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In Vivo monitoring of dopamine in the nucleus accumbens during intravenous self-administration of D-Amphetamine… Di Ciano, Patricia 1993

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IN VIVO MONITORING OF DOPAMINE IN THE NUCLEUSACCUMBENS DURING INTRAVENOUS SELF-ADMINISTRATION OFD-AMPHETAMINE BY THE RATByPATRICIA DI CIANOB.A. (Hons.), Queen’s University, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF ARTSinTHE FACULTY OF GRADUATE STUDIESDepartment of PsychologyWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1993©Patricia Di Ciano, 1993tCciQQ+111A VI S UNHf CDIn 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__________________The University of British ColumbiaVancouver, CanadaDate Q /DE-6 (2/88)AbstractCurrent theory suggests that mesolimbic dopamine (DA) in the nucleusaccumbens is involved in the self-administration of drugs such as cocaineand amphetamine. The present thesis was conducted to determine some ofthe behavioural and in vivo dopaminergic correlates of intravenous (i.v.)self-administration of amphetamine in the rat. Experiment one tested thehypothesis that rats will self-administer amphetamine to maintain anoptimal extracellular level of DA in the brain that exceeds a criticalthreshold. In vivo measurements of DA oxidation currents(electrochemical studies) and DA dialysate concentrations (microdialysisstudy) were used to determine changes in extracellular DA concentrationsin rats permitted to self-administer 12 infusions of d-amphetamine a day ata given dose. During all self-administration sessions, distinct ‘loading’ and‘maintenance’ phases were seen. The loading phase was characterized byrapid responding at the start of the session which slowed during themaintenance phase to a constant level for the duration of the self-administration session. DA extracellular concentrations in the nucleusaccumbens increased at the start of the self-administration session and thenlevelled off around a stable mean for the duration of the session. Aninverse dose-response relationship between rate of responding foramphetamine and the dose of drug administered (0.05, 0.10 or 0.20mg/infusion) was seen. No dose-related changes in the maximal increase inDA oxidation currents or dialysate concentrations during selfadministration was evident. Experiment two tested the hypothesis thatincreased DA levels associated with the reinitiation of responding after anabstinence serve as a negative reinforcer by increasing DA levels that areIIItheoretically depleted during an abstinence (Dackis and Gold, 1985). Whenpermitted continuous access to 0.10 mg/infusion of amphetamine for 48hours, response rates and in vivo DA oxidation currents were similar tothose in experiment one and remained steady during the course of theentire self-administration session. After at least 24 hours, all ratsabstained from self-administering amphetamine for a single period of timelasting at least two hours. During this time, a decrease in DAconcentrations was seen. Reinitiation of responding for amphetamine didnot occur when DA concentrations were at their lowest, but was correlatedwith an immediate increase in DA concentrations as measured by in vivoelectrochemistry. In summary, the findings of experiment one and twosuggest that rats titer self-administered i.v. amphetamine to maintain asteady optimal extracellular level of DA in the brain that exceeds areinforcement threshold.ivTable of ContentsAbstract iiList of Figures viList of Tables ixAcknowledