1991; Dykstra et al.,1992). It is also noteworthy that morphine produces a decrease in the rate of responding forcocaine (Stretch, 1977). Given that "speedballs" (cocaine + heroin or other opioids) are apopular form of illicit drug use (Kosten et al., 1987) and that subjects in a clinical studyreported a greater degree of euphoria following the administration of d-amphetamine andmorphine than when they received either drug alone (Jasinski & Nutt, 1972), it is reasonableto assume that morphine-induced decreases in responding for cocaine are due to asummation of the rewarding properties of these two drugs. Decreases in the self-administration of cocaine following BUP may similarly be due to additive effects of BUP-and cocaine-induced reward.(E) NotesNote 1: In Experiments I and II in vivo microdialysis has been used to measure theinterstitial concentrations of DA, DOPAC, HVA and 5-HIA A. Because the efflux of DAhas been shown to be TTX-sensitive and Ca 2+-dependent under appropriate conditions(Benveniste and Hilttemeier, 1990; Brown et al., 1991; Santiago and Westerink, 1990), it iswidely accepted that dialysate concentrations of DA reflect activity-dependent neuronalrelease.Note 2: The concentrations of DA and the metabolites (fmol/min) presented in ExperimentsI and II are dialysate values uncorrected for probe recovery.46III. COCAINE-INDUCED CONDITIONED LOCOMOTION: ABSENCE OFASSOCIATED INCREASES IN DOPAMINE RELEASE(A) IntroductionAn important aspect of the behavioural properties of cocaine involves the classicalconditioning of its unconditioned neurochemical effects with specific environmental stimuli(Barr et al., 1983; Stewart et al., 1984; Tatum and Seevers, 1929). This property of cocaineis of major significance with respect to its abuse potential, as intense craving can be evokedby stimuli previously associated with the act of taking the drug (Gawin, 1991; Johanson andFischman, 1989; O'Brien et al., 1992). The magnitude of these conditioned cravings can beoverwhelming and can result in previously abstinent abusers resuming the use of cocaine.Stewart et al. (1984) have proposed that stimuli associated with the administration ofopiates or stimulants can come to elicit neural states that are similar to those produced bythe drugs themselves. Given the large body of evidence implicating the mesolimbic DAsystem in the reinforcing properties of these drugs of abuse (Di Chiara and Imperato, 1988a;Fibiger and Phillips, 1987; Lyness et al., 1979; Roberts et al., 1977; Roberts et al., 1989) itis possible that conditioned stimuli associated with drug administration may also produceincreases in dopaminergic transmission. Earlier studies have provided evidence forconditioned dopaminergic activity with a variety of drugs, such as morphine, amphetamineand apomorphine (Lal et al., 1976; Perez-Cruet, 1976; Shiff, 1982). However, Barr et al.(1983) reported that presentation of environmental stimuli associated with the injection ofcocaine elicited behaviours similar to the drug itself, but noted an absence of conditionalneurochemical changes in the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) orhomovanillic acid (HVA). Other investigators have also failed to observe conditioneddopaminergic activity following conditioning with a variety of agents such as fentanyl(Finlay et al., 1988), morphine (Walter and Kuschinsky, 1989), and apomorphine (Moller,471987). Although these recent studies reported an absence of conditional neurochemicaleffects, all found significant conditioned behavioural changes.One shortcoming of the previous neurochemical studies is that changes indopaminergic transmission were inferred from changes in tissue concentrations of DAmetabolites. Although this approach can be useful, it also possesses certain limitations andpotential confounds (Commissiong, 1987). The advent of in vivo microdialysis has allowed amore direct assessment of the release of DA. The present study utilized this in vivotechnique to determine if environmental stimuli paired with cocaine administration produceincreases in DA release in the nucleus accumbens.(B) Materials and MethodsSubjects and DrugsSubjects were 61 male Long Evans rats (Charles River, Quebec), weighing 250 - 350g at the beginning of the experiments. The rats were group housed (2 - 4 per cage), on a12-hr light/12-hr dark cycle (lights on 08:00), with food and water available ad libitum. Allsubjects were handled periodically for one week prior to the experiments. All experimentalprocedures were conducted at approximately the same time each day, during the animal'slight phase.Both cocaine hydrochloride (10 mg/kg, dissolved in isotonic saline , BDH) and 0.9 °/osaline were injected i.p. in a volume of 1 ml/kg. The dose of cocaine is expressed as theweight of the salt.ApparatusLocomotor Activity (Experiment 2)Five circular (61 cm diameter) activity cages (BRS/LVE), each transected by sixinfra-red beams, were used to measure locomotor activity. Photocell beam interruptions48occurring more than 0.5 s apart were recorded with a NOVA IV (Data General)minicomputer equipped with MANX (GC Controls) software and interface.Conditioning Box (Experiment 3)The "conditioning environment" for this experiment was a Plexiglas cage (34 x 26 x36 cm) with 2 cm wide black and white vertical stripes on the walls and a floor of parallelmetal bars (1 cm apart). A black Plexiglas lid , with a small stimulus light (CM-47, ChicagoMiniature Lamp) mounted in its center, was held 1 cm above the top of the conditioningbox. The space between the box and the lid was designed to allow free movement of thedialysis inlet and outlet tubings during testing. A tray of corncob bedding (bed-o' cobs,The Andersons) was placed beneath the floor of the cage, and was replaced between eachrat. The conditioning box was placed within a sound attenuating chamber equipped with acontinuously operating fan.Locomotor Activity (Experiment 4)The conditioning box for this experiment was a Plexiglas cage (40 x 31 x 42 cm)with red acetate (Behnsen's Graphic Supplies) covering the lower portion (21 cm) of the boxand 1 cm wide black and white horizontal stripes covering the upper portion (21 cm) of thebox. The bottom of the cage was filled (3 cm deep) with cat litter (99% Dust-Free, KittyLitter Brand). No lid was used. A buzzer (SMB-24, Star Microtronics, 15 V, 60-70 dB)was continuously activated while the rat was in the box.This box was placed within a Digiscan Animal Activity Monitor [modelRXYZCM(16); Omnitech Electronics, Inc.] to measure locomotor activity in 10 min blockscorresponding to the 10 min dialysate samples.49Surgery and microdialysisRats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), mounted in astereotaxic instrument, and a vertical microdialysis probe was implanted into the nucleusaccumbens (AP: +3.6 mm; ML: -1.5 mm; DV: -8.2 mm from dura; relative to bregma;Pellegrino et al., 1979). The microdialysis probe was a variant of the concentric verticaldesign (outer diameter = 320 pm, molecular weight cut-off < 60,000 Dalton; AN69, Hospal).The active surface was 2 mm and was 0.2 mm from the tip of the probe.After surgery, the rats were individually housed for 48 hr before testing. On thetest day, the appropriate experimental manipulations were performed on each rat after astable baseline had been established (not more than 10% variation for three consecutivesamples). Histological verification of the probe placement was conducted after testing.Specifically, rats were administered a lethal dose of pentobarbital (120 mg/kg, i.p.) andtranscardially perfused with 4% formalin. Coronal sections (50 pm) were collected andstained with cresyl violet.Microdialysis experiments were conducted on-line as described in Chapter II, exceptthat samples were automatically injected into a HPLC system with electrochemical detectionevery 10 min.Biochemical AssayThe concentrations of DA, DOPAC, HVA and 5-HIAA were determined with HPLCin conjunction with electrochemical detection utilizing the same experimental protocol asdescribed in Chapter II.ProcedureThe first experiment involved the determination of the acute neurochemical effectsof saline and cocaine on DA and its metabolites in the nucleus accumbens using in vivomicrodialysis. On the test day, subjects were injected with saline (1 ml/kg, i.p.), followed5060 min later by cocaine (10 mg/kg, i.p.). Microdialysis sampling continued for 3 hr afterthe administration of cocaine.The classical conditioning of the locomotor stimulant effects of cocaine with aspecific environment was examined in the second experiment. Thirty rats were randomlyassigned to 1 of 3 groups: conditioned, pseudoconditioned or control. Conditioned subjectswere injected with cocaine (10 mg/kg, i.p.) and then placed into one of the circular activitycages (as previously described) for 30 min. After the training session, subjects werereturned to their homecages, where they were injected with saline (1 ml/kg, i.p.) 4 hr later.Pseudoconditioned rats were exposed to an identical procedure except that the order ofadministration of cocaine and saline was reversed. Specifically, these subjects were injectedwith saline prior to being placed in the activity cages and later with cocaine in theirhomecages. Control subjects were exposed to the same procedure as the other groups,except that they were injected with saline in both environments and never received cocaine.Each subject was assigned to a particular locomotor cage for the duration of the experiment.Training was conducted daily for 7 days at approximately the same time each day. On thetest day (48 hr after the final training session), subjects were placed in the locomotor cagesand activity was monitored for 30 min. No injections were given on the test day.The third experiment examined if the previous training regimen, which had beenshown to produce conditioned locomotion, also produced changes in indices of dopaminergictransmission. Subjects were randomly assigned to either conditioned or pseudoconditionedgroups, and trained following the same procedure as in Experiment 2. The only notabledifference was that a different cage (as described above) was used. Three to 4 hrs after thefinal injection of the training procedure, animals were anesthetized and implanted with avertical microdialysis probe into the nucleus accumbens. On the test day (approximately 45hr after the final training session), dialysis was initiated while the rats remained in their"post-operative" home cages. After a stable baseline had been obtained, each subject was51moved to the testing room and placed in the conditioning box, where it remained for 50min.In order to validate the results of the previous two experiments, an additionalconditioning experiment was undertaken in which both behaviour and neurochemistry weremonitored simultaneously during the exposure of rats to the previously conditionedenvironment. The training procedure and surgery for this experiment was similar to thethird experiment, with the exception of the different conditioning box (as noted above). Onthe test day (approximately 45 hrs after the final training session), baseline dialysis sampleswere collected while the subject remained in its homecage. After a stable baseline had beenobtained, the rat was placed into the conditioning box for 50 min. Behaviour was monitoredthroughout the period the rat remained in the conditioning box.Statistical AnalysisThe data from the dialysis experiments (percent values) were evaluated using aunivariate analysis of variance with repeated measures (Huynh-Feldt adjustment of degreesof freedom). Between group differences for both the dialysis data from Experiments 3 and4 and the locomotor data from Experiments 2 and 4 were evaluated with a two-way analysisof variance with repeated measures (Huynh-Feldt adjustment of degrees of freedom).Additional comparisons of differences in locomotor counts between groups at specific timepoints were made using the Bonferoni t statistic. Comparisons of absolute concentrations(fmol/min) of DA, DOPAC, HVA and 5-HIAA between the different experimental groupswere made using a univariate analysis of variance.52(C) ResultsThe average baseline output of DA (± SEM., n = 29) was 6.20 ± 0.51 fmol/min anddid not differ significantly between the various experimental groups [F(4,24) = 1.00](Conditioned subjects (n=13): 6.5 ± 0.9 fmol/min, pseudoconditioned subjects (n=12): 5.4 ±0.6 fmol/min, acute cocaine subjects (n=4): 7.7 + 1.4 fmol/min). The average basal output(± SEM, n = 29) for DOPAC, HVA and 5-HIAA were 683 ± 49, 377 ± 29 and 261 ± 14fmol/min, respectively. Basal values of DOPAC [F(4,24) = 2.67], HVA [F(4,24) = 1.85] and5-HIAA [F(4,24) = 1.80] did not differ significantly between experimental groups. Figure10 is a photomicrograph of a typical histological section (50 pm, cresyl violet stain)illustrating the placement of the dialysis probe within the nucleus accumbens.Cocaine produced a rapid increase in interstitial concentrations of DA [F(10.02,30.07)= 67.21, p < 0.001] that returned to baseline within 140 to 180 min (Figure 11). Cocainealso significantly decreased DOPAC [F(1.92, 5.77) = 9.89, p < 0.02], HVA [F(4.00, 12.00) =16.16, p < 0.001] and 5-HIAA [F(2.09, 6.28) = 8.82, p < 0.02]. The dramatic increase ininterstitial DA produced by cocaine is contrasted by the lack of effect of salineadministration [F(2.78, 8.33) = 0.88]. Saline also failed to affect DOPAC [F(3.83, 11.48) =0.64], HVA [F(1.84, 5.52) = 0.78] or 5-HIAA [F(6.00, 18.00) = 1.44].The conditioning procedure employed in Experiment 2 produced a notableconditional behavioural effect (Figure 12). A significant difference in locomotor countsbetween the conditioned and pseudoconditioned groups is demonstrated by significant group[F(1,18) = 12.6, p < 0.005] and group x time [F(2.00,36.00) = 6.25, p < 0.01] effects. Theconditioned group exhibited significantly more locomotor counts during the first [t(18) =3.82, p < 0.005] and second [418) = 3.43, p < 0.005] 10 min periods. This conditioned effectis directly contrasted by the lack of a significant group [F(1,18) = 0.00] or group x time[F(2.00,36.00) = 0.51] difference between the pseudoconditioned and control groups.53Figure 10. Photomicrograph of a coronal section (50 pm) through the nucleus accumbens ofa rat implanted with a dialysis probe. Inset: Schematic representation of thelocation of the dialysis probe.55Figure 11. The effect of acute administration of saline (I ml/kg) and cocaine (10 mg/kg,i.p.) on the microdialysis output of DA (o), DOPAC (0), HVA (A) and 5-HIAA (o)in the nucleus accumbens (n=4). Values represent the group mean ± SEM.Percentage values of dialysate concentrations were based on an average of threesamples before the injection of saline.o — Dopamine^ — DOPACA - HVAO — 5—HIAACocainet1 11111II180 240SalineI . I^1 ^—30 0 60^120TIME (min)56300 -a)c•_733^.tnco^_03 _o 200 -aei-na_i-n0 wo -57Figure 12. Locomotor counts of conditioned (Cs+), pseudoconditioned (Cs -) and controlsubjects following one week of conditioning with cocaine (10 mg/kg, i.p.). Testingoccurred 48 hrs after the last training session, and was conducted in the sameapparatus as used for the training procedure. Values represent the group mean ±SEM (n=10/group). Inset: Total number of locomotor counts for the conditioned,pseudoconditioned and control subjects for the 30 min test period. * p < 0.05compared to pseudoconditioned controls.aoC.=0C.)4001...0....0s000—J300i10 20130300 -Cl)4■0C=0 200 —L-00E000 100 ——J50 -o — Cs+ GroupNo Cond. Cs —.a° 150 -^ — Cs— GroupA — No ConditioningCs +200--=E500 0ce,250 -58Time (min)59Although the data from Experiment 2 indicated that the conditioning paradigmproduced significant conditional changes in behaviour, the exposure of conditioned subjectsto the cocaine-paired environment did not produce a change in interstitial DA [F(4.97,54.71)= 0.40] (Figure 13-A), DOPAC [F(3.54,38.89) = 0.47] or HVA [F(2.72,29.9) = 0.30] (Figure13-B) that differed from the pseudoconditioned controls. The limited increase in DAobserved in both groups (approximately 10%) failed to reach significance [F(4.97,54.71) =0.90]; however, a significant increase in both DOPAC [F(3.54, 38.89) = 4.17, p < 0.01] andHVA [F(3.15, 34.70) = 2.93, p < 0.05] was observed in both groups.The data from the fourth experiment strongly substantiated the results from theprevious two experiments. Exposure of conditioned subjects to the drug-paired environmentdid not produce a change in interstitial DA that was significantly different from thepseudoconditioned controls [F(4.87, 48.65) = 0.80] (Figure 14-A). Moreover, neitherDOPAC [F(4.09, 40.94) = 1.22] nor HVA [F(5.00, 50.00) = 0.64] differed between theconditioned or pseudoconditioned groups (Figure 14-B). Although no group x timedifferences were present, significant effects over time were observed for DA [F(4.87, 48.65)= 10.12, p < 0.001], DOPAC [F(4.09, 40.94) = 9.83, p < 0.001] and HVA [F(5.00, 50.00) =7.05, p < 0.001]. The lack of neurochemical differences between the conditioned andpseudoconditioned subjects is contrasted by the significant group [F(1, 10) = 11.14, p < 0.01]and group x time [F(1.73, 17.27) = 5.59, p < 0.05] effects observed for the locomotor activityof these subjects (Figure 15). The conditioned subjects exhibited significantly morelocomotor activity than the pseudoconditioned controls during the first [t(10) = 2.81, p <0.05] and third [t(10) = 4.34, p < 0.05] 10 min periods of the test session.60Figure 13. Experiment 3 A. Interstitial concentrations of DA in the nucleus accumbensprior to and during exposure to an environment paired with cocaine (Cs+ group, n=6)or saline (Cs - group, n=7). B. Interstitial concentrations of DOPAC and HVA inthe nucleus accumbens prior to and during exposure to an environment paired withcocaine (Cs+ group, n=6) or saline (Cs - group, n=7). Values represent the groupmean ± SEM. Percentage values of dialysate concentrations were based on anaverage of three samples before exposure to the conditioned environment.1^I^IConditioned EnvironmentI^I^I^I ^1120 -117=O(044 115 --0"6110 -bei-a. 105 -I-0< 100 -0o — Cs+ Group (n=6)• — Cs — Group (n=7)61AConditioned Environment I1^1^I^I—30 —20 —10^0^10^20^30^40^501^I^I95 -3.E 115 -mNta_o' 46 110  -a ei--EL 105 -I-0a)7-0  100 -N76095 -^ — DOPAC 1A - HVA f Cs+ Group (n=6)■ — DOPAC 1A - HVA^Cs— Group (n=7)B—30 —20 —10^0^10^20^30^40^50TIME (min)62Figure 14. Experiment 4 A. Interstitial concentrations of DA in the nucleus accumbensprior to and during exposure to an environment paired with cocaine (Cs+ group, n=6)or saline (Cs - group, n=6). B. Interstitial concentrations of DOPAC and HVA inthe nucleus accumbens prior to and during exposure to an environment paired withcocaine (Cs+ group, n=6) or saline (Cs - group, n=6). Values represent the groupmean ± SEM. Percentage values of dialysate concentrations were based on anaverage of three samples before exposure to the conditioned environment.120 -13cliito 115 -KJ75110 -I-Da. 105 -I--D0< 100 -095 -I^.^Io — Cs+ Group (n=6)• — Cs — Group (n=6)AConditioned EnvironmentI^I^I^I1Conditioned Environment50—30 —20 —10^0^10^20^30^40A - HVA^ — DOPAC } Cs+ Group■ — DOPAC 1 Cs — GroupA — HVAB—30 —20 —10^0^10^20^30^40^5063li;.E 115 -a50Iu_a"a 110 -atI-D0_ 105 -I-D0a)t; 100  -t a>ft(UEl95 —TIME (min)Figure 15. Locomotor activity (horizontal distance) of conditioned (Cs+, n=6) andpseudoconditioned (Cs - , n=6) subjects from Experiment 4. Values represent thegroup mean ± SEM. Inset: Total horizontal distance for the conditioned andpseudoconditioned subjects for the 30 min test period. * p < 0.05 compared topseudoconditioned controls.64- 7500E0- 5000 0z4CF-cna- 2500 _1<F-0I-010^20^30^40^50o — Cs+ Group• — Cs — Group*TIME (min)655000 —Ec.)w 4000 -0z<1— 3000 -coa<- 2000 -I—z0N 1000 -CC0o -66(D) DiscussionThe first experiment was designed to assess the unconditioned neurochemical effectsof cocaine. The findings of this experiment confirm that cocaine produces an increase ininterstitial DA concentrations in the nucleus accumbens (Chapter II; Di Chiara and Imperato,1988a; Kalivas and Duffy, 1990). The ability of cocaine to increase dopaminergictransmission appears to be critically involved in its reinforcing properties (De Wit and Wise,1977; Ritz et al., 1987; Roberts et al., 1977; Roberts et al., 1989). Although it has beensuggested that the dopaminergic projection to the medial prefrontal cortex may also play animportant role in the reinforcing properties of intracranially administered cocaine (Goedersand Smith, 1983), a variety of behavioural and neurochemical studies suggests that thedopaminergic innervation of the nucleus accumbens is necessary for the reinforcingproperties of systemically administered cocaine (Martin-Iverson et al., 1986; Moghaddamand Bunney, 1989a; Roberts et al., 1977).Cocaine also produced decreases in the DA metabolites, DOPAC and HVA, and thisis consistent with the proposed neurochemical actions of cocaine (Heikkila et al., 1975;Nomikos et al., 1990). These decreases are thought to be a direct result of the inhibition ofDA uptake, resulting in less DA being available for intraneuronal metabolism by monoamineoxidase (Brown et al., 1991; Roffler-Tarlov et al., 1971; Soares-da-Silva and Garrett, 1990).Although some earlier dialysis studies reported limited effects of cocaine on these DAmetabolites (Di Chiara and Imperato, 1988a; Hurd and Ungerstedt, 1989), the presentfindings are consistent with our previous results (Chapter II), as well as recent data fromother laboratories (Kalivas and Duffy, 1990; Maissonneuve et al., 1990).The results of the second experiment confirmed that behavioural effects of cocainecan be classically conditioned to environmental stimuli (Barr et al., 1983; Beninger and Herz,1986; Tatum and Seevers, 1929). Specifically, when subjects were tested in the environmentthat had previously been paired with cocaine they exhibited significantly more locomotor67activity than subjects that had received cocaine in their homecage. The classicalconditioning of a drug to environmental cues is not unique to cocaine and has been reportedwith other stimulants, as well as with opiates (Beninger and Hanh, 1983; Carey, 1992; Willeret al., 1987; Stewart et al., 1984; Walter and Kuschinsky, 1989). This type of conditioninghas important clinical significance because conditioned cues can evoke intense craving, andthese conditioned cravings play a substantial role in the relapse into cocaine use (Gawin,1991; O'Brien et al., 1992).Given the demonstration of the classical conditioning of cocaine to environmentalstimuli, as well as the large unconditioned effect of cocaine on interstitial DA in the nucleusaccumbens, the third experiment was designed to determine if the conditional change inbehaviour was associated with an increase in DA release. No evidence for conditioneddopaminergic activity was found, as the exposure of conditioned subjects to the cocaine-paired environment failed to produce a change in interstitial DA or its metabolites, DOPACor HVA, that was significantly different from pseudoconditioned controls. Although the 10% increase in DA for the two groups of subjects failed to reach significance, the increasesin DOPAC and HVA were significant. The increase in the DA metabolites suggests thatexposure to the conditioning environment did influence these dopaminergic neurons;however, the lack of differentiation between the groups indicates that the increase was notrelated to the conditioning.The final experiment served as a validation and extension of Experiment 3, in whichneurochemistry and behaviour were simultaneously monitored in the experimental subjectson the test day. In agreement with the results of the previous experiment, exposure of theconditioned subjects to the cocaine-paired environment did not produce an increase in DA,DOPAC or HVA that differed from the pseudoconditioned subjects. This negativeneurochemical finding is directly contrasted by the significantly greater amount oflocomotor behaviour exhibited by the conditioned subjects. Taken as a whole, these datasuggest that there is a clear dissociation between the behavioural and the neurochemical68responses of the two groups of subjects. Both the conditioned and pseudoconditionedgroups exhibited a significant increase in DA, DOPAC and HVA when they were placed inthe conditioning apparatus, suggesting that dopaminergic tone was increased. However, thefinding that this increase did not differ between the groups indicate that this effect isunrelated to the conditioning. One difference between the findings of Experiments 3 and 4is that there was a significant increase in DA in the later. Although the increase in DA wassimilar in both studies (approximately 10% in Experiment 3 and 15% in Experiment 4), thelarger standard error in Experiment 3 resulted in the failure of this effect to reachsignificance. It is also important to note that the magnitude of the increase in DA, or itsmetabolites, is of minor interest, as it is the hypothesized difference between theconditioned and pseudoconditioned subjects that is of primary concern.The present data do not support the hypothesis that conditioned stimuli associatedwith cocaine arouse similar neural states as the drug itself. The first caveat about thisconclusion is that absence of evidence cannot be equated with evidence of absence, and it ispossible that microdialysis is not sufficiently sensitive to detect small and/or transientneurochemical changes associated with cocaine-conditioning. It is also possible that a smallsubset of dopaminergic projections to the accumbens is responsible for the conditionalchange in behaviour, and that these discrete changes are being masked by the surroundingunresponsive neurons. An additional possibility is that, despite the fact that the nucleusaccumbens has been strongly implicated in stimulant-induced locomotor activity (Delfs etal., 1990; Joyce and Koob, 1981; Kelly et al., 1975), dopaminergic projections to otherstructures may also play an important role. Although the present data cannot fully discountthese possibilities, the results of other neurochemical and pharmacological studies, discussedbelow, cast doubt on these alternative interpretations.It is unlikely that microdialysis is not sensitive enough to detect behaviourallyrelevant changes in DA, as data from this laboratory has illustrated that the consumption ofa palatable meal (Nomikos et al., in preparation) and sexual behaviour (Pfaus et al., 1990)69increase interstitial DA in the nucleus accumbens in a robust and reliable fashion.Moreover, a significant increase in DA in the final conditioning experiment was reported.Although this increase was similar in the conditioned and pseudoconditioned groups, it doesillustrate the sensitive nature of the presently employed microdialysis procedure. A similarmagnitude increase in accumbens DA has been reported by Nomikos et al. (in preparation)as a result of transfering a rat from one cage to another. The increase in DA and itsmetabolites observed in the present experiments may also simply reflect this manipulation.The present data are supported by a previous neurochemical study which failed tofind a difference in tissue DA turnover between conditioned and pseudoconditioned subjectsupon presentation of a cocaine conditioned environment (Barr et al., 1983). Otherinvestigators have also failed to find changes in dopaminergic transmission associated withconditional changes in behaviour following conditioning with a variety of agents such asfentanyl (Finlay et al., 1988), morphine (Walter and Kuschinsky, 1989), or apomorphine(Willer et al., 1987). Although Barrett and Nader (1990) have recently concluded that "...there appears to be a reasonable amount of evidence to indicate that neurochemical changescan be conditioned using Pavlovian procedures", important aspects of some of the earlyneurochemical studies that gave rise to this conclusion are flawed. For example, anelevation in tissue HVA levels over control values has been taken as evidence of aconditioned increase in DA metabolism in all of the studies that reported this effect (Lal etal., 1976; Perez-Cruet, 1976; Shiff, 1982). Although tissue concentrations of DA metabolitescan provide an indirect measure of dopaminergic transmission, this is of questionable valuein the absence of concomitant information about DA concentrations (Commissiong, 1985;Westerink, 1985). This limitation aside, an additional factor that limits the value of the dataof both Perez-Cruet (1976) and Lal et al. (1976) is that these studies lacked adequate datademonstrating that their conditioning procedures affected behaviour. Specifically, there wasno evidence that conditioning had occurred. Taken as a whole, the neurochemical evidencefor conditioned dopaminergic activity is weak.70In addition to the absence of convincing neurochemical support for a dopaminergicrole in the cocaine-induced conditional locomotion, a number of investigators have reportedthat while DA receptor antagonists block both the acute unconditioned behavioural effectsof cocaine and the development of environment-specific conditioning, they are much lesseffective in attenuating the conditioned locomotor effects of cocaine and other psychomotorstimulants (Beninger and Hanh, 1983; Beninger and Herz, 1986; Carey, 1992; Weiss et al.,1989). It is noteworthy that pimozide also fails to block the conditioned response to food-paired stimuli (Horvitz and Ettenberg, 1991). It should be noted, however, that someinvestigators have suggested that the mesolimbic DA system is involved in the conditionalresponse to stimulants (Drew and Glick, 1990; Gold et a/., 1988). Gold et al. (1988)demonstrated that the conditioned locomotor response to amphetamine was significantlyattenuated by 6-OHDA lesions of the nucleus accumbens when the lesion was made prior toor following the conditioning procedure. The authors interpret their results to indicate thatthe mesolimbic DA system may be responsible for both the unconditioned and conditionedlocomotor responses to psychomotor stimulant drugs. This conclusion, however, is notjustified given that these lesions also produced significant depletions in noradrenaline.Moreover, the effect of these lesions on catecholamine concentrations in regions other thanthe nucleus accumbens were not reported. Given the lack of specificity of these lesions itcannot be assumed that the attenuation of conditioned locomotion was the result of DAdepletion in the nucleus accumbens. Drew and Glick (1990) have reported that both DI andD2 DA receptor antagonists can attenuate amphetamine-induced conditioned circling.However, the absence of pseudoconditioned or control subjects in this study make itimpossible to determine if conditioning had occured. Taken as a whole, the previouspharmacological results, together with the present in vivo neurochemical data, stronglysuggest that although the development of cocaine-induced environment-specific conditioningis DA -dependent, the neurochemical events associated with the expression of theconditioned response are not.71Given the clinical relevance of conditioned cocaine cues, the elucidation of theneurobiology of this phenomenon is of obvious importance. Furthermore, the availability ofa pertinent animal model makes this a feasible and worthwhile avenue of investigation.Investigations of the role of the amygdala in drug-induced environment-specificconditioning may prove to be particularly fruitful. For example, Post et al. (1988) havereported that although lesions of the amygdala (electrolytic and 6-OHDA) did not affectcocaine-induced hyperactivity they greatly attenuated environment-specific cocainesensitization. Moreover, a role for the amygdala in the development of stimulus-rewardassociations has been demonstrated in a variety of experimental paradigms (Cador et al.,1989; Everitt et al., 1991; Hiroi and White, 1991a; Robbins et al., 1989).72IV. EVIDENCE FOR CONDITIONAL NEURONAL ACTIVATION FOLLOWINGEXPOSURE TO A COCAINE-PAIRED ENVIRONMENT: ROLE OF FOREBRAIN LIMBICSTRUCTURES(A) IntroductionAs discussed previously, the classical conditioning of cocaine's behavioural effectswith specific environmental stimuli is an important aspect of its actions (Barr et al., 1983;Stewart et al., 1984; Tatum and Seevers, 1929). This property of cocaine is of majorsignificance with respect to its abuse potential, as intense craving can be evoked by stimulipreviously associated with the act of taking the drug (Gawin, 1991; Johanson and Fischman,1989; O'Brien et al., 1992). Given the large body of evidence implicating the mesolimbicDA system in the reinforcing properties of drugs of abuse (Di Chiara and Imperato, 1988a;Fibiger and Phillips, 1987; Lyness et al., 1979; Roberts et al., 1977, 1989) it is possible thatconditioned stimuli associated with drug administration may also produce increases indopaminergic transmission (Stewart et al., 1984). However, the data from Chapter III failedto provide evidence for this hypothesis. Specifically, the presentation of an environmentthat had been repeatedly paired with cocaine administration did not produce an increase ininterstitial DA concentrations in the nucleus accumbens that was significantly greater thanthat observed in pseudoconditioned controls. This negative neurochemical finding wassharply contrasted by the significantly greater amount of locomotor behaviour exhibited bythe conditioned subjects. Previous investigators have also reported an absence of evidencefor conditioned dopaminergic activity following conditioning with a variety of agents suchas cocaine (Barr et al., 1983), fentanyl (Finlay et al., 1988), morphine (Walter andKuschinsky, 1989) and apomorphine (Moller et al., 1987). Although the findings of thesestudies indicated an absence of conditional DA release, all found significant conditionedbehavioural changes.73Recent studies have demonstrated that cocaine increases neuronal expression of Fos,the product of the proto-oncogene c-Jos, within the striatum and nucleus accumbens(Graybiel et al., 1990; Young et al., 1991), in agreement with the importance of thesestructures in many of the behavioural properties of cocaine. The transient expression of Fosin response to a variety of physiological and pharmacological manipulations suggests that c-fos induction may be used as a marker of neuronal activation (Dragunow and Robertson,1987; Fu and Beckstead, 1992; Hunt et al., 1987; Morgan and Curran, 1991; Robertson etal., 1989, 1991; Rusak et al., 1990). It has therefore been proposed that Fosimmunohistochemistry might be utilized as a cellular metabolic marker, similar to 2-deoxyglucose (Dragunow and Faull, 1989; Hunt et al., 1987; Morgan and Curran, 1989; Sagaret al., 1988). Given the ability of cocaine to increase Fos expression and the proposed useof this proto-oncogene product as a marker of neuronal activation, the present study utilizedFos immunohistochemistry to determine if environmental stimuli paired with cocaineadministration produce increases in Fos expression in the basal ganglia and various limbicregions.(B) Materials and MethodsSubjects and DrugsSubjects were 28 male Long Evans rats (Charles River, Quebec), weighing 300 - 400g at the beginning of the experiments. The rats were group housed (3 per cage), on a 12-hrlight/12-hr dark cycle (lights on 08:00), with food and water available ad libitum. Allsubjects were handled periodically for one week prior to the experiments. All experimentalprocedures were conducted at approximately the same time each day, during the animals'light phase.74Both cocaine hydrochloride (10 mg/kg, dissolved in isotonic saline , BDH) and 0.9 %saline were injected i.p. in a volume of 1 ml/kg. The dose of cocaine is expressed as theweight of the salt.Apparatus and Behavioural ProcedureFour circular (61 cm diameter) activity cages (BRS/LVE), as described in ChapterIII, were used to measure locomotor activity.The first experiment involved the characterization of the acute effects of cocaine (10mg/kg) and saline on locomotor behaviour and Fos expression in the rat forebrain. Ratswere randomly assigned to one of the activity cages and habituated for 90 min on fourconsecutive days. One day following the final habituation period rats were injected witheither cocaine (n=4) or saline (n=4) and immediately placed into the activity cage, andlocomotor counts were monitored for 90 min. Following the test session, the subjects weredeeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfusedwith isotonic saline followed by 4% paraformaldehyde in phosphate-buffered (0.1 M) saline.The effect of the presentation of an environment that had previously been pairedwith cocaine administration on locomotor behaviour and Fos expression in the rat forebrainwas examined in the second experiment. Rats were randomly assigned to 1 of 3 groups:conditioned (n=7), pseudoconditioned (n=7) or control (n=6). Conditioned subjects wereinjected with cocaine (10 mg/kg, i.p.) and then placed into one of the circular activity cagesfor 30 min. After the training session, subjects were returned to their homecages, wherethey were injected with saline (1 ml/kg, i.p.) 4 hr later. Pseudoconditioned rats wereexposed to an identical procedure except that the order of administration of cocaine andsaline was reversed. Specifically, these subjects were injected with saline prior to beingplaced in the activity cages and later with cocaine in their homecages. Control subjectswere exposed to the same procedure as the other groups, except that they were injected withsaline in both environments and never received cocaine. Each subject was assigned to a75particular locomotor cage for the duration of the experiment. Training was conducted dailyfor 10 consecutive days at approximately the same time each day. On the test day (48 hrafter the final training session), subjects were placed in the locomotor cages (no injectiongiven) and activity was monitored for 90 min. Following the test session, the subjects weredeeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfusedwith isotonic saline followed by 4% paraformaldehyde in phosphate-buffered (0.1 M) saline.Fos ImmunohistochemistryFollowing a 24 - 48 hr postfixative period, 30 pm sections were cut from each brainusing a Vibratome (Pelco). The sections were washed (10 min) three times with 0.02 Mphosphate buffer (PB) before being incubated in phosphate buffered 0.3% hydrogenperoxide for 10 min to remove endogenous peroxidase activity. Sections were then washedthree times in PB and incubated with the Fos primary antisera (1:2000 dilution; alsocontained 0.3% Triton-X and 0.02% Na azide in PB) for 48 hr. A polyclonal sheep antibody(Cambridge Research Biochemicals; CRB 0A-11-823) directed against residues 2 - 16 of theN-terminal region of the Fos protein was used in the present studies. The sections werethen washed three times in PB and incubated with a biotinylated rabbit anti-sheep secondaryantibody (Vector Laboratories; 1:500 dilution; also contained 0.3% Triton-X in PB) for 1 hr.Sections were washed three times in PB before being incubated with 0.3% Triton-X and0.5% avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) in PB for 1hr. Sections were then washed two times with PB and once with an acetate buffer (0.1 M,pH 6.0) before being visualized by the glucose oxidase-DAB-nickel method (Shu et al.,1988). The reaction was terminated by washing the sections in acetate buffer (0.1 M, pH6.0). Sections were mounted on chrome-alum coated slides, dehydrated and prepared formicroscopic examination. A more detailed description of this technique has been publishedpreviously (Robertson and Fibiger, 1992). It should be noted that immunoblot analysis(CRB technical data sheet) indicates that the CRB antibody recognizes Fos (62 kDa) as well76as other Fos-related antigens (48/49 and 70 kDa). However, the delayed onset of the Fos-related antigens (Morgan and Curran, 1989) would suggest that Fos is responsible for theimmunoreactivity quantified in the present study. The time chosen for perfusion (90 minafter the initiation of all experimental manipulations) approximately coincides with the peaklevels of Fos antigens (Morgan and Curran, 1989).Quantification of Fos Positive CellsFos expression within the cingulate cortex, claustrum, piriform cortex, nucleusaccumbens, dorsomedial striatum, lateral septal nucleus and paraventricular nucleus of thethalamus was quantified by counting the number of Fos-positive nuclei within a 510 x 510pm grid placed over the area at 107 X magnification. The number of Fos-positive nucleiwithin the amygdala was also quantified; the grid for these counts was 1290 x 1290 pm at 43X magnification. Camera lucida drawings illustrate the specific regions sampled and APposition of the sections used (Figure 16). In addition to the aforementioned regions, thenumber of Fos-positive nuclei within the lateral habenula was also examined. However, dueto rostro-caudal variation of the sections available from this region it was not deemedappropriate to quantify the data in the same manner as was used for the other regions. Toincrease the reliability of the quantification, cell counts were made from two separatesections of each region from a given subject. In addition, duplicate counts of each sectionwere conducted by two independent observers. This procedure, therefore, resulted in a totalof four determinations of the number of Fos-positive nuclei within a specified region foreach subject. The average of these four determinations was utilized for the subsequentstatistical analysis.77Statistical AnalysisBetween group differences in locomotor counts and the number of Fos-positivenuclei within specified regions from the conditioning experiment subjects were evaluatedusing univariate analyses of variance. Post hoc comparisons between control,pseudoconditioned and conditioned subjects were conducted using Tukey's HSD. Univariateanalyses of variance were also conducted to assess locomotor differences and the number ofFos-positive nuclei within specified regions between acute saline- and cocaine-treatedsubjects.(C) ResultsThe brain areas quantified in the present study reflect those areas that weredetermined to have positive responses to the acute administration of cocaine or thepresentation of the cocaine-paired environment. These initial observations were performedby a "blind" observer who scanned coronal sections throughout the forebrain to determineregions that exhibited increased Fos-immunoreactivity. No other forebrain regions exhibitedreliable responses to the present treatments.As expected, compared to saline treated controls the acute administration of cocaineproduced a significant increase in locomotor counts [F(1,6)=45.42, p<0.005] (Figure 17).This behavioural effect was accompanied by significant increases in the number of Fos-positive nuclei in the cingulate cortex [F(1,6)=10.69, p<0.05], claustrum [F(1,6)=50.26,p<0.001], piriform cortex [F(1,6)=49.98, p<0.001], nucleus accumbens [F(1,6)=82.81,p<0.001], dorsomedial striatum [F(1,6)=40.83, p<0.005], lateral septum [F(1,6)=134.32,78Figure 16. Camera lucida drawings of representative sections used for the counting of Fos-positive nuclei in the cingulate cortex (1), claustrum (2), piriform cortex (3), nucleusaccumbens (4), dorsomedial striatum (5), lateral septum (6), paraventricular nucleusof the thalamus (7) and the amygdala (8). The area of quantification for areas I - 7was 510 x 510 pm. The quantification of the amygdala (8) encompassed a 1290 x1290 pm area.7980Figure 17. Total number of locomotor counts for control (n=6), pseudoconditioned (Cs - ,n=7) and conditioned (Cs+ , n=7) subjects following 10 days of conditioning withcocaine (10 mg/kg, i.p.). Testing occurred 48 hr after the last training session, andwas conducted in the same apparatus as used for the training procedure. Locomotorcounts are also shown for unconditioned subjects that received acute injections ofsaline (1 ml/kg, n=4) or cocaine (10 mg/kg, n=4). Values represent the group mean± SEM. * P < 0.005 compared to pseudoconditioned controls. f P < 0.005compared to saline treated controls.Control^Cs-^Cs+^Saline Cocaine*t1600 --,=° 1400 -0 1200 -o)co 1000 -c^-=o 800 -00 600 -4 ;E 400 -ooo 200 -J■08182Figure 18. Number of Fos-positive nuclei within a 510 x 510 pm area in the cingulatecortex, claustrum, piriform cortex and nucleus accumbens for control (n=6),pseudoconditioned (Cs - , n=7), conditioned (Cs + , n=7), saline treated (n=4) andcocaine treated (n=4) subjects. Values represent the group mean + SEM. * P < 0.05compared to pseudoconditioned controls. f P < 0.05 compared to saline treatedcontrols.83110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -01101009080706050403020100Nucleus AccumbenstI Claustrum Control Cs—^Cs+^Saline CocaineCingulate CortextControl Cs-^Cs+^Saline CocainePiriformCortexControl cs-tCs+^Saline CocainetSaline Cocaine—o0z00_0U-00z1101009080706050403020100—o0z0-440C/)0LL0L0Ez110100908070605040302010084Figure 19. Number of Fos-positive nuclei within a 510 x 510 pm area in the dorsomedialstriatum, lateral septum and paraventricular nucleus of the thalamus and within a1290 x 1290 pm area in the amygdala for control (n=6), pseudoconditioned (Cs - ,n=7), conditioned (Cs+ , n=7), saline treated (n=4) and cocaine treated (n=4) subjects.Values represent the group mean ± SEM. * P < 0.05 compared topseudoconditioned controls. f P < 0.05 compared to saline treated controls.851101009080706050403020100Lateral Septum1101009080706050403020100Dorsomedial Striatumt T C s+Control CC Saline Cocaine Control Cs—^Cs+^Saline Cocaine*Tt110 -100 -90 -80 -.70 -.60 -..50 740 -.30 -_,20 -.10 -0110 -100 -90 -80--70  -60 -50 -40 -30 -20 -10 -0ThalamicParaventricularNucleust*^IControl CC^Cs+^Saline CocainelAmygdala*Control CS-^CS+^Saline Cocaine86p<0.001], paraventricular nucleus of the thalamus [F(1,6)=80.46, p<0.001] and amygdala[F(1,6)=28.3, p<0.005], as compared to saline treated controls (Figures 18, 19, 20 and 21).The conditioning procedure employed in the present study produced a clearconditional behavioural effect [F(2,17)=10.35, p<0.005] (Figure 17). Post hoc comparisonsdemonstrated that conditioned subjects exhibited significantly more locomotor counts thaneither control or pseudoconditioned controls (p<0.05). This conditioned effect stands incontrast to the lack of a significant group difference between the pseudoconditioned andcontrol subjects (Figure 17).In addition to producing a significant behavioural effect, the conditioning procedureresulted in increased Fos expression within specific brain regions (Figures 18, 19, 20 and21). Significant group effects were observed for the cingulate cortex [F(2,17)=14.19,p<0.0005], claustrum [F(2,17)=12.56, p<0.0005], lateral septal nucleus [F(2,17)=6.05, p<0.02],paraventricular nucleus of the thalamus [F(2,17)=32.12, p<0.0001] and amygdala[F(2,17)=8.33, p<0.005]. Post hoc comparisons revealed that conditioned subjects exhibitedsignificantly more Fos-positive nuclei than control or pseudoconditioned subjects in thecingulate cortex, claustrum, lateral septal nucleus, paraventricular nucleus of the thalamusand amygdala (p<0.05), while no differences were observed between control andpseudoconditioned subjects (Figures 18 and 19). In contrast, the number of Fos-positivenuclei within the nucleus accumbens [F(2,17)=1.51], dorsomedial striatum [F(2,17)=1.04] andpiriform cortex [F(2,17)=1.29] did not differ significantly between the control,pseudoconditioned or conditioned subjects.Although the conditioned subjects exhibited an enhanced locomotor response, themagnitude of this effect was considerably less than that observed following an acuteinjection of cocaine [F(1,9)=18.68, p<0.005] (Figure 17). In accordance with this behaviouraldifference, conditioned subjects exhibited significantly fewer Fos-positive nuclei thananimals treated acutely with cocaine in the cingulate cortex [F(1,11)=11.29, p<0.01],claustrum [F(1,11)=40.45, p<0.001], piriform cortex [F(1,11)=46.38, p<0.001], nucleusFigure 20. Photomicrographs of Fos-immunoreactivity in the cingulate cortex fromrepresentative saline (A) and cocaine (B) treated subjects, as well aspseudoconditioned (C) and conditioned (D) subjects. Scale Bar = 100 pm.87Figure 21. Photomicrographs of Fos-immunoreactivity in the lateral habenula fromrepresentative saline (A) and cocaine (B) treated subjects, as well aspseudoconditioned (C) and conditioned (D) subjects. Scale Bar = 100 pm.8991accumbens [F(1,11)=44.89, p<0.001], dorsomedial striatum [F(1,11)=37.82, p<0.001],paraventricular nucleus of the thalamus [F(1,11)=20.61, p<0.005] and amygdala[F(1,11)=22.66, p<0.005] (Figures 18 and 19). Interestingly, the number of Fos-positivenuclei within the lateral septal nucleus was equivalent between the conditioned and cocainetreated subjects [F(1,11)=0.13] (Figure 19).It is noteworthy that the number of Fos-positive nuclei within the cingulate cortex[F(1,8)=1.23], claustrum [F(1,8)=0.02], piriform cortex [F(1,8)=0.01], nucleus accumbens[F(1,8)=0.58], dorsomedial striatum [F(1,8)=2.89], lateral septal nucleus [F(1,8)=0.01],paraventricular nucleus of the thalamus [F(1,8)=0.08] or amygdala [F(1,8)=1.08] were notsignificantly different between control subjects from the conditioning experiment andsubjects acutely injected with saline (Figures 18 and 19).In addition to the aforementioned regions, compared to saline treated controlscocaine produced an increase in the number of Fos-positive nuclei in the medial portion ofthe lateral habenula (Figure 21). Conditioned subjects also exhibited a large and robustincrease in Fos expression within this area, while virtually no Fos-positive nuclei wereobserved within this region of the pseudoconditioned controls. Due to variations in theanterior-posterior coordinates of the sections obtained from this region, reliablequantification was not possible. However, all of the acute cocaine and conditioned subjectsthat were examined displayed reliable increases in Fos expression, while none of the control,pseudoconditioned or saline treated subjects exhibited substantial Fos expression in thisstructure.92(D) DiscussionIn agreement with earlier studies (Graybiel et al., 1990; Young et al., 1991), acuteadministration of cocaine elevated Fos-immunoreactivity in the striatum and nucleusaccumbens. In addition, cocaine enhanced Fos-immunoreactivity in the cingulate cortex,claustrum, piriform cortex, lateral septal nucleus, paraventricular nucleus of the thalamus,medial portion of the lateral habenula and amygdala. The results of the second experimentconfirmed that the unconditioned locomotor stimulatory effects of cocaine can be classicallyconditioned to environmental stimuli (Barr et al., 1983; Beninger and Herz, 1986; Tatum andSeevers, 1929). Moreover, this conditioning was accompanied by changes in Fosimmunoreactivity within specific limbic regions, suggesting a conditional increase inneuronal activity within these areas and that these limbic regions are involved in mediatingthe conditioned behaviour.The ability of cocaine to increase Fos expression in the striatum and nucleusaccumbens is thought to be mediated through the activation of D1 DA receptors (Graybiel etal., 1990; Young et al., 1991). This finding may be anticipated from the ability of cocaineto increase dopaminergic transmission (Chapters II and III; Di Chiara and Imperato, 1988a;Pettit and Justice, 1991) and the heavy dopaminergic innervation of the nucleus accumbensand striatum (BjOrklund and Lindvall, 1984). The present results illustrate that cocaine alsoproduces increases in Fos in the cingulate cortex, claustrum, piriform cortex, lateral septalnucleus, paraventricular nucleus of the thalamus, medial portion of the lateral habenula andamygdala. Although each of these structures receive at least a modest dopaminergicinnervation (B jOrklund and Lindvall, 1984), it is possible that the increase in Fos-positivenuclei within some or all of these regions is not dopaminergically mediated. Specifically,cocaine blocks the uptake of noradrenaline and serotonin, in addition to its well studiedactions on DA (Richelson and Pfenning, 1984; Ritz et al., 1987). Furthermore, the presenceof noradrenergic and serotonergic projections to many of these areas precludes the93assumption that the observed effects were dopaminergically mediated (Moore and Card,1984; Steinbusch, 1984). It is noteworthy that a recent study has provided evidence thatclozapine-induced increases in Fos expression within the nucleus accumbens are DA-dependent, while increases in the cingulate cortex and lateral septum are not (Robertson andFibiger, 1992). Finally, it is also possible that cocaine-induced activation of Fos expressionin some areas was one or more synapses "downstream" from one of the primary sites ofaction of cocaine. Studies utilizing selective neurotoxins and receptor antagonists would helpelucidate the neurotransmitter(s) involved in the cocaine-induced increases in Fos expressionwithin these limbic regions.In agreement with previous studies, the results from the second experimentdemonstrate that the behavioural effects of cocaine can be classically conditioned toenvironmental stimuli (Barr et al., 1983; Beninger and Herz, 1986; Tatum and Seevers,1929). The classical conditioning of a drug to environmental cues is not unique to cocaineand has been reported with other stimulants, as well as with opiates (Beninger and Hahn,1983; Carey, 1992; Willer et al., 1987; Stewart et al., 1984; Walter and Kuschinsky, 1989).The clinical significance of this drug-environment conditioning is considerable, asconditioned cues can evoke intense cravings, which play a substantial role in the relapse intococaine use (Gawin, 1991; O'Brien et al., 1992).Cocaine-induced conditioned locomotion was accompanied by a conditional increasein Fos expression within specific limbic nuclei. Given the proposed use of Fosimmunohistochemistry to map functional pathways in the brain (Dragunow and Faull, 1989;Hunt et al., 1987; Morgan and Curran, 1989; Sagar et al., 1988) and the ability of variousbehavioural and physiological manipulations to increase c-fos expression in the centralnervous system (Campeau et al., 1991; Chastrette et al., 1991; Hunt et al., 1987; Rusak etal., 1990; Sharp et al., 1991) the present findings may reflect at least a portion of the nucleiand regions involved in the conditioned response. It is noteworthy that a recent report hasalso demonstrated a conditioned activation of c-fos. Campeau et al. (1991) have reported94that conditioned fear produces dramatic increases in c-fos expression within the amygdala, aresult supported by previous electrophysiological findings (Applegate et al., 1982; Pascoe andKapp, 1985).The claustrum or orbitofrontal area and the cingulate cortex, both components of theprefrontal cortex (Groenwegen, 1988), exhibited an increase in Fos expression in conditionedsubjects when they were exposed to the environment in which they had previously receivedcocaine. This finding is fully compatible with previous electrophysiological and lesionstudies that demonstrate the importance of the cingulate cortex in classical conditioning(Buchanan and Powell, 1982; Gabriel et al., 1980; Gabriel and Sparenborg, 1987; Powell etal., 1990). Moreover, the diverse afferent connections of the prefrontal cortex from limbic,as well as sensory areas, allow for the potential integration of multimodal sensoryinformation necessary to produce the associations involved in conditioning (Groenwegen etal., 1990; Lopez da Silva, 1990). Although both the acute administration of cocaine and thepresentation of the cocaine-paired environment produced increases in Fos-positive nuclei inthe claustrum and cingulate cortex, it is not possible to ascertain from the present data ifthese different treatments produce their effects by the activation of the same afferentprojections.Conditional Fos expression was also observed in the lateral septal nucleus.Interestingly, this was the only area examined where the magnitude of the conditionedresponse was equivalent to the acute drug effect (Figure 19). This unique response maysuggest a primary role for the lateral septum in the conditioned behaviour. With afferentsfrom the amygdala, ventral tegmental area, and hippocampus and major efferent pathwaysto the mammillary bodies, lateral hypothalamus and medial septum, the lateral septumexhibits connectivity that is consistent with its role in the formation of associations betweenaffective states and environmental stimuli (Krettek and Price, 1978; Raisman, 1966; Swansonand Cowan, 1979). Consistent with this proposal, electrophysiological studies havedemonstrated that the activity of neurons in the lateral septal nucleus is sensitive to the95presentation of conditioned stimuli (Thomas, 1988; Thomas et al., 1991; Thomas and Yadin,1980). Based on these and other findings, it has been hypothesized that the lateral septum isinvolved in the inhibition of aversive affective states (Thomas, 1988). In view of thepresent findings, future examination of the potential role of this structure in appetitiveclassical conditioning is warranted.Given the large body of data suggesting that various amygdaloid nuclei participate inPavlovian conditioning (Applegate et al., 1982; Dunn and Everitt, 1988; Kapp et al., 1981;Mishkin and Aggleton, 1981; Pascoe and Kapp, 1985; Roozendaal et al., 1990), it is notsurprising that a conditional increase in Fos-positive nuclei was observed within thisstructure in the present study. These data are also consistent with the proposed role of theamygdala in the development of stimulus-reward associations, as demonstrated in a varietyof experimental paradigms (Cador et al., 1989; Everitt et al., 1991; Hiroi and White, 1991a;Robbins et al., 1989). Furthermore, Post et al. (1988) have reported that although lesions ofthe amygdala (electrolytic and 6-OHDA) did not affect cocaine-induced hyperactivity theygreatly attenuated environment-specific cocaine sensitization. Given these previousfindings, the present results are fully consistent with the proposed role of the amygdala inconditioning in general, and in stimulus-reward associations in particular.One shortcoming of the present data is that Fos-immunoreactivity was not localizedto specific amygdaloid nuclei. Although the area of quantification was centered around thecentral and basolateral nuclei, the present histological material was not suitable for theunequivocal delineation of the boundaries of the amygdaloid nuclei. Given the anatomicaland physiological diversity of the specific amygdaloid nuclei, future studies should examinethe role of particular nuclei.Two additional areas that exhibited a conditional increase in Fos-immunoreactivitywere the lateral habenula and the paraventricular nucleus of the thalamus. Although theabsence of quantifiable data precludes any strong conclusions being put forward regardingthe effect observed in the lateral habenula, it is noteworthy that it has recently been96reported that the medial portion of the lateral habenula exhibits an increase in Fos-immunoreactivity in response to restraint stress (Chastrette et al., 1991). Interestingly, thedistribution of Fos-positive nuclei observed in this study (Figure 21) and that of Chastretteet al. (1991) corresponds to the dopaminergic innervation of the lateral habenula (Phillipsonand Pycock, 1982). Given the proposed role of this nucleus in a feedback loop from thefrontal cortex to the ventral tegmental area (Lisoprawski et al., 1980; Phillipson and Pycock,1982) and its purported significance in motivated behaviour (Sutherland, 1982), furtherexamination of its role in conditioned behaviour is warranted.A variety of stressors have been shown to increase Fos-immunoreactivity in theparaventricular nucleus of the thalamus (Chastrette et al., 1991; Sharp et al., 1991). Thepresent data indicate that this midline thalamic nucleus is also responsive to the presentationof an appetitive conditioned stimulus. Although the functional significance of theparaventricular nucleus is unclear, it is apparent that it is not a "nonspecific" nucleus(Bentivoglio et al., 1991). The dopaminergic innervation of the paraventricular nucleus andits dense efferent projections to the amygdala, cingulate cortex and the nucleus accumbensare consistent with a "limbic" role for this midline thalamic nucleus and are in agreementwith its hypothesized role in learning and memory (Bentivoglio et al., 1991).Although the conditional activation of Fos expression was observed within many ofthe regions that were affected by acute cocaine, the nucleus accumbens, striatum andpiriform cortex did not exhibit an increased number of Fos-positive nuclei in theconditioned subjects. The absence of conditional Fos expression within the nucleusaccumbens supports our results from Chapter II that demonstrated that cocaine-inducedconditioned locomotion is not accompanied by an increase in DA release in this structure.Despite the fact that the nucleus accumbens has been strongly implicated in stimulant-induced locomotor activity (Delfs et al., 1990; Joyce and Koob, 1981; Kelly et al., 1975),dopaminergic projections to other limbic structures may play a critical role in environment-specific conditioned locomotion. This possibility is consistent with the conditional increase97in Fos expression in the aforementioned limbic structures, such as the cingulate cortex,lateral septum and amygdala, that have been demonstrated to participate in classicalconditioning and to receive dopaminergic innervation (B jOrklund and Lindvall, 1984).Although a number of investigators have reported that DA receptor antagonists areineffective in attenuating the conditioned locomotor effects of cocaine and otherpsychomotor stimulants (Beninger and Hahn, 1983; Beninger and Herz, 1986; Weiss et al.,1989), the antagonists used in these studies are primarily directed against the D2 receptor,leaving the possibility that DA could be acting through D1 receptors.Irrespective of the potential dopaminergic involvement in the conditional behaviouraleffect, the present data indicate that the neurobiological substrates for a conditionedresponse can differ from those of the unconditioned response. As noted previously, thedopaminergic projection to the nucleus accumbens appears to be necessary for theunconditioned locomotor effects of cocaine (Delfs et al., 1990; Joyce and Koob, 1981; Kellyet al., 1975) and the acute reinforcing effects of cocaine and other psychomotor stimulants(Lyness et al., 1979; Roberts et al., 1977, 1980; Pettit et al., 1984). However, the presentfindings fail to provide evidence of increased Fos expression in the nucleus accumbensassociated with the conditioned response. This absence of effect occurs in spite of the largenumber of other regions that displayed a conditioned response and the finding that thisnucleus exhibits a robust increase in Fos expression in response to cocaine itself. This resultis in general agreement with previous neurochemical studies that have reported no evidencefor an increase in dopaminergic transmission in the nucleus accumbens in response to thepresentation of a cocaine-paired environment (Chapter III; Barr et al., 1983). The presentresults, together with the previous neurochemical data, strongly suggest that although thedevelopment of cocaine-induced environment-specific conditioning is dependent on thedopaminergic projection to the nucleus accumbens, the expression of the conditionedresponse is not. Taken as a whole, it appears that cocaine-induced environment-specificlocomotion is not simply reflective of an increase in dopaminergic transmission to the98nucleus accumbens, but is associated with increased neuronal activation within variousforebrain limbic structures known to be critically involved in emotion and learning.Consequently, the results of the present study suggest that this specific form of conditioninginvolves similar neural circuits as other forms of learning (Mishkin and Aggleton, 1981).99V. DIFFERENTIAL EFFECTS OF EXCITOTOXIC LESIONS OF THE AMYGDALA ONCOCAINE-INDUCED CONDITIONED LOCOMOTION AND CONDITIONED PLACEPREFERENCE(A) IntroductionA significant clinical feature of cocaine abuse is the occurrence of environmentallycued craving (Gawin, 1991; O'Brien et al., 1992). These conditioned cravings are theproduct of the repeated pairing of objects, places or events with the administration ofcocaine and its subsequent euphoric effects. This clinical observation is supported by anumber of laboratory studies that have demonstrated that cocaine's behavioural effects canreadily become classically conditioned with specific environmental stimuli (Chapter III andIV; Barr et al., 1983; Stewart et al., 1984; Tatum and Seevers, 1929). Given the prevalenceof these conditioned responses and their clinical significance, a better understanding of theneurobiology of this phenomenon may assist in the development of more rational treatmentsfor cocaine abuse.Although there is a large body of evidence implicating the mesolimbic DA system inthe acute rewarding properties of cocaine (Di Chiara and Imperato, 1988a; Fibiger andPhillips, 1987; Roberts et al., 1977, 1989; Ritz et al., 1987), the conditioned responses tococaine and other psychomotor stimulants is apparently DA-independent (Beninger andHahn, 1983; Beninger and Herz, 1986; Weiss et al., 1989; Carey, 1992). In Chapter III itwas demonstrated that there was no evidence for a conditional increase in DA release in thenucleus accumbens in response to the presentation of a drug paired environment. This isconsistent with an absence of evidence for conditioned dopaminergic activity followingconditioning with a variety of agents such as cocaine (Barr et al., 1983), fentanyl (Finlay etal., 1988), morphine (Walter and Kuschinsky, 1989) and apomorphine (MOIler et al., 1987).100Although the findings of these studies indicated an absence of conditional DA release, allfound significant conditioned behavioural changes.In Chapter IV it was reported that exposure to a cocaine-paired environmentproduces a conditional increase in c-fos expression in various forebrain limbic regions, suchas the cingulate cortex, claustrum, lateral septal nucleus and the amygdala. In agreementwith our previous neurochemical findings, no conditional activation was observed in thenucleus accumbens. Taken as a whole these data suggested that cocaine-induced conditionedlocomotion is associated with increased neuronal activation within various forebrain limbicstructures known to be involved in emotion and learning (Powell et al., 1990; Thomas et al.,1991; Pascoe and Kapp, 1985; Lopez da Silva et al., 1990; Davis, 1992). Consequently, it ispossible that this specific form of classical conditioning involves similar neural circuits asother forms of learning (Mishkin and Aggleton, 1981).Of the areas exhibiting an increase in Fos-positive neurons, the amygdala may be ofparticular importance. A large body of evidence suggests that amygdaloid nuclei areinvolved in stimulus-reward associations (Weiskrantz, 1956; Jones and Mishkin, 1972;Mishkin and Aggleton, 1981; Gaffan and Harrison, 1987; Cador et al., 1989; Everitt et al.,1991; Hiroi and White, 1991a; Kentridge et al., 1991). Furthermore, Post et al. (1988) havereported that although lesions of the amygdala (electrolytic and 6-OHDA) do not affectcocaine-induced hyperactivity, they greatly attenuate environment-specific cocainesensitization. Given these findings, the present study investigated the role of the amygdalain cocaine-induced conditional locomotion and cocaine-induced CPP. As previous studieshave demonstrated that damage to fibres of passage within the amygdaloid complex producesbehavioural effects that are unrelated to destruction of the amygdala (Riolobos and Garcia,1987; Dunn and Everitt, 1988), lesions were made using the fibre-sparing excitotoxinquinolinic acid.(B) Materials and MethodsSubjects and DrugsSubjects were 66 male Long Evans rats (Charles River, Quebec), weighing 270 - 310g at the beginning of the experiments. The rats were group housed (3 per cage), on a 12-hrlight/12-hr dark cycle (lights on 08:00), with food and water available ad libitum. Allsubjects were handled periodically for one week prior to surgery. All experimentalprocedures were conducted at approximately the same time each day, during the animals'light phase.Both cocaine hydrochloride (10 mg/kg, dissolved in isotonic saline , BDH) and 0.9 %saline were injected i.p. in a volume of 1 ml/kg. The dose of cocaine is expressed as theweight of the salt.Surgical ProcedureSubjects were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.; BDH) andxylazine (5 mg/kg, i.p.; Haver) and mounted in a stereotaxic frame (incisor bar: + 5.0 mm;David Kopf Instruments). Bilateral lesions of the amygdala were produced by the infusionof 0.5 pi of quinolinic acid (0.12 M, in phosphate buffer, pH 7.1 - 7.4; RBI) at each of fourinjection sites (AP: +0.2 and -0.8 mm; ML: ±4.7 mm; DV: -7.9 from dura; relative tobregma; Pellegrino et al., 1979). Control subjects received 0.5 pl infusions of phosphatebuffer (0.12 M, pH 7.4). Infusions were made through 30-gauge stainless steel cannulaeattached to pump driven (Harvard Apparatus) 5 pl syringes (Hamilton) by PE-10 tubing(Clay Adams). All infusions were made at a rate of 0.1 pl/min, and cannulae remained atthe injection site for an additional 5 min following the injection to allow for the diffusionof the excitotoxin. Solutions of quinolinic acid were prepared immediately prior to eachinfusion to ensure the potency of the excitotoxin. Following the removal of the cannulae,topical antibiotic (Rifocin, Lepetit) was applied to the wound and the incision was sutured101102with 4-0 silk. To reduce potential post-operative hypophagia and hypodipsia, the liquid dietSustacal (Mead Johnson) was made available to all subjects for 7 - 10 days followingsurgery. During the 3 - 4 week post-operative recovery period subjects were handled daily.Subjects that exhibited spontaneous seizures or failed to resume normal feeding weresacrificed with sodium pentobarbital (100 mg/kg, i.p.).ApparatusSix circular (61 cm diameter) activity cages (BRS/LVE), as described in Chapter III,were used to measure locomotor activity during the training and testing of subjects in thecocaine-induced conditioned locomotion experiment.Place preference conditioning was conducted in the same four identical shuttle boxesas utilized in the experiments in Chapter II.ProcedureThe effect of excitotoxic lesions of the amygdala on the classical conditioning of thelocomotor stimulant effects of cocaine to a specific environment was examined in the firstexperiment. Both lesion and control rats were randomly assigned to 1 of 3 groups:conditioned, pseudoconditioned or control. Subjects were conditioned according to the sameprocedure as described in Chapter II, except that on the test day (48 hr after the finaltraining session), subjects were placed in the locomotor cages and activity was monitored for30 min.Following completion of the conditioned locomotion experiment, both the lesionedand non-lesioned control (i.e. drug naive) subjects were tested for cocaine-induced CPP.The procedure for this experiment was identical to that utilized in Chapter II, except thatthe dose of cocaine utilized was 10 mg/kg, i.p.103HistologyFollowing the completion of the second experiment, the subjects were deeplyanaesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused withisotonic saline followed by 4% paraformaldehyde in phosphate-buffered (0.1 M) saline.Following the perfusion, brains remained in 4% paraformaldehyde for 24 - 48 hr beforebeing transferred to a solution of 10% dimethylsulfoxide and 0.02% Na-azide in Naphosphate buffer (0.1 M) for 48 - 72 hr. Fifty pm coronal sections were cut on a freezingmicrotome and every fourth section was mounted on chrome-alum coated slides and stainedwith cresyl violet.The extent of the quinolinic acid lesions was examined microscopically. Subjectsthat were found to possess incomplete or misplaced lesions were excluded (n=5).Statistical AnalysisDifferences in locomotor counts during the conditioning trials were evaluated using athree-way (group x lesion x trial) analysis of variance with repeated measures (Huynh-Feldtadjustment of degrees of freedom). Separate univariate analyses were also performed toevaluate potential group differences between and within lesioned and non-lesioned subjects.Reverse Helmert contrasts were performed to further assess potential group differences.The conditioned locomotion results were evaluated using a two-way analysis (group xlesion). Additional univariate analyses were performed on the lesioned and non-lesionedsubjects to evaluate potential group differences. These analyses also included ReverseHelmert group contrasts to further assess these data. Post-hoc comparisons were made usingScheffe's procedure. Paired t-tests were used to assess the time spent in the drug pairedenvironment before and after place preference conditioning. These within-subjectcomparisons are justified, given that the preconditioning values for the time spent on thenon-start side did not differ between the two groups [t(21) = 0.99, n.s.]. All statisticalanalyses were performed using SPSS:X version.3 software (SPSS:X User's Guide, 1988).(C) ResultsHistologyQuinolinic acid lesions produced extensive damage to the amygdaloid complex(Figure 22). A schematic representation of coronal sections from a representative lesionedsubject are shown in Figure 23. Only subjects that exhibited extensive bilateral amygdalalesions were included in this study. The most rostral portion of the lesion generallyincluded the anterior amygdaloid area, with some subjects exhibiting damage to thesubstriatal region and the ventral endopiriform nucleus. Generally, the central, lateral,basolateral and basomedial nuclei were almost completely destroyed in all subjects.However, the medial and cortical nuclei were partially spared in some subjects. As a resultof the magnitude of these lesions, most subjects exhibited a degree of extra-amygdalardamage. Due to their close proximity to the amygdala, the dorsal and ventral endopiriformnuclei were damaged in almost all rats. A small number of subjects exhibited damage to theventral portion of the caudate putamen. Many lesioned subjects also displayed damage tothe piriform cortex. No subject included in the study appeared to possess damage to thehippocampus.In addition to the extensive gliosis produced by the quinolinic acid lesions, manysubjects exhibited enlargement of the inferior horn of the lateral ventricle. This result mayhave been due to liquifactive necrosis as a consequence of neuronal destruction. In apreliminary study, these ventricular enlargements were not observed in lesioned subjects thatwere perfused 2 - 3 weeks after surgery. This finding suggests that the 2 month intervalbetween surgery and histology may have contributed to this result. In agreement with otherinvestigators (Bermudez-Ratoni and McGaugh, 1991; Cahill and McGaugh, 1990; Jellestadand Cabrera, 1986), limited lesion-induced cavities were present in some subjects. Thisfinding may also be a consequence of the long interval between surgery and histology, assubjects that were perfused 2 - 3 weeks after the infusion of quinolinic acid did not exhibit104105Figure 22. Photomicrogragh of the amygdaloid region of a representative subject followinginfusions of quinolinic acid. This 50 pm section is approximately 2.8 mm posteriorto bregma, according to the atlas of Paxinos and Watson (1986). Scale bar = 1 mm.106107Figure 23. Schematic of a representative bilateral lesion of the amygdala following infusionsof quinolinic acid. The stipled area represents the area of neuronal loss. The valuesto the upper right of each coronal section indicate the anterior/posterior distancefrom bregma, according to the altar of Paxinos and Watson (1986).-2.30- 3.80108-2.80-1.80 -3.30this histological feature. An additional factor worth noting is that quinolinic acid wasprepared fresh before each infusion. Preliminary findings suggested that this procedureproduced greater neurotoxicity than when toxin was prepared prior to surgery.Conditioned LocomotionAmygdala lesions did not alter basal or cocaine-induced locomotion as no significantdifference in locomotor counts was observed between lesioned and non-lesioned subjects forany of the experimental groups [F(1,60)=0.09, n.s.] (Figure 24). Moreover, individualunivariate analyses demonstrated that locomotor counts did not differ between lesioned andnon-lesioned subjects that received saline, such as the control [F(1,21)=0.75, n.s.] orpseudoconditioned [F(1,19)=0.00, n.s.] groups, or the conditioned subjects that receivedcocaine [F(1,20)=0.01, n.s.]. However, a significant group effect was observed [F(2,60)=90.34, p < 0.001]. This group effect was due to the cocaine-induced locomotor counts ofthe conditioned subjects, as a Reverse Helmert analysis of this group effect revealed thatthere was no significant difference between the control and pseudoconditioned groups[F(1,60)=0.01, n.s.], although a highly significant difference between these control groupsand the conditioned group [F(1,60)=180.67, p < 0.001] was present. A nonsignificant groupx lesion interaction [F(2,60)=0.03, n.s.] further illustrates that the amygdala lesions failed toreliably alter locomotor activity. Although no significant effect of trials was observed[F(6.78, 407.01)=0.79, n.s.], the group x trial interaction was significant [F(13.57.,401.01)=67.79, p < 0.001]. Separate univariate analyses for each of the three groupsdemonstrated that this effect was due to a significant decrease in locomotor counts overtrials for the control [F(6.37, 133.79)=17.48, p < 0.001] and pseudoconditioned [F(8.73,165.91)=20.63, p < 0.001] groups. The conditioned subjects just failed to exhibit asignificant increase in locomotor counts over trials [F(8.06, 161.18)=1.92, p < 0.06].The conditioning procedure produced a notable conditional behavioural effect, asindicated by a significant group effect [F(2,60)=20.87, p < 0.001] (Figure 25). A Reverse109110Helmert contrast further indicated that there was no significant difference between thecontrol and pseudoconditioned groups [F(1,60)=0.46, n.s.], but that the conditioned groupshad significantly greater locomotor counts than other groups [F(1,60)=41.29, p < 0.001].This behavioural effect was not affected by amygdala lesions as there was no significanteffect of lesion [F(1,60)= 0.12, n.s.] or group x lesion interaction [F(2,60)=0.60, n.s.].Individual analyses demonstrated that amygdala lesions did not affect locomotor counts forcontrol [F(1,21)=2.19, n.s.], pseudoconditioned [F(1,19)=0.02, n.s.] or conditioned[F(1,20)=0.07, n.s.] subjects. Moreover, separate univariate analyses demonstrated thatsignificant group effects were apparent for both non-lesioned [F(2,27)=17.71, p < 0.001] andlesioned [F(2,33)=7.162, p < 0.005] subjects. In addition, Reverse Helmert contrastsdemonstrated that control and pseudoconditioned groups do not differ significantly fromeach other for either non-lesioned [F(1,27)=1.27, n.s.] or lesioned [F(1,33)=0.04, n.s.]subjects, while conditioned subjects exhibited significantly more locomotor counts than thesecontrols for both non-lesioned [F(1,27)=34.15, p < 0.001] and lesioned [F(I,33)=14.33, p <0.001] subjects. Post-hoc comparisons revealed that both non-lesioned and lesionedconditioned subjects exhibited significantly more locomotor counts than either control orpseudoconditioned subjects (p < 0.05; Figure 25).Conditioned Place PreferenceNon-lesioned subjects exhibited a robust cocaine-induced CPP, as illustrated by asignificant increase in the time spent on the drug-paired side of the apparatus [t(9)=6.20, p <0.001] (Figure 26). In contrast, lesioned subjects did not exhibit an increase in time spenton the drug-paired side of the apparatus [t(12)=0.33, n.s.] (Figure 26), suggesting thatcocaine-induced CPP was blocked by the amygdala lesions.111Figure 24. Total locomotor counts during the 10 days of locomotor conditioning for non-lesioned control (LI, n=10), pseudoconditioned (^, n=10) and conditioned (o, n=10)subjects, as well as lesioned control (A, n=13), pseudoconditioned (N, n=11) andconditioned (o, n=12) subjects. Conditioned subjects received cocaine (10 mg/kg,i.p.) prior to being placed in the locomotor apparatus, while pseudoconditioned andcontrol subjects were injected with saline. Values represent the group mean .t SEM.1200 -H 1000 -4•••0=00 800 -L.,08E 600 -0o^_0-J400 -200 -1121 1 1 i 1 1 11^2^3^4^5^6^7^8^9^10Days113Figure 25. Total locomotor counts for non-lesioned (o) control (n=10), pseudoconditioned(n=10) and conditioned (n=10) subjects, as well as lesioned (U) control (n=13),pseudoconditioned (n=11) and conditioned (n=12) subjects following 10 days ofconditioning with cocaine (10 mg/kg, i.p.). Testing occurred 48 hr after the lasttraining session, and was conducted in the same apparatus as used for the trainingprocedure. Values represent the group mean + SEM. * p < 0.05 compared to controlor pseudoconditioned subjects.Control ConditionedPseudocond.^ Non—LesionedLesionedCO 400 -o- 300 -4-,0EO 200 -c.)0*114115Figure 26. Effect of cocaine (10 mg/kg, i.p.) on the time spent in the drug-paired(nonstart) compartment before (0) and after (■) conditioning for non-lesioned (n=10)and lesioned (n=13) subjects. Values represent the group mean + SEM. * indicates asignificant within-group difference (p < 0.05) of pre- vs. post-conditioning scores.*650 -a)N-.....w 600 -0o 550 -wcc500 -I0ccD 450 -0? 400 -F-zU-I 350 -cr)w 300 -I=^ Pre-Conditioning111 Post-ConditioningNon-Lesioned^Lesioned116(D) DiscussionIn agreement with previous reports (Post et al., 1988; Cador et al., 1989), excitotoxiclesions of the amygdala did not affect basal or cocaine-induced locomotor activity. Thisfinding indicates that the amygdaloid complex is not a substrate for the unconditionedpsychomotor stimulant effects of cocaine. Lesions of the amygdaloid complex also failed toaffect cocaine-induced conditional locomotion, a somewhat unexpected result given previousdata implicating these nuclei in this form of classical conditioning (Chapter IV; Post et al.,1988). Despite an absence of effect of these excitotoxic lesions on the unconditionedlocomotor effects of cocaine, as well as cocaine-induced conditional locomotor activity,destruction of the amygdaloid complex blocked cocaine-induced CPP.Conditioned LocomotionThe failure of amygdala lesions to alter cocaine-induced conditional locomotion wasunanticipated given that electrolytic and 6-OHDA lesions of the amygdala have beenreported to greatly attenuate environment-specific cocaine-sensitization (Post et al., 1988)and that exposure to a cocaine-paired environment produces an increase in c-fos expressionin the amygdala (Chapter IV). Moreover, a large body of evidence strongly implicatesvarious nuclei of the amygdala in stimulus-reward learning (Weiskrantz, 1956; Jones andMishkin, 1972; Mishkin and Aggleton, 1981; Gaffan and Harrison, 1987; Cador et al., 1989;Everitt et al., 1991; Hiroi and White, 1991a; Kentridge et al., 1991). The second experimentdemonstrated that cocaine-induced CPP was blocked by excitotoxic lesions of the amygdala,indicating that destruction of the amygdala can affect certain forms of cocaine-inducedconditioning.One explanation for the failure of amygdala lesions to affect cocaine-inducedconditional locomotion relates to the learning demands of this paradigm. Although lesionsof the amygdaloid complex clearly can produce impairments in learning of stimulus-reward117118associations (Weiskrantz, 1956; Jones and Mishkin, 1972; Gaffan and Harrison, 1987; Cadoret al., 1989; Kesner et al., 1989; Cahill and McGaugh, 1990; Kentridge et al., 1991), thedeficits are evident only in certain situations. For example, both Weiskrantz (1956) andJones and Mishkin (1972) have noted that amygdala lesions do not completely or irreversiblyblock the learning of all stimulus-reward associations. Specifically, Jones and Mishkin(1972) suggest that "... only when associative learning demands are high, as in discriminationreversal, for example, will the animal show a severe and prolonged impairment." There isconsiderable experimental support for this proposal as many investigators have reported thatamygdala lesions fail to produce substantial deficits in certain tasks that are based on theformation of associations between specific stimuli and biologically relevant events(Schwartzbaum, 1965; Pelligrino, 1968; Shuckman et al., 1969; Slotnick, 1985; Cahill andMcGaugh, 1990; Kentridge et al., 1991). It is noteworthy that the conditioned locomotionprocedure utilized in the present study involved multiple cocaine-environment pairings, andrequired a rudimentary discrimination involving multimodal stimuli (homecage versustestcage), and a contextual conditioned stimulus for cocaine administration. Although nosingle critical factor appears to predict if amygdala lesions will produce deficits in learning,the design of the present conditioned locomotion experiment was not biased in favor ofobserving a lesion effect, given the long post-operative recovery period before conditioning,the relative simplicity of the task and the number of conditioning trials. Similar factors mayalso contribute to the discrepancy between the present findings and those of Post et al.(1988), who reported that amygdala lesions attenuate environment-specific cocainesensitization. These investigators used a one-trial learning paradigm, which may have biasedtheir result in favor of observing a lesion effect. It is also important to note that thisinterpretation of the present results does not preclude the possibility that the amygdala isinvolved in this form of conditioning in non-lesioned subjects, given that recovery offunction via other structures and pathways is possible (Jones and Mishkin, 1972).119Conditioned Place PreferenceAlthough lesions of the amygdaloid complex failed to attenuate cocaine-inducedconditional locomotion, the destruction of this region blocked cocaine-induced CPP. Thisresult is in agreement with previous reports that destruction of specific amygdaloid nucleiattenuate amphetamine- (Hiroi and White, 1991a) and food- (Everitt et al., 1991) inducedCPP. Although the findings of Hiroi and White (1991a) and Everitt et al. (1991) implicatedifferent amygdaloid nuclei as being critically involved in CPP, the extensive pathwaysbetween amygdaloid nuclei (Krettek and Price, 1978; Nitecka et al., 1981; Ottersen, 1982;Aggleton, 1985; Smith and Millhouse, 1985) suggest that several nuclei may be involved inthe learning of associations between specific stimuli and biologically relevant events. Forexample, in addition to the well documented role of the central nucleus in conditioned fear(Applegate et al., 1982; Pascoe and Kapp, 1985; Hitchcock and Davis, 1986, 1987; Davis,1992), it has recently been demonstrated that the lateral and/or basolateral nuclei of theamygdala play a role in fear-potentiated startle responses (Sananes and Davis, 1992). Theimportance of a given nucleus in stimulus-reward associations may depend on the modalityof the incoming sensory information, the specific demands of the associations and thecharacteristics of the response. Although the present findings demonstrate that lesions ofthe amygdala abolish cocaine-induced CPP, it cannot be determined from the present resultsif this effect is due to the blockade of the aquisition or expression of conditioning. Hiroiand White (1991a) have obtained data that are consistent with a role for the amygdala in theexpression of amphetamine-induced CPP, as well as a potential role in the acquisition ofconditioning. This is consistent with other data showing a role of the amygdala in both theacquistion and expression of stimulus-reward associations (Jones and Mishkin, 1972; Mishkinand Aggleton, 1981; Murray, 1991; Davis, 1992; Helmstetter, 1992). The present CPP datasupport the proposal that nuclei within the amygdaloid complex can play an essential role inthe association of environmental stimuli with reward.120The Role of the Amygdala in Cocaine-induced ConditioningAlthough both cocaine-induced conditional locomotion and CPP involve theformation and expression of environmental stimulus-drug associations, only cocaine-inducedCPP was affected by amygdala lesions. This differential effect may reflect the distinctlearning demands of the two paradigms. For example, subjects in the CPP experiment wererequired to discriminate between two highly similar environments, while subjects in theconditioned locomotion experiment were only required to discriminate a novel environmentfrom their homecage. However, these results may also reflect the fact that stimulant-induced conditional locomotion and CPP are behaviourally distinct phenomena, and hencecould be subserved by different neural circuits. Stimulant-induced conditional locomotion isclearly a form of classical conditioning, with the conditioned response resembling theunconditioned response. The conditioned response of CPP, however, appears to be a formof approach/orienting behaviour and does not resemble the unconditioned response tococaine, and hence cannot be explained in terms of traditional Pavlovian conditioning (Wise,1989). It is noteworthy that a recent study has demonstrated that lesions of the centralnucleus of the amygdala can differentially affect different classes of appetitivelyconditioned behaviours (Gallagher et al., 1990). Specifically, destruction of the centralnucleus impaired the acquisition of conditioned orienting responses, but produced no deficitin the conditioning of behaviours originally evoked by the unconditioned stimulus. Thedifferential effect of amygdala lesions on conditional locomotion and CPP is remarkablysimilar to these findings, and may provide support for the proposal that the acquisition ofthese two classes of appetitively conditioned responses are subserved by distinct neuralmechanisms (Holland, 1984). Taken as a whole, the data from these experiments support theproposal that nuclei within the amygdaloid complex can play a role in the association ofenvironmental stimuli with reward. However, the importance of this structure appears to bedependent on the demands of the learning paradigm and/or the responses examined.121VI. General DiscussionThe preceding chapters have presented the findings of experiments that have assessedvarious aspects of the neurobiology of cocaine's behavioural actions in relation to its abusepotential. The first series of experiments (Chapter II) evaluated the behavioural andneurochemical interactions between buprenorphine and cocaine. The resulting datasuggested that buprenorphine, like other opioids, can increase the rewarding effects ofcocaine. Moreover, the ability of buprenorphine to potentiate the cocaine-induced increasesin interstitial DA in the nucleus accumbens provides a potential explanation for thisbehavioural effect.Given the large body of evidence that implicates the mesolimbic dopaminergicsystem in the unconditioned behavioural effects of cocaine, the second series of experiments(Chapter III) examined whether stimuli previously paired with cocaine administration canelicit increases in interstitial DA in the nucleus accumbens that are similar to theunconditioned effects of this drug. When administered acutely, cocaine produced a potentunconditioned increase in interstitial DA concentrations in the nucleus accumbens.Although repeated pairing of cocaine with a specific environment produced conditionedlocomotion upon subsequent presentation of that environment, there was no concomitantconditional increase in DA release. These data do not support the hypothesis that stimulipaired with cocaine produce their behavioural effects by eliciting similar neurochemicaleffects as cocaine.To further elucidate the neurobiology of cocaine-induced environment-specificconditioning, expression of Fos, a putative marker of neuronal activity, was examined in theforebrain of rats exposed to an cocaine-paired environment (Chapter IV). Consistent withits stimulant actions, cocaine produced an increase in locomotion that was accompanied byan increase in Fos expression within specific limbic regions (cingulate cortex, claustrum,piriform cortex, lateral septal nucleus, paraventricular nucleus of the thalamus, lateralhabenula, and amygdala), as well as the basal ganglia (dorsomedial striatum and nucleus122accumbens). In agreement with our previous results, exposure of rats to the cocaine-pairedenvironment produced a conditional increase in locomotion. In addition to this behaviouraleffect, conditioned subjects exhibited a significant increase in the number of Fos-positiveneurons within the cingulate cortex, claustrum, lateral septal nucleus, paraventricular nucleusof the thalamus, lateral habenula and the amygdala, suggesting increased neuronal activitywithin these regions. In contrast to the dramatic effects observed in these structures, noconditional activation was observed within the piriform cortex, nucleus accumbens, or dorsalstriatum, suggesting that these brain regions are not involved in the conditioned locomotorresponse. These findings suggest that specific limbic regions exhibit increased neuronalactivation during the presentation of cocaine-paired cues and may be involved in theformation of associations between cocaine's stimulant actions and the environment in whichthe drug administration occurred. Although the nucleus accumbens is necessary for thereinforcing and locomotor effects of cocaine, it did not exhibit a conditional Fos response,further suggesting that different neural circuits are involved in the unconditioned andconditioned locomotor effects of cocaine.The final series of experiments (Chapter V) evaluated the role of the amygdaloidcomplex in cocaine-induced conditional locomotion and CPP. Quinolinic acid lesions of theamygdaloid complex did not affect basal or cocaine-induced locomotion, suggesting that theamygdala does not mediate the unconditioned psychomotor stimulant effects of this drug.Preconditioning lesions also failed to affect cocaine-induced conditional locomotion. Thislack of effect was contrasted by a complete blockade of cocaine-induced CPP by theamygdaloid lesions. These data demonstrate that cocaine-induced stimulus-rewardconditioning can be differentially affected by lesions of the amygdala.The impetus for the first series of experiments (Chapter II) was a report thatbuprenorphine suppresses cocaine self-administration by rhesus monkeys, and thereforemight be useful in the pharmacotherapy of cocaine abuse (Mello et al., 1989). However, thepresent findings, as well as the results of two recent double-blind, controlled studies123(Johnson et al., 1992; Kosten et al., 1992), suggest that the buprenorphine will not be aneffective pharmacotherapy for cocaine abuse. In addition to the aforementioned studies thathave examined the efficacy of buprenorphine in the treatment of cocaine abuse, numerousclinical and preclinical studies have evaluated a variety of drugs as potentialpharmacotherapies for cocaine abuse. Although it is possible that this approach may resultin the discovery of a "magic bullet" for treating cocaine abuse, present understanding of theneurobiology of cocaine's behavioural actions suggests that this goal is unlikely to berealized, particularly given the theoretical orientation of the majority of recentinvestigations, as will be discussed.As discussed previously, a large body of evidence indicates that the mesolimbic DAsystem plays a fundamental role in the reinforcing properties of cocaine (Di Chiara andImperato, 1988; Fibiger et al., 1992; Fibiger and Phillips, 1987; Johanson and Fischman,1989; Roberts et al., 1977, 1989). Accordingly, the majority of preclinical and clinicalstudies of potential pharmacotherapeutic treatments for cocaine abuse have been directed ataltering dopaminergic transmission. However, the normal biological function of themesolimbic dopaminergic projection appears to be intimately involved in the rewardingand/or incentive motivational effects of natural stimuli such as food and sex (Fibiger, inpress; Fibiger and Phillips, 1987; Nomikos et al., in preparation; Pfaus et al., 1990; Wise andRompre, 1989). Therefore, drug therapies that block or attenuate dopaminergictransmission, although highly effective in reducing the reinforcing effects of cocaine in bothrats and non-human primates (Bergman et al., 1990; Roberts et al., 1989), also decrease therewarding or hedonic value of natural stimuli (Fibiger, in press; Fibiger and Phillips, 1987;Willner et al., 1991). This hypothesis is supported by the findings that neuroleptics producedysphoria in normal subjects (Belmaker and Wald, 1977; Heninger et al., 1965; Simonsone,1964; Willner, 1983) and that these DA receptor antagonists are considered unacceptable bypatients seeking treatment for cocaine abuse (Gawin, 1986; Gawin and Kleber, 1986; Shereret al., 1989). As the mesolimbic DA system plays a critical role in the reinforcing effects of124both cocaine and natural stimuli, such as sex and food, it is improbable that a drug can bedeveloped that will decrease the actions of cocaine, without also attenuating the hedonicand/or incentive motivational properties of natural reinforcers.As DA receptor antagonists appear to be unsuitable in the pharmacotherapy ofcocaine abuse, researchers have also examined the potential use of both direct and indirectDA receptor agonists to treat cocaine abuse, hypothesizing that they could be utilized in amanner similar to methadone in the treatment of heroin abuse. One obvious problem withthis approach is that many indirect DA agonists, such as amphetamine, methylphenidate,GBR 12909 and mazindol, also possess considerable abuse liability (Bergman et al., 1989;Johanson and Fischman, 1989; Mansbach and Balster, 1993; Ritz et al., 1987; Roberts, 1993;Sannerud and Griffiths, 1992). The use of these cocaine-like drugs is also problematicinsofar as they may act as powerful cues that could lead to increased drug craving, andtherefore a return to drug use (Jaffe et al., 1989; O'Brien et al., 1992; Stewart et al., 1984).In addition to the aforementioned practical difficulties in the use of dopaminergicagonists in the pharmacotherapy of cocaine abuse, the theoretical basis for this approach ishighly questionable. The basic premise behind this hypothesis is that cocaine craving is theconsequence of a decrease in dopaminergic transmission as a result of abstinence fromcocaine (Dackis and Gold, 1985; Spealman, 1992). This hypothesis is based on a withdrawalmodel of drug abuse, similar to those that have been proposed to explain opioid abuse (Wiseand Bozarth, 1987). The general inadequacies of these models is discussed extensively byWise and Bozarth (1987), and therefore only the principal flaw of this model in relation tococaine abuse will be reviewed.A clear prediction of the DA depletion hypothesis of cocaine abuse (Dackis andGold, 1985) and other withdrawal theories is that craving should be alleviated by takingcocaine. In contrast, clinical and laboratory results indicate that craving for cocaine isreported to be most intense following drug use, while blood levels of cocaine are stillelevated (Gawin and Kleber, 1986; Jaffe et al., 1989). It is noteworthy that similar results125have also been reported for heroin (Meyer and Mirin, 1979). These findings are inagreement with results that indicate that drug self-administration is stimulated by thepresence of the drug, not its absence (De Wit and Stewart, 1981, 1983; Gerber and Stretch,1975; Stewart et al., 1984; Wise et al., 1990). These data clearly fail to provide support forthe use of DA receptor agonists to treat cocaine abuse. In summary, the aforementioneddata suggest that because the mesolimbic DA system is intimately involved in the reinforcingand/or incentive motivational properties of natural rewarding stimuli, drugs that attenuatecocaine's primary rewarding effects are likely to interfere with the rewarding properties ofnatural reinforcers, and hence be unacceptable in the treatment of substance abuse. Also,there is virtually no support for the hypothesis that treatment with cocaine-like drugs willdecrease craving for cocaine; on the contrary, these drugs appear to increase cocainecraving.Although the DA projection to the nucleus accumbens is strongly implicated in thereinforcing properties of cocaine, the neurobiology of cocaine-induced conditioned responsesis largely unknown. As discussed previously, this aspect of cocaine's action appears to be animportant component in its abuse liability (Gawin, 1991; O'Brien et al., 1992). In additionto its potential clinical importance, a better comprehension of the neurobiology of thisphenomenon may also directly contribute to our understanding of the neural circuitryinvolved in the classical conditioning of natural rewards to environmental stimuli. Taken asa whole, the present findings (Chapters III, IV and V) suggest that the neural circuitsunderlying the conditioned response to the presentation of cocaine-paired stimuli differsomewhat from those responsible for the unconditioned response to cocaine. Moreover,these data suggest that limbic structures involved in the stimulus-reward conditioning ofnatural reinforcers may also be involved in cocaine-induced conditioned responses.The apparent DA-independent nature of the conditioned locomotor response tococaine-paired stimuli (Chapter III and IV) has potential implications for the treatment forcocaine abuse; however, the generalizability of these findings to other abused substances and126natural reinforcers is not clear. Although there is strong support for a dopaminergic role inthe acquisition of various stimulus-reward associations (Beninger and Hahn, 1983; Beningerand Herz, 1986; Beninger and Phillips, 1980; Hiroi and White, 1989; Horvitz and Ettenberg,1989; Spyraki et al., 1982b; Weiss et al., 1989), the role of DA in the expression of theseassociations remains uncertain. In support of a non-dopaminergic mechanism for theexpression of these conditioned behaviors, the presentation of stimuli that signal theavailability of food have not been found to increase interstitial DA concentrations in thenucleus accumbens, as measured by in vivo microdialysis, despite increases in locomotoractivity or lever pressing (Hernandez and Hoebel, 1988; Nomikos et al., in preparation;Radhakishun et al., 1988). In agreement with these data, the DA receptor antagonistpimozide fails to block the conditioned locomotor response to the presentation of food-paired stimuli (Horvitz and Ettenberg, 1991). Moreover, responding for conditionedrewarding stimuli is unaffected by 6-OHDA lesions of the nucleus accumbens, leading theauthors to suggest that "the information about the conditioned reinforcer is not directlydependent upon activity in the ventral striatal DA system" (Robbins et al., 1989). Theresults from these conditioning studies employing non-drug reward are in agreement withthe present results (Chapter III and IV), as well as previous studies that have failed toobserve conditional dopaminergic activity following conditioning with a variety of drugs(Barr et al., 1983; Finlay et al., 1988; Moller et al., 1987; Walter and Kuschinsky, 1989).In contrast to those studies that suggest that conditioned stimuli can activate behaviorthrough non-dopaminergic pathways, Blackburn et al. (1989b) found that presentation ofdiscrete stimulus that predicted the availability of food produced an increase in theDOPAC/DA ratio, which suggests an increase in DA turnover. Moreover, responding to thisconditioned stimulus was reduced following administration of DA receptor antagonists(Blackburn et al., 1987, 1989a). The activity of DA neurons have also been demonstrated toincrease in anticipation of a food reward; however, this apparent conditional responsedecreases with continued training (Ljungberg et al., 1992). Based on the ability of intra-127accumbens a-flupenthixol to attenuate the expression of amphetamine-induced CPP, Hiroiand White (1990) suggest that "when animals encounter environmental cues which havepreviously been paired with a primary reward, dopamine is released in the nucleusaccumbens". These authors have also reported that systemically administered D1 and D2receptor antagonists block the expression of amphetamine-induced CPP (Hiroi and White,1991b). Finally, the local administration of amphetamine or DA into the nucleus accumbenscan increase responding for a stimulus previously paired with a natural reward, suggestingthat DA can affect this conditioned response (Taylor and Robbins, 1984, 1986; Robbins etal., 1989). In summary, these data suggest that dopaminergic transmission can affect theexpression of responses to stimuli previously paired to primary rewards.Although there is support for the hypothesis that the expression of conditionedstimulus-reward associations is not dependent on an increase in dopaminergic transmission inthe nucleus accumbens, other data appear to suggest that DA does play a critical role in theconditioned stimulus activation of these behaviors. A number of factors may account forthese discrepancies, such as differences in the learning paradigms or responses measured(e.g. responding for a conditioned reinforcer vs. conditioned locomotion vs. CPP), the natureof the unconditioned reward (e.g. drug vs. non-drug) or the characteristics of theconditioned stimulus (e.g. explicit vs. contextual cues), and clearly future studies shouldattempt to evaluate the importance of these specific considerations. It is possible thatincreased DA release is a critical component for the expression of certain forms of stimulus-reward learning, while others are independent of an increase in dopaminergic transmission.However, it should also be noted that some of the data that have been proposed to indicatethat enhanced DA release is essential for the expression of stimulus-reward associations donot provide direct support for this hypothesis. Specifically, the ability of DA receptorantagonists to reduce or block a behavioural response does not provide evidence thatenhanced DA release is associated with the production of the behaviour; rather, these datasimply suggest that there is a DA-dependent component to the expression of these128behaviours. The competitive blockade produced by DA receptor antagonists suggests thatthese drugs may produce their largest effects when dopaminergic transmission is reduced,not elevated. Therefore, the reduction of a conditioned response following theadministration of a DA receptor antagonist may reflect the attenuation of necessarymodulatory effects of basal DA release. Specifically, the blockade of dopaminergictransmission may alter the efficacy of transmission of neural signals from the amygdala orsubiculum, for example, to the subpallidal area via the nucleus accumbens. This proposal iscompatible with electrophysiological studies that provide evidence for a neuromodulatoryrole of DA in the nucleus accumbens (Yim and Mogenson, 1982, 1986, 1988). A similarhypothesis has been proposed to account for the finding that 6-OHDA lesions block thefacilitation of responding for a conditioned reinforcer produced by intra-accubmensamphetamine, but fail to affect basal responding for the conditioned stimulus (Robbins etal., 1989). Specifically, Robbins and colleagues (1989) have proposed that the informationregarding the association between the primary reward and the conditioned stimulus isdependent on projections from the amygdala and other limbic structures, such as thesubiculum and the prefrontal and entorhinal cortex, to the nucleus accumbens, whereas themesolimbic dopaminergic projection plays a modulatory role, enhancing the performance ofthose behaviours resulting from presentation of the conditioned stimulus. Despite the factthat the ability of neuroleptics to affect the performance of conditioned response does notnecessarily indicate enhanced dopaminergic transmission, the fact remains that additionalresults also provide evidence for conditional dopaminergic activity (Blackburn et a!., 1989b;Ljungberg et al., 1992). Future studies using in vivo techniques, such as microdialysis,should be utilized to resolve these discrepancies by directly examining if enhanced DArelease is associated with the performance of specific conditioned behaviors.Given the aforementioned findings that indicate that stimuli paired to either drug ornatural rewards can apparently produce conditioned responses in the absence of increasedDA release in the nucleus accumbens and perhaps through non-dopaminergic pathways, the129cocaine-induced conditional responses examined in the present studies (Chapters II, IV andV) may be mediated by the same neural mechanism involved in the classical conditioning ofnatural rewards to environmental stimuli (Robbins et al., 1989). This proposal is furthersupported by the fact that the presentation of a cocaine-paired environment increased Fosexpression in the amygdala and that lesions of the amygdala blocked cocaine-induced CPP,which are in agreement with a large body of evidence that suggests the amygdala is involvedin stimulus-reward learning (Weiskrantz, 1956; Jones and Mishkin, 1972; Mishkin andAggleton, 1981; Gaffan and Harrison, 1987; Cador et al., 1989; Everitt et al., 1991; Hiroiand White, 1991a; Kentridge et al., 1991). To further evaluate this proposal the directexamination of single- or multi-unit activity in the amygdala and other regions implicatedin stimulus-reward associations could be examined during the acquisition and expression ofboth cocaine- and natural reward-induced conditional behaviours. This examination duringresponding for a cocaine-paired stimulus would also be a highly worthwhile area ofinvestigation. One distinct advantage of the use of this paradigm is that the data fromprevious studies that have used natural rewards, such as food, water and receptive sexualpartners, could be directly compared to the results obtained using cocaine as the reward(Robbins et al., 1989). Moreover, the use of a different paradigm would allow for theexamination of the generalizability of the present findings to other forms of stimulus-rewardconditioning.Cocaine-related stimuli can elicit conditioned responses, including drug-likephysiological changes and reports of craving, in individuals who abuse cocaine (Ehrman etal., 1992; O'Brien et al., 1992). Although craving is a subjective state, it appear to behighly correlated to verbal and physiological measures (Pickens and Johanson, 1992).Assuming that the neural mechanisms involved in the conditioned responses of cocaineabusing individuals and the subjects in the present studies are similar, the proposed neuralmechanisms underlying the cocaine-induced conditioned responses provide a potentialframework for understanding cocaine craving, as well as predictions regarding treatment.130The DA-independent nature of the conditioned response suggests that craving should notnecessarily be associated with euphoria or positive feelings elicited by cocaine itself. Thisprediction is supported by the finding that less than half of the subjects who reportedincreases in craving in response to cocaine related cues indicated that this was associatedwith feelings of a "cocaine high" (Ehrman et al., 1992). A related prediction is that cocainecraving should be relatively resistant to neuroleptic treatment. Although no publishedreports have directly examined this possibility, it remains a testable hypothesis for futureinvestigation.The other major conclusion of the present findings, as discussed previously (ChaptersIII, IV and V), is that cocaine-induced conditional responses are potentially subserved by thesame neural mechanisms involved in the formation of associations between specific stimuliand other classes of biologically relevant events. This suggests that the established principlesof classical conditioning should also be applicable to cue-induced cocaine craving. Althoughthis has not been directly investigated, the importance of generalization and spontaneousrecovery in extinction-based treatment of cocaine abusing individuals has been recognized(Hammersley, 1992; O'Brien et al., 1992). However, the profound resistance to extinctionexhibited by chronic cocaine users related to the presentation of cocaine related cues appearsunusually protracted (O'Brien et al., 1992). Although this pattern of extinction may simplyreflect the large number of cocaine-stimulus pairings that have occurred prior topresentation for treatment, this uncharacteristic resistance to extinction may alternativelyreflect the supraphysiological stimulation of the meso-accumbens pathway produced bycocaine during the acquisition of this stimulus-reward association. Future studies thatevaluate the relationship between dopaminergic stimulation during the acquisition ofstimulus-reward associations and its subsequent effect on rate of extinction and number ofspontaneous recoveries will directly address this hypothesis. Finally, the present hypothesisconcerning the neurobiology of cocaine-induced conditioned responses would suggest thatthe development of a pharmacotherapy to reduce cravings is unlikely. If cravings are the131result of the same neural processes involved in the expression of other stimulus-rewardassociations, it is highly improbable that a drug will be developed to "erase" the memory ofcocaine.VII. ReferencesAggleton J.P. (1985) A description of intra-amygdaloid connections in the old worldmonkeys. Exper. Brain Res. 57, 390-399.Anthony J.C. (1992) Epidemiological research on cocaine use in the USA. In: Cocaine:Scientific and Social Dimensions (Bock G.R. and Whelan J., eds.), pp 20-33.Chichester: John Wiley and Sons.Applegate C.D., Frysinger R.C., Kapp B.S. and Gallagher M. (1982) Multiple unit activityrecorded from the amygdala central nucleus during Pavlovian heart rate conditioningin rabbit. Brain Res. 238, 457-462.Bals-Kubik R., Ableitner A., Herz A. and Shippenberg T. (1993) Neuroanatomical sitesmediating the motivational effects of opioids as mapped by the conditioned placepreference paradigm in rats. J. Pharmacol. Exper. Ther. 264, 489-495.Balster R.L., Mansbach R.S., Gold L. and Harris L.S. (1992) Preclinical methods for thedevelopment of pharmacotherapies for cocaine abuse. In: Problems of drugdependence, 1991, National Institute on Drug Abuse Research Monograph 119(Harris L.S., ed.), pp. 160-164. Washington: Committee on Problems of DrugDependence, Inc.Bardo M.T., Neisewander J.L. and Miller J.S. (1986) Repeated testing attenuatesconditioned place preference with cocaine. Psychopharmacol. 89, 239-243.Barr G.A., Sharpless N.S., Cooper S. and Schiff S.R. (1983) Classical conditioning, decayand extinction of cocaine-induced hyperactivity and stereotypy. Life Sci. 33, 1341-1351.Barrett J.E and Nader M.A. (1990) Neurochemical correlates of behavioural processess.Drug Dev. Res. 20, 313-335.Belmaker R.H. and Wald D. (1977) Haloperidol in normals. Br. J. Psychiatry 131, 701-707.Beninger R.J. and Hanh B.L. (1983) Pimozide blocks the establishment but not expressionof amphetamine-produced environment-specific conditioning. Science (Wash. D.C.)220, 1304-1306.Beninger R.J. and Herz R.S. (1986) Pimozide blocks the establishment but not expression ofcocaine-produced environment-specific conditioning. Life Sci. 38, 1425-1431.Beninger R.J. and Phillips A.G. (1980) The effect of pimozide on the establishmentconditioned reinforcement. Psychopharmacol. 68, 147-153.Benowitz N.L. (1992) How toxic is cocaine? In: Cocaine: Scientific and Social Dimensions(Bock G.R. and Whelan J., eds.), pp 125-143. Chichester: John Wiley and Sons.Bentivoglio M., Balercia G. and Kruger L. (1990) The specificity of the nonspecificthalamus: The midline nuclei. In: Progress in brain research, Vol 85 (UylingsH.B.M., Van Eden C.G., De Bruin J.P.C., Corner M.A. and Feenstra M.G.P., eds.),pp 53-80. New York: Elsevier.132133Benveniste H. and Hiittemeier P.C. (1990) Microdialysis-Theory and application. Prog.Neurobiol. 35, 195-215.Bergman J., Kamien J.B. and Spealman R.D. (1990) Antagonism of cocaine self-administration by selective dopamine Di and D2 antagonists. Behay. Pharmacol. 1,355-363.Bergman J., Madras B.K., Johnson S.E. and Spealman R.D. (1989) Effects of cocaine andrelated drugs in nonhuman primates. III. Self-administration by squirrel monkeys.J. Pharmacol. Exp. Ther. 251, 150-155.Bermudez-Rattoni F. and McGaugh J.L. (1991) Insular cortex and amygdala lesionsdifferentially affect acquistion on inhibitory and condtioned taste aversion. BrainRes. 549, 165-170.Bjarklund A. and Lindvall 0. (1984) Dopamine-containing systems in the CNS. In:Handbook of Chemical Neuroanatomy: Volume 2, Classical neurotransmitters in theCNS (BjOrklund A. and Htikfelt T., eds.), pp 55-122. New York: Elsevier.Blackburn J.R., Phillips A.G. and Fibiger H.0 (1987) Dopamine and preparatory behavior:I. Effects of pimozide. Behay. Neurosci. 101, 352-360.Blackburn J.R., Phillips A.G. and Fibiger H.0 (1989a) Dopamine and preparatory behavior:III. Effects of metoclopramide and thioridazine. Behay. Neurosci. 103, 903-906.Blackburn J.R., Phillips A.G., Jakubovic A. and Fibiger H.0 (1989b) Dopamine andpreparatory behavior: II. A neurochemical analysis. Behay. Neurosci. 103, 15-23.Bozarth M.A. and Wise R.A. (1981b) Heroin reward is dependent on a dopaminergicsubstrate. Life Sci. 29, 1881-1886.Bradberry C.W. and Roth R.H. (1989) Cocaine increases extracellular dopamine in the ratnucleus accumbens and ventral tegmental area as shown by in vivo microdialysis.Neurosci. Lett. 103, 97-102.Britt M.D. and Wise R.A. (1983) Ventral tegmental site of opiate reward: Antagonism by ahydrophillic receptor blocker. Brain Res. 258, 105-108.Brown E.E., Damsma G., Cumming P. and Fibiger H.C. (1991) Interstitial 3-methoxytyramine reflects striatal dopamine release: An in vivo microdialysis study.J. Neurochem. 57, 701-707.Buchanan S.L. and Powell D.A. (1982) Cingulate cortex: its role in Pavlovian conditioning.J. Comp. Physiol. Psycho!. 96, 755-774.Cador M., Robbins T.W. and Everitt B.J. (1989) Involvement of the amygdala in stimulus-reward associations: Interactions with the ventral striatum. Neuroscience 30, 77-86.Cahill L. and McGaugh J.L. (1990) Amygdaloid complex lesions differentially affectretention of tasks using appetitve and aversive reinforcement. Behay. Neurosci. 104,532-543.134Campeau S., Hayward M.D., Hope B.T., Rosen, J.B., Nestler E.J. and Davis M. (1991)Induction of c-fos proto-oncogene in rat amygdala during unconditioned andconditioned fear. Brain Res. 565, 349-352.Carey R.J. (1992) Pavlovian conditioning of L-dopa induced movement.Psychopharmacology 107, 203-210.Carr G.D., Fibiger H.C. and Phillips A.G. (1989) Conditioned place preference as a measureof drug reward. In: The Neuropharmacological Basis of Reward (Liebman J.M. andCooper S.J., eds.), pp 264-319. Oxford: Clarendon Press.Carroll M.E., Carmona G.N., May S.A., Buzalsky S. and Larson C. (1992) Buprenorphine'seffects on self-administration of smoked cocaine base and orally deliveredphencyclidine, ethanol and saccharin in rhesus monkeys. J. Pharmacol. Exper. Ther.261, 26-37.Carroll M.E. and Lac S.T (1992) Effects of buprenorphine on self-administration of cocaineand a nondrug reinforcers in rats. Psychopharm. 106, 439-446.Chambers C.D., Taylor W.J.R. and Moffett A.D. (1972) The incidence of cocaine abuseamong methadone maintenance patients. Int. J. Addict. 7, 427-441.Chastrette N., Pfaff D.W. and Gibbs R.B. (1991) Effects of daytime and nighttime stress onFos-like immunoreactivity in the paraventricular nucleus of the hypothalamus, thehabenula, and the posterior paraventricular nucleus of the thalamus. Brain Res. 563,339-344.Chowdhury A.N. and Chowdhury S. (1990) Buprenorphine abuse: report from India. Br. J.Addiction 85, 1349-1350.Colpaert F.0 (1978) Discriminative stimulus properties of narcotic analgesic drugs.Pharmacol. Biochem. Behay. 9, 863-867.Commissiong J.W. (1985) Monoamines metabolites: their relationship and lack ofrelationship to monoaminergic neuronal activity. Biochem. Pharmacol. 34, 1127-1131.Cowan A., Dettmar P.W. and Walter D.S. (1976) The effect of acute doses of buprenorphineon concentrations of homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA)and 3-methoxy-4-hydroxyphenylglycol (MHPG) in the rat forebrain. Proc. Brit.Pharmacol. Soc. 58, 275P.Cowan A., Lewis J.W. and MacFarlane I.R. (1977) Agonist and antagonist properties ofbuprenorphine, a new antinociceptive agent. Br. J. Pharmacol. 60, 537-545.Dackis C.A. and Gold M.S. (1985) New concepts in cocaine addiction: the dopaminedepletion hypothesis. Neurosci. Biobehay. Rev. 9, 469-477.Damsma G., Boisvert D.P., Mudrick L.A., Wenkstern D. and Fibiger H.C. (1990) Effects oftransient forebrain ischemia and pargyline on extracellular concentrations ofdopamine, serotonin and their metabolites in the rat striatum as determined by invivo microdialysis. J. Neurochem. 54, 801-808.135Davis M. (1992) The role of the amygdala in fear and anxiety. Annual Rev. Neurosci. 15,353-375.De Wit H. and Stewart J. (1981) Reinstatement of cocaine-reinforced responding in the rat.Psychopharmacol. 75, 134-143.De Wit H. and Stewart J. (1983) Reinstatement of heroin-reinforced responding in the rat.Psychopharmacol. 79, 29-31.De Wit H. and Wise R.A. (1977) Blockade of cocaine reinforcement in rats with thedopamine receptor blocker pimozide, but not with the noradrenergic blockersphentolamine and phenoxybenzamine. Can. J. Psychol. 31, 195-203.Delfs J.M., Schreiber L. and Kelley A.E. (1990) Microinjections of cocaine into the nucleusaccumbens elicits locomotor activation in the rat. J. Neurosci. 10, 303-310.Di Chiara G. and Imperato A. (1988a) Drugs abused by humans preferentially increasesynaptic dopamine concentrations in the mesolimbic system of freely moving rats.Proc. Natl. Acad. Sci. 85, 5274-5278.Di Chiara G. and Imperato A. (1988b) Opposite effects of mu and kappa opiate agonists ondopamine release in the nucleus accumbens and in the dorsal caudate of freelymoving rats. J. Pharmacol. Exp. Ther. 244, 1067-1080.Dragunow M. and Faull R. (1989) The use of c-fos as a metabolic marker in neuronalpathway tracing. J. Neurosci. Methods 29, 261-265.Dragunow M. and Robertson, H.A. (1987) Kindling stimulation induces c-fos protein(s) ingranule cells of the rat dentate gyrus. Nature 329, 441-442.Drew K.L. and Glick S.D. (1990) Role of D-1 and D-2 receptor stimulation in sensitizationto amphetamine-induced circling behavior and in expression and extinction of thePavlovian conditioned response. Psychopharmacology 101, 465-471.DSM-III-R (1987) Diagnostic and Statistical Manual of Mental Disorders, ed 3, revised.Washington, D.C.: American Psychiatric Association.Dum J.E. and Herz A. (1981) In vivo receptor binding of the opiate agonist buprenorphine,correlated with its agonist and anatagonist actions. Br. J. Pharmacol. 74, 627-633.Dunn L.T. and Everitt B.J. (1988) Double dissociations of the effects of amygdala andinsular cortex lesions on conditioned taste aversions, passive avoidance, andneophobia in the rat using the excitotoxin ibotenic acid. Behay. Neurosci. 102, 3-22.Dykstra, L.A., Doty, P., Johnson A.B. and Picker M.J. (1992) Discriminative stimulusproperties of cocaine, alone and in combination with buprenorphine, morphine andnaltrexone. Drug and Alcohol Depen. 30, 227-234.Ehrman R.N., Robbins S.J., Childress A.R. O'Brien C.P. (1992) Conditioned responses tococain-related stimuli in cocaine abuse patients. Psychopharmacology 107, 523-529.Ettenberg A., Pettit H.O., Bloom F.E. and Koob G.F. (1982) Heroin and cocaineintravenous self-administration in rats: Mediation by seperate neural systems.Psychopharmacology 78, 204-209.136Everitt B.J., Morris K.A., O'Brien A. and Robbins T.W. (1991) The basolateral amygdala-ventral striatal system and conditioned place preference: Further evidence of limbic-striatal interactions underlying reward-related processes. Neuroscience 42, 1-18.Fibiger H.C. and Phillips A.G. (1987) Role of catecholamine transmitters in brain rewardsystems: Implications for the neurobiology of affect. In: Brain Reward Systems andabuse (Engel J. and Oreland L., eds.), pp 61-74. New York: Raven Press.Fibiger H.C. Mesolimbic dopamine: An analysis of its role in motivated behavior. Seminarsin Neuroscience, in press.Finlay J.M., Jakubovic A., Phillips A.G. and Fibiger H.C. (1988) Fentanyl-inducedconditional place preference: lack of associated conditional neurochemical events.Psychopharmacology 96, 534-540.Fu L. and Beckstead R.M. (1992) Cortical stimulation induces fos expression in striatalneurons. Neuroscience 46, 329-334.Gabriel M. and Sparenborg E. (1987) Posterior cingulate cortex lesions eliminate learning-related unit activity in the anterior cingulate cortex. Brain Res. 409, 151-157.Gabriel M., Foster K. and Orona E. (1980) Interactions of laminae of the cingulate cortexwith the anteroventral thalamus during behavioral learning. Science (Wash. D.C.)203, 1050-1052.Gaffan D. and Harrison S. (1987) Amygdalectomy and disconnection in visual learning forauditory secondary reinforcement by monkeys. J. Neuroscience 7, 2285-2292.Gallagher M., Graham P.W. and Holland P.C. (1990) The amygdala central nucleus andappetitive Pavlovian conditioning: Lesions impair one class of conditioned behavior.J. Neuroscience 10, 1906-1911.Gastfriend D.R., Mendelson J.H., Mello N.K and Teoh S.K.. (1992) Preliminary results ofan open trial of buprenorphine in the outpatient treatment of combined heroin andcocaine dependence. In: Problems of Drug Dependence, 1991, National Institute onDrug Abuse Research Monograph 119 (Harris L.S., ed.), pg. 461. Washington:Committee on Problems of Drug Dependence, Inc.Gawin F.H. (1986) Neuroleptic reduction of cocaine-induced paranoia but not euphoria?Psychopharmacol. 90, 142-143.Gawin F.H. (1991) Cocaine addiction: Psychology and neurophysiology. Science (Wash.D.C.) 251, 1580-1586.Gawin F.H. and Ellinwood E.H. (1988) Cocaine and other stimulants: Actions, abuse, andtreatment. N. Engl. J. Med. 318, 1173-1182.Gawin F.H. and Kleber H.D. (1986) Abstinence symptomology and psychiatric diagnosis incocaine abusers. Arch. Gen. Psychiat. 43, 107-113.Gerber G.J. and Stretch R. (1975) Drug-induced reinstatement of extinguished self-administration behavior in monkeys. Pharmacol. Biochem. Behay. 3, 1055-1061.137Glysing K. and Wang R.Y. (1983) Morphine-induced activation of A10 dopamine neuronsin the rat. Brain Res. 277, 119-127.Goeders N.E. and Smith J.E. (1983) Cortical involvement in cocaine reinforcement. Science(Wash. D.C.) 221, 773-775.Gold L.H., Swerdlow N.R. and Koob G.F. (1988) The role of mesolimbic dopamine inconditioned locomotion produced by amphetamine. Behay. Neurosci. 102, 544-552.Graybiel A.M., Moratalla, R. and Robertson H.A. (1990) Amphetamine and cocaine inducedrug-specific activation of the c-fos gene in striosome-matrix compartments andlimbic subdivisions of the striatum. Proc. Natl. Acad. Sci. 87, 6912-6916.Grinspoon L. and Bakalar J.B. (1980) Drug dependence: non-narcotic agents. In:Comprehensive Textbook of Psychiatry, 3rd edn. (Kaplan H.I., Freedman A.M. andSadock B.J., eds.), pp 1621-1622. Baltimore: Williams and Wilkins.Groenewegen H.J (1988) Organization of the afferent connections of the mediodorsalthalamic nucleus in the rat, related to the mediodorsal-prefrontal topography.Neurosci. 24, 379-431.Groenewegen H.J., Berendse H.W, Wolters J.G. and Lohman A.H.M. (1990) The anatomicalrelationship of the prefrontal cortex with the striatopallidal system, the thalamus andthe amygdala: evidence for a parallel organization. In: Progress in Brain Research,Vol 85 (Uylings H.B.M., Van Eden C.G., De Bruin J.P.C., Corner M.A. and FeenstraM.G.P., eds.), pp 95-118. New York: Elsevier.Hambrook J.M. and Rance M.J. (1976) The interaction of buprenorphine with the opiatereceptor: Lipophilicity as a determining factor in drug-receptor kinetics. In:Opiates and Endogenous Opioid Peptides (Kosterlitz H.W., ed.), pp.295-301.Amsterdam: Elsevier.Heikkila R.E., Orlansky H. and Cohen G (1975) Studies on the distinction between uptakeinhibition and release of [3H] dopamine in rat brain slices. Biochem. Pharmacol. 24,847-852.Helmstetter F.J. (1992) Contribtution of the amygdala to learning and performance ofconditioned fear. Physiol. and Behay. 51, 1271-1276.Heninger G., DiMascio A. and Klerman G.I. (1965) Personality factors in variability ofresponse to phenothiazines. Amer. J. Psychiat. 121, 1091-1094.Herling S., Downs D.A. and Woods J.H. (1979) Cocaine, d-amphetamine, and pentobarbitaleffects on responding maintained by food or cocaine in rhesus monkeys.Psychopharmacology 64, 261-269.Hernandez L and Hoebel B.G. (1988) Feeding and hypothalamic stimulation increasedopamine turnover in the accumbens. Physiol. Behay. 44, 599-606.Hiroi N. and White N.M. (1989) Conditioned stereotypy: behavioral specification of theUCS and pharmacological investigation of the neural change. Pharmacol. Biochem.Behay. 32, 249-258.138Hiroi N. and White N.M. (1990) The reserpine-sensitive dopamine pool mediates (+)-amphetamine-conditioned reward in the place preference paradigm. Brain Res. 510,33-42.Hiroi N. and White N.M. (1991a) The lateral nucleus of the amygdala mediates expressionof the amphetamine-produced conditioned place preference. J. Neurosci. 11, 2107-2116.Hiroi N. and White N.M. (1991b) The amphetamine conditioned place preference:differential involvement of dopamine receptor subtypes and two dopaminergicterminal areas. Brain res. 552, 141-152.Hitchcock J. and Davis M. (1986) Lesions of the amygdala, but not of the cerebellum orred nucleus, block conditioned fear as measured with the potentiated startleparadigm. Behay. Neuroscience 100, 11-22.Hitchcock J. and Davis M. (1987) Fear potentiated startle using an auditory conditionedstimulus: Effect of lesions of the amygdala. Physiol. and Behay. 39, 403-408.Holland P.C. (1984) Origins of behavior in Pavlovian conditioning. Psychol. Learn. Motiv.18, 129-174.Horvitz J.C. and Ettenberg A. (1989) Haloperidol blocks the response-reinstating effects offood reward: A methodology for seperating neuroleptic effects on reinforcement andmotor processes. Pharmacol. Biochem. Behay. 31, 861-865.Horvitz J.C. and Ettenberg A. (1991) Conditioned incentive properties of a food-pairedconditioned stimulus remain intact during dopamine receptor blockade. Behay.Neurosci. 105, 526-541.Hubner C., Bain G.T. and Kornetsky C. (1987) The combined effect of morphine and d-amphetamine on the threshold for brain stimulation reward. Pharmacol. Biochem.Behay. 28, 311-315.Hubner C.B. and Kornetsky C. (1988) The reinforcing properties of the mixed agonist-antagonist buprenorphine as assessed by brain-stimulation reward. Pharmacol.Biochem. Behay. 30, 195-197.Hunt S.P., Pini A. and Evan G. (1987) Induction of c-fos-like protein in spinal cordneurons following sensory stimulation. Nature 328, 632-634.Hurd Y.L. and Ungerstedt U. (1989) Cocaine: An in vivo microdialysis evaluation of itsaction on dopamine transmission in rat striatum. Synapse 3, 48-54.Izenwasser S. and Kornetsky C. (1989) The effect of amphonelic acid or nisoxetine incombination with morphine on brain-stimulation reward. Pharmacol. Biochem.Behay. 32, 983-986.Jaffe J.H. (1989) Drug dependence: opioids, non-narcotics, nicotine (tobacco), andcaffeine. In: Comprehensive Textbook of Psychiatry, 5th edn. (Kaplan H.I. andSadock B.J., eds.), pp 642-686. Baltimore: Williams and Wilkins.139Jasinski D.R. and Nutt J.G. (1972) Progress report on the assessment program on the NIMHaddiction center. In: Report of the Thirty-Fourth Annual Scientific MeetingCommittee on Problems of Drug Dependence, pp. 442-477. Michigan: NIMH.Jasinski D.R., Pevnick J.S. and Griffith J.D. (1978) Human pharmacology and abusepotential of the analgesic buprenorphine. Arch. Gen. Psychiatry 35, 501-516.Jellestad F.K. and Cabrera I.C. (1986) Exploration and avoidance learning after ibotenicacid and radio-frequency lesions in the rat amygdala. Behay. Neural Biol. 46: 195-215.Johanson C.E. and Fischman M.W. (1989) The pharmacology of cocaine related to its abuse.Pharmacol. Rev. 41, 3-52.Johnson R.E., Jaffe J.H. and Fudala P.J. (1992) A controlled trial of buprenorphine foropioid dependence. JAMA 267, 2750-2755.Jones R.T. (1990) The pharmacology of cocaine smoking in humans. In: Research Findingson Smoking of Abused Substances, National Institute on Drug Abuse ResearchMonograph 99 (Chiang C.N. and Hawks R.L., eds.), pp. 30-41. Washington: USGovernment Printing Office.Jones B. and Mishkin M. (1972) Limbic lesions and the problem of stimulus-reinforcementassociations. Exp. Neurol. 36, 362-377.Joyce E.M. and Koob G.F. (1981) Ampetamine-, scopolamine- and caffeine-inducedlocomotor activity following 6-hydroxydopamine lesions of the mesolimbic dopaminesystems. Psycopharmacol. 73, 311-313.Kalivas P.W. and Duffy P. (1990) The effects of acute and daily cocaine treatment onextracellular dopamine in the nucleus accumbens. Synapse 5, 48-58.Kamien J.B. and Spealman R.D. (1991) Modulation of the discriminative-stimulus effects ofcocaine by buprenorphine. Behay. Pharmacol. 2, 517-520.Kapp B.S., Gallager M., Frysinger R.C. and Applegate C.D. (1981) The amygdala, emotionand cardiovascular conditioning. In: Amygdaloid Complex (Ben-Ari Y., ed.), pp355-366. Amsterdam: Elsevier.Kelly P.H., Seviour P.W. and Iversen S.D. (1975) Amphetamine and apomorphine responsesin the rat following 6-OHDA lesions of the nucleus accumbens septi and corpusstriatum. Brain Res. 94, 4507-522.Kentridge R.W., Shaw C. and Aggleton J.P. (1991) Amygdaloid lesion and stimulus-rewardassociations in the rat. Behay. Brain Res. 42, 57-66.Kesner R.P., Walser R.D. and Winzenried G. (1989) Central but not basolateral amygdalamediates memory for positive affective experiences. Behay. Brain Res. 33, 189-195.Koob G.F., Le H.T. and Creese I. (1987a) D-1 receptor antagonist SCH 23390 increasescocaine self-administration in the rat. Neurosci. Lett. 78, 315-321.140Koob G.F., Vaccarino F.J., Amalric M. and Bloom F.E. (1987b) Positive reinforcementproperties of drugs: search for neural substrates. In: Brain Reward Systems andAbuse (Oreland L. and Engel J., eds.), pp. 35-50. New York: Raven Press.Kosten T.R., Kleber H.D. and Morgan C. (1989) Treatment of cocaine abuse withbuprenorphine. Biol. Psychiatry 26, 637-639.Kosten T.R., Rounsaville B.J., Gawin F.H. and Kleber H.D. (1986) Cocaine abuse amongopioid addicts: demographics and diagnostic characteristics. Am. J. Drug AlcoholAbuse 12, 1-16.Kosten T.R., Rounsaville B.J. and Kleber H.D. (1987a) A 2.5 year follow-up of cocaine useamong treated opioid addicts. Arch. Gen. Psychiatry 44, 281-284.Kosten T.R., Schottenfeld R.S., Morgan C.H., Falcioni J. and Ziedonis D. (1992)Buprenorphine vs. methadone for opioid and cocaine dependence. In: Problems ofDrug Dependence, 1991, National Institute on Drug Abuse Research Monograph 119(Harris L.S., ed.), pg. 359. Washington: Committee on Problems of DrugDependence, Inc.Kosten T.R., Schumann B., Wright D., Carney M.K. and Gawin F.H. (1987b) A preliminarystudy of desipramine in the treatment of cocaine abuse in methadone maintenancepatients. J. Clin. Psychiatry 48, 442-444.Krettek J.E. and Price J.L. (1978) Amygdaloid projections to subcortical structures withinthe basal forebrain in the rat and cat. J. Comp. Neurol. 178, 225-254.Lal H., Miksic S., Drawbaugh R., Numan R. and Smith N. (1976) Alleviation of narcoticwithdrawal syndrome by conditional stimuli. Pavlov J. Biol. Sci. 11, 252-262.Lewis J.W. (1985) Buprenorphine. Drug and Alcohol Dependence 14, 363-372.Ljungberg T., Apicella P. and Schultz W. (1992) Responses of dopamine neurons duringlearning of behavioral reactions. J. Neurophysiol. 67, 145-163.Lisoprawski A., Herve D., Blanc G., Glowinski J. and Tassin J.P. (1980) Selectiveactivation of the mesocortico-frontal dopaminergic neurons induced by lesions of thehabenula in the rat. Brain Res. 183, 229-234.Lopez da Silva F.H, Witter M.P., Boeijinga P.H. and Lohman A.H.M. (1990) Anatomicorganization and physiology of the limbic cortex. Physiological Rev. 70, 453-511.Lukas S.E., Brady J.V. and Griffiths , R.R. (1986) Comparison of opioid self-injection anddisruption of schedule controlled performance in the baboon. J. Pharmacol. Exp.Ther. 238, 924-931.Lyness W.H., Friedle N.M. and Moore K.E. (1979) Destruction of dopaminergic nerveterminals in nucleus accumbens: Effects on d-amphetamine self-administration.Pharmacol. Biochem. Behay. 11, 553-556.Mackey W.B. and van der Kooy, (1985) Neuroleptics block the positive reinforcing effectsof amphetamine, but not of morphine as measured by place conditioning.Pharmacol. Biochem. and Behay. 22, 101-105.141Maissonneuve I.M., Keller R.W. and Glick S.D. (1990) Similar effects of d-amphetamineand cocaine on extracellular dopamine levels in medial prefrontal cortex of rats.Brain Res. 535, 221-226.Mansbach R.S. and Balster R.L. (1993) Effects of mazidol on behavior maintained oroccasioned by cocaine. Drug Alcohol Dep. 31, 183-191.Martin-Iverson M.T., Szostak C. and Fibiger H.C. (1986) 6-Hydroxydopamine lesions of themedial prefrontal cortex fail to influence intravenous self-administration of cocaine.Psychopharmacology 88, 310-314.Mello N.K. and Mendelson J.H. (1980) Buprenorphine supresses heroin use by heroinaddicts. Science (Wash, D.C.) 207, 657-659.Mello N.K., Bree M.P. and Mendelson J.H. (1981) Buprenorphine self-administration byrhesus monkey. Pharmacol. Biochem. Behay. 15, 215-225.Mello N.K., Bree M.P. and Mendelson J.H. (1983) Comparison of buprenorphine andmethadone effects on opiate self-administration in primates. J. Pharmacol. Exp.Ther. 225, 378-386.Mello N.K., Kamien J.B., Lukas S.E., Mendelson J.H., Drieze J.M. and Sholar J.W. (1993)Effects of intermittent buprenorphine administration on cocaine self-administrationby rhesus monkeys. J. Pharmacol. Exper. Ther. 264, 530-541.Mello N.K., Lukas S.E., Kamien J.B., Mendelson J.H., Drieze J.M. and Cone E.J. (1992)The effect of chronic buprenorphine treatment on cocaine and food self-administration by rhesus monkeys. J. Pharmacol. Exper. Ther. 260, 1185-1193.Mello N.K., Mendelson J.H. and Kuehnle J.C. (1982) Buprenorphine effects on human self-administration: an operant analysis. J. Pharmacol. Exp. Ther. 223, 30-39.Mello N.K., Mendelson J.H., Bree M.P. and Lukas S.E. (1989) Buprenorphine supressescocaine self-administration by rhesus monkeys. Science (Wash. D.C.) 245, 859-862.Mello N.K., Mendelson J.H.,Bree M.P. and Lukas S.E. (1990) Buprenorphine and naltrexoneeffects on cocaine self-administration by rhesus monkeys. J. Pharmacol. Exper.Ther. 254, 926-939.Mendelson J.H., Mello N.K., Teoh S.K., Kuehnle J., Sintavanarong P. and Dooley-CoufosK. (1991) Buprenorphine treatment for concurrent heroin and cocaine dependence:Phase I study. In: Problems of Drug Dependence, 1990, National Institute on DrugAbuse Research Monograph 105 (Harris L.S., ed.), pp. 196-202. Washington:Committee on Problems of Drug Dependence, Inc.Mendelson J.H., Teoh S.K., Mello N.K. and Ellingboe J. (1992) Buprenorphine attenuatesthe effects of cocaine on Adrenocorticotropin (ACTH) secretion and mood states inman. Neuropsychopharmacol. 7, 157-162.Meyer R.E and Mirin S.M. (1979) The Heroin Stimulus. New York: Plenum Press.Mishkin M. and Aggleton J. (1981) Multiple functional contributions of the amygdala in themonkey. In: Amygdaloid Complex (Ben-Ari Y., ed.), pp 409-420. Amsterdam:Elsevier.142Moghaddam B. and Bunney B.S. (1989a) Differential effect of cocaine on extracellulardopamine in rat medial prefrontal cortex and nucleus accumbens: Comparison toamphetamine. Synapse 4, 156-161.Moghaddam B. and Bunney B.S. (1989b) Ionic composition of microdialysis perfusingsolution alters the phamacological responsiveness and basal outflow of striataldopamine. J. Neurochem. 53, 652-654.M011er H.-G., Nowak K. and Kuschinsky K. (1987) Conditioning of pre- and post-synapticbehavioural responses to the dopamine receptor agonist apomorphine in rats.Psychopharmacology 91, 50-55.Moore R.Y. and Card J.P. (1984) Noradrenaline-containing neuron systems. In: Handbookof Chemical Neuroanatomy: Volume 2, Classical Neurotransmitters in the CNS(Bjiirklund A. and Hiikfelt T. eds.), pp 123-156. New York: Elsevier.Morency M.A. and Beninger R.J. (1987) Dopaminergic substrates of cocaine-induced placeconditioning. Brain Res. 399, 33-41.Morgan I. and Curran T. (1989) Stimulus-transcription coupling in neurons: role of cellularimmediate-early genes. Trends Neurosci. 12, 459-462.Mucha R.F. and Herz A. (1985) Motivational properties of kappa and mu opioid receptoragonists studied with place and taste preference conditioning. Psychopharmacology86, 274-280.Mucha R.F., van der Kooy D., O'Shaughnessy M. and Bucenieks P. (1982) Drugreinforcement studies by the use of place conditioning in rat. Brain Res. 243, 91-105.Murray E.A. (1991) Contributions of the amygdalar complex to behavior in macaquemonkeys. In: Progress in Brain Research, Vol 87 (Holstege G., ed.), pp 167-180.Amsterdam: Elsevier.Musto D.F. (1992) Cocaine's history, especially the American experience. In: Cocaine:Scientific and Social Dimensions (Bock G.R. and Whelan J., eds.), pp 7-14.Chichester: John Wiley and Sons.Nitecka L., Amerski L. and Narkiewicz 0. (1981) The organization of intraamygdaloidconnections: An HRP study. J. fur Hirnforschung 22, 3-7.Nomikos G.G., Damsma G., Wenkstern D. and Fibiger H.C. (1990) In vivo characterizationof locally applied dopamine uptake inhibitors by striatal microdialysis. Synapse 6,106-112.Nomikos G.G. and Spyraki C. (1988) Cocaine-induced place conditioning: importance ofroute of administration and other procedural variables. Psychopharmacol. 94, 119-125.Nowycky M.C., Walters J.R. and Roth R.H. (1978) Dopaminergic neurons: effect of acuteand chronic morphine administration on single cell activity and transmittermetabolism. J. Neural Trans. 42, 99-116.143O'Brien C.P., Childress A.R., McLellan A.T. and Ehrman R. (1992) Classical conditioningin drug-dependent humans. In: The Neurobiology of Drug and Alcohol Addiction,Vol. 654 (Kalivas P.W. and Samson H.H., eds.), pp 400-415.. New York: New YorkAcademy of Sciences.O'Connor J.J., Moloney E., Travers R. and Campbell A. (1988) Buprenorphine abuse amongopiate addicts. Br. J. Addiction 83, 1085-1087.Ottersen O.P. (1982) Connections of the amygdala of the rat: IV Corticoamygdaloid andintramygdaloid connections as studied with axonal transport of horseradishperoxidase. J. Comp. Neurol. 205, 30-48.Pascoe J.P. and Kapp B.S. (1985) Electrophysiological characteristics of amygdaloid centralnucleus during Pavlovian fear conditioning in the rabbit. Behay. Brain Res. 16, 117-133.Paxinos G. and Watson C. (1986) The rat brain in stereotaxic coordinates. Orlando:Academic Press.Pelligrino L. (1968) Amygdaloid lesions and behavioral inhibition in the rat. J. Comp.Physiol. Psychol. 65, 483-491.Pelligrino L.K., Pelligrino A.A. and Cushman A.J. (1979) A stereotaxic atlas of the ratbrain. New York: Plenum Press.Perez-Cruet J. (1976) Conditioning of striatal dopamine metabolism with methadone,morphine or bulbocapnine as an unconditioned stimulus. Pavlov J. Biol. Sci. 11,237-250.Pettit H.O. and Justice J.B. Jr. (1989) Dopamine in the nucleus accumbens during cocaineself-administration as studied by in vivo microdialysis. Pharmacol. Biochem. Behay.34, 899-904.Pettit H.O. and Justice J.B. Jr (1991) Effect of dose on cocaine self-administration behaviorand dopamine levels in the nucleus accumbens. Brain Res. 539, 94-102.Pettit H.O., Ettenberg A., Bloom F.E. and Koob G.F. (1984) Destruction of dopamine inthe nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats. Psychopharmacology 84, 167-173.Pfaus J.G., Damsma G., Nomikos G.G., Wenkstern D.G., Blaha C.D., Phillips A.G. andFibiger H.C. (1990) Sexual behavior enhances central dopamine transmission in themale rat. Brain Res. 530, 345-348.Phillips A.G. and Broekkamp C.L.E. (1980) Inhibition of intravenous cocaine self-administration by rats after microinjections of spiroperidol into the nucleusaccumbens. Soc. Neurosci. Abstr. 6, 105.Phillips A.G. and LePiane F.G. (1980) Reinforcing effects of morphine microinjections intothe ventral tegmental area. Pharmacol. Biocem. Behay. 12, 965-968.Phillips A.G. and LePiane F.G. (1982) Reward produced by microinjections of (d-ala)-metenkephalinamide into the ventral tegmental area. Behay. Brain Res. 5, 225-229.144Phillipson O.T. and Pycock C.J. (1982) Dopamine neurones of the ventral tegmentumproject to both medial and lateral habenula. Exp. Brain Res. 45, 89-94.Pickens R.W. and Johanson C.E. (1992) Craving: consensus of status and agenda for futureresearch. Drug Alcohol Depend. 30, 127-131.Post R.M., Weiss S.R.B. and Pert A. (1988) Cocaine-induced behavioral sensitization andkindling: Implications for the emergence of psychopathology and seizures. In: TheMesocortical Dopamine System, Vol. 537 (Kalivas P.W. and Nemeroff C.B., eds.), pp292-308. New York: New York Academy of Sciences.Powell D.A., Buchanan S.L and Gibbs C.M. (1990) Role of the prefrontal-thalamic axis inclassical conditioning. In: Progress in Brain Research, Vol 85 (Uylings H.B.M., VanEden C.G., De Bruin J.P.C., Corner M.A. and Feenstra M.G.P., eds.), pp 433-466.New York: Elsevier.Radhakishun F.S., VAN Ree J.M. and Westerink B.H.C. (1988) Scheduled eating increasesdopamine release in the nucleus accumbens and ventral tegmental area in the rat:measurement by in vivo microdialysis. Neurosci. Lett. 85, 351-3561.Raisman G. (1966) The connexions of the septum. Brain 89, 317-348.Rance M.J. and Shillingford J.S. (1976) The role of the gut in the metabolism of stronganalgesics. Biochem. Pharm. 25, 735-741.Reirez J., Mena M.A., Bazan E., Muradds V., Lerma J., Delgado J.M.R. and DeYebenes J.G.(1989) Temporal profile of levels of monoamines in striata of rats implanted withdialysis tubes. J. Neurochem. 53, 789-792.Richelson E. and Pfenning M. (1984) Blockade by antidepressants and related compounds ofbiogenic amine uptake into rat brain synaptosomes: Most antidepressants selectivelyblock norepinephrine uptake. Eur. J. Pharmacol. 104, 277-286.Riolobos A.S. and Garcia A.I.M. (1987) Open field activity and passive avoidance responsesin rats after lesions of the central amygdaloid nucleus by electrocoagulation andibotenic acid. Physiol. Behay. 39, 715-720.Ritz M.C., Lamb R.J., Goldberg S.R. and Kuhar M.J. (1987) Cocaine receptors ondopamine transporters are related to the self-administration of cocaine. Science(Wash. D.C.) 237, 1219-1223.Robbins T.W., Cador M., Taylor J.R. and Everitt B.J. (1989) Limbic-striatal interactions inreward-related processess. Neuroscience & Biobehavior Rev. 13, 155-162.Roberts D.C.S. (1993) Self-administration of GBR 12909 on a fixed ratio and progressiveratio schedule in rats. Psychopharmacology 111, 202-206.Roberts D.C.S. and Vickers G. (1984) Atypical neuroleptics increase self-administration ofcocaine: an evaluation of a behavioral screen for antipsychotic activity.Psychopharmacology 82, 135-139.145Roberts D.C.S., Corcoran M.E. and Fibiger H.C. (1977) On the role of ascendingcatecholaminergic systems in intravenous self-administration of cocaine. Pharmacol.Biochem. Behay. 6, 615-620.Roberts D.C.S., Klonoff P., Koob G.F. and Fibiger H.C. (1980) Extinction and recovery ofcocaine self-administration following 6-hydroxydopamine lesions of the nucleusaccumbens. Pharmacol. Biochem. Behay. 12, 781-787.Roberts D.C.S. and Koob G.F. (1982) Disruption of cocaine self-administration following6-hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol.Biochem. Behay. 17, 901-904.Roberts D.C.S., Loh E.H. and Vickers G. (1989) Self-administration of cocaine on aprogressive ratio schedule in rats: dose-response relationship and effect ofhaloperidol pretreatment. Psychopharmacology 97, 535-538.Robertson G.S. and Fibiger H.C. (1992) Neuroleptics increase c-fos expression in theforebrain: contrasting effects of haloperidol and clozapine. Neurosci. 46, 315-328.Robertson G.S., Herrera D.G., Dragunow M. and Robertson H.A. (1989) L-Dopa activatesc-fos in the striatum ipsilateral to a 6-hydroxydopamine lesion of the substantianigra. Eur. J. Pharmacol. 159, 99-100.Robertson G.S., Pfaus J.G., Atkinson L.J., Matsumara H., Phillips A.G. and Fibiger H.C.(1991) Sexual behavior increases c-fos expression in the forebrain of the male rat.Brain Res. 564, 352-357.Roffler-Tarlov S., Sharman D.F. and Tegerdine P. (1971) 3,4-Dihydroxyphenylacetic acidand 4-hydroxy-3-methoxyphenylacetic acid in the mouse striatum: a reflection ofintra- and extra-neuronal metabolism of dopamine? Br. J. Pharmacol. 42, 343-351.Roozendaal B., Oldenburger W.P., Strubbe J.H., Koolhass J.M. and Bohus B. (1990) Thecentral amygdala is involved in the conditioned but not the meal-induced cephalicinsulin response in the rat. Neurosci. Lett. 116, 210-215.Rusak B., Robertson H.A., Wisden W. and Hunt S.P. (1990) Light pulses that shift rhythmsinduce gene expression in the suprachiasmatic nucleus. Science (Wash. D.C.) 248,1237-1240.Sadee W., Rosenbaum J.S. and Herz A. (1982) Buprenorphine: Differential interaction withopiate receptor subtypes in vivo. J. Pharmacol. Exp. Ther. 223, 157-162.Sagar S.M., Sharp F.R. and Curran T. (1988) Expression of c-fos protein in brain:metabolic mapping at the cellular level. Science (Wash. D.C.) 240, 1328-1331.Sananes C.B. and Davis M. (1992) N-methyl-D-aspartate lesions of the lateral andbasolateral nuclei of the amygdala block fear-potentiated startle and shocksensitization of startle. Behay. Neuroscience 106, 72-80.Sannerud C.A. and Griffiths R.R. (1992) Evaluation of the reinforcing effects of mazindolin baboons. In: Problems of Drug Dependence, 1991, National Institute on DrugAbuse Research Monograph 119 (Harris L.S., ed.), pg. 393. Washington: Committeeon Problems of Drug Dependence, Inc.146Santiago M. and Westerink B.H.C. (1990) Characterization of the in vivo release ofdopamine as recorded by different types of intracerebral microdialysis probes.Naunyn-Schmiedebergs Arch. Pharmacol. 342, 407-414.Satel S.L., Southwick S.M. and Gawin F.H. (1991) Clinical features of cocaine-inducedparanoia. Am. J. Psychiat. 148, 495-498.Schuckman H., Kling A. and Orbach J. (1969) Olfactory discrimination in monkeys withlesions in the amygdala. J. Comp. Physiol. Psychol. 67, 212-215.Schulz R. and Herz A. (1976) The guinea-pig ileum as an in vitro model to analysedependence liability of narcotic drugs. In: Opiates and Endogenous Opioid Peptides(Kosterlitz H.W., ed.), pp.319-326. Amsterdam: Elsevier.Schwartzbaum J.S. (1965) Discrimination behavior after amygdalectomy in monkeys: Visualand somaesthetic learning and perceptual capacity. J. Comp. Physiol. Psychol. 60,314-319.Sharp F.R., Sagar S.M., Hicks K., Lowenstein D. and Hisanaga K. (1991) c-fos mRNA,Fos, and Fos-related antigen induction by hypertonic saline stress. J. Neurosci. 11,2321-2331.Shiff S.R. (1982) Conditioned dopaminergic activity. Biol. Psychiat. 17, 135-154.Sherer M.A., Kumor K.M. and Jaffe J.H. (1989) Effects of intravenous cocaine arepartially attenuated by haloperidol. Psychiat. Res. 27, 117-125.Shippenberg T.S., Bals-Kubik R. and Herz A. (1993) Examination of the neurochemicalsubstrates mediating the motivational effects of opioids: Role of the mesolimbicdopamine system and D-1 vs. D-2 dopamine receptors. J. Pharmacol. Exper. Ther.265, 53-59.Shu S., Ju G. and Fan L. (1988) The glucose oxidase-DAB-nickel method in peroxidasehistochemistry of the nervous system. Neurosci. Lett. 85, 169-171.Simonson M. (1964) Phenothiazine depressive reaction. J. Neuropsychiat. 5, 259-265.Slotnick B.M. (1985) Olfactory discriminations in rats with anterior amygdala lesions.Behay. Neuroscience 99, 956-963.Smith B.S. and Millhouse O.E. (1985) The connections between basolateral and centralnuclei. Neuroscience Lett. 56, 307-309.Soares-da-Silva P. and Garrett M.C. (1990) A kinetic study of the rate of formation ofdopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) inthe brain of the rat: Implications for the origin of DOPAC. Neuropharmacology 29,869-874.Spanagel R., Herz A. and Shippenberg T.S. (1990) The effects of opioid peptides ondopamine release in the nucleus accumbens: An in vivo microdialysis study. JNeurochem. 55, 1734-1740.Spealman R.D. (1992) Use of cocaine-discrimination techniques for preclinical evaluation ofcandidate therapeutics for cocaine dependence. In: Problems of Drug Dependence,1471991, National Institute on Drug Abuse Research Monograph 119 (Harris L.S., ed.),pp 175-179. Washington: Committee on Problems of Drug Dependence, Inc.SPSS:X User's Guide, 3rd Edition (1988). Chicago: SPSS Inc.Spyraki C., Fibiger H.C. and Phillips A.G. (1982a) Cocaine-induced place conditioning:lack of effects of neuroleptics and 6-hydroxydopamine lesions. Brain Res. 253, 195-203.Spyraki C., Fibiger H.C. and Phillips A.G. (1982b) Attenuation by haloperidol of placepreference conditioning using food reinforcement. Psychopharmacol. 77, 379-382.Spyraki C., Fibiger H.C. and Phillips A.G. (1983) Attenuation of heroin reward in rats bydisruption of mesolimbic dopamine system. Psychopharmacology 79, 278-283.Spyraki C., Nomikos G.G. and Varonos D.D. (1987) Intravenous cocaine-induced placepreference: attenuation by haloperidol. Behay. Brain Res. 26, 57-62.Steinbusch H.W.M. (1984) Serotonin-immunoreactive neurons and their projections in theCNS. In: Handbook of Chemical Neuroanatomy: Volume 3, Classical Transmittersand Transmitter Receptors in the CNS (BjOrklund A., HOkfelt T. and Kuhar M.J.eds.), pp 68-125. New York: Elsevier.Stewart J., De Wit H. and Eikelboom R. (1984) Role of unconditioned and conditioned drugeffects in the self-administration of opiates and stimulants. Psychol. Rev. 91, 251-268.Stretch R. (1977) Discrete-trial control of cocaine self-injection behavior in squirrelmonkeys: effects of morphine, naloxone, and chlorpromazine. Can. J. Physiol.Pharmacol. 55, 778-790.Strang J. (1985) Abuse of buprenorphine. Lancet ii, 725.Sutherland R.J. (1982) The dorsal diencephalic conduction system: A review of theanatomy and functions of the habenular complex. Neurosci. and Biobehay. Rev. 6, 1-13.Swanson L.W. and Cowan W.M. (1979) The connections of the septal region in the rat. J.Comp. Neurol. 186, 621-656.Tatum A.L. and Seevers M.H. (1929) Experimental cocaine addiction. J. Pharmacol. Exp.Ther. 36, 401-410.Taylor J.R. and Robbins T.W. (1984) Enhanced behavioural control by conditionedreinforcers following microinjections of d-amphetamine into the nucleus accumbens.Psychopharmacol. 84, 405-412.Taylor J.R. and Robbins T.W. (1986) 6-Hydroxydopamine lesions of the nucleusaccumbens, but not the caudate nucleus, attenuate enhanced responding with reward-related stimuli produced by intra-accumbens d-amphetamine. Psychopharmacol. 90,390-397.Teoh S.K., Sintavanarong P., Kuehnle J., Mendelson J.H., Hallgring E., Rhoades E. andMello N.K. (1992) Buprenorphine's effects on morphine and cocaine challenges inheroin and cocaine dependent men. In: Problems of Drug Dependence, 1991,148National Institute on Drug Abuse Research Monograph 119 (Harris L.S., ed.), pg.460. Washington: Committee on Problems of Drug Dependence, Inc.Thomas E. (1988) Forebrain mechanisms in the relief of fear: the role of the lateralseptum. Psychobiology 16, 36-44.Thomas E. and Yadin E. (1980) Multiple unit activity in the septum during Pavlovianaversive conditioning: evidence for an inhibitory role of the septum. Exp. Neurol.69, 50-60.Thomas E., Yadin E. and Strickland C.E. (1991) Septal unit activity during classicalconditioning: a regional comparison. Brain Res. 547, 303-308.Van Dyke C. and Byck R. (1982) Cocaine. Sci. Am. 246, 108-119.Vitti T.G. and Boni R.L. (1985) Metabolism of cocaine. In: Pharmacokinetics andPharmacodynamics of Psychoactive drugs: A Research Monograph (Barnett G. andChiang C.N., eds.), pp. 427-440. California: Biomedical Publications.Walter S. and Kuschinsky K. (1989) Conditioning of morphine-induced locomotor activityand stereotyped behaviour in rats. J. Neural Trans. 78, 231-247.Weeks J.R. (1962) Experimental morphine addiction: method for automatic intravenousinjections in unrestrained rats. Science (Wash. D.C.) 138, 143-144.Weiskrantz L.(1956) Behavioral changes associated with ablation of the amygdaloid complexin monkeys. J. Comp. Physiol. Psychol. 49, 381-391.Weiss S.R.B., Post R.M., Pert A., Woodland R. and Murman D. (1989) Context-dependentcocaine sensitization: Differential effect of haloperidol on development versusexpression. Pharmacol. Biochem. Behay. 34, 655-661.Welzl H., Kuhn G. and Huston J.P. (1989) Self-administration of small amounts ofmorphine through glass micropipettes into the ventral tegmental area of the rat.Neuropharmacology 28, 1017-1023.Westerink B.H.C. (1985) Sequence and significance of dopamine metabolism in the ratbrain. Neurochem.Int. 7, 221-227.Westerink, B.H.C. Hofsteede H.M., Damsma G. and de Vries J.B. (1988) The significance ofextracellular calcium for the release of dopamine, acetylcholine and amino acids inconscious rats, evaluated by brain microdialysis. Naunyn-Schmiedeberg's Arch.Pharmacol. 337, 373-378.Willner P. (1983) Dopamine and depression: A review of recent evidence. I. Empiricalstudies. Brain Res. Rev. 6, 211-224.Willner P., Phillips G. and Muscat R.T. (1991) Supression of rewarded behaviour byneuroleptic drugs: Can't or won't, and why? In: The Mesolimbic Dopamine System:From Motivation to Action (Willner P. and Scheel-Kruger J., eds.), pp 251-272.Colchester: Wiley.Wilson M.C. and Schuster C.R. (1974) Aminergic influences on intravenous cocaine self-administration by rhesus monkeys. Pharmacol. Biochem. Behay. 2, 563-571.149Winger G., Skjoldager P. and Woods J.H. (1992) Effects of buprenorphine and other opioidagonists and antagonists on alfentanil- and cocaine-reinforced responding in rhesusmonkeys. J. Pharmacol. Exper. Ther. 261, 311-317.Wise R.A. (1989) The brain and reward. In: The Neuropharmacological Basis of Reward(Liebman J.M. and Cooper S.J., eds.), pp 377-424. Oxford: Clarendon Press.Wise R.A., Murray A. and Bozarth M.A. (1990) Bromocriptine self-administration andbromocriptine-reinstatement of cocaine-trained and heroin-trained lever pressing inrats. Psychopharmacol. 100, 355-360.Wise R.A. and Rompre P.P. (1989) Brain dopamine and reward. Ann. Rev. Psychol. 40,191-225.Woods J.H. (1977) Narcotic-reinforced responding: A rapid screening procedure. In:Proceedings of the 39th Meeting of the Committee on Problems of DrugDependence, pp. 420-449. Cambridge: NIMH.Woolverton W.L. (1987) Evaluation of the role of norepinephrine in the reinforcing effectsof psychomotor stimulants in rhesus monkeys. Pharmacol. Biochem. Behay. 26, 835-839.Yanagita T., Katoh S., Wakasa Y. and Oinuma N. (1982) Dependence potential ofbuprenorphine studied in rhesus monkeys. In: Problems of Drug Dependence, 1981,National Institute on Drug Abuse Research Monograph 41 (Harris L.S., ed.), pp. 208-214. Washington: Committee on Problems of Drug Dependence, Inc.Yim C.Y. and Mogenson G.J. (1982) Response of nucleus accumbens neurons to amygdalastimulation and its modification by dopamine. Brain Res. 239, 401-415.Yim C.Y. and Mogenson G.J. (1986) Mesolimbic dopamine projection modulates amygdala-evoked EPSP in nucleus accumbens neurons: an in vivo study. Brain Res. 369, 347-352.Yim C.Y. and Mogenson G.J. (1988) Neuromodulatory action of dopamine in the nucleusaccumbens: an in vivo intracellular study. Neuroscience 26, 403-415.Young A.M., Stephens K.R., Hein D.W. and Woods J.H. (1984) Reinforcing anddiscriminative stimulus properties of mixed agonist-antagonist opioids. J. Pharmacol.Exp. Ther. 229, 118-126.Young S.T., Porrino L.J.T., Porrino L.J. and Iadarola, M.J. (1991) Cocaine induces striatalc-Fos-immunoreactivity proteins via dopaminergic D1 receptors. Proc. Natl. Acad.Sci. 88, 1291-1295.Zis A.P., Nomikos G.G., Damsma G. and Figiber H.C. (1991) In vivo neurochemical effectsof electroconvulsive shock studied by microdialysis in the rat striatum.Psychopharmacol. 103, 343-350.

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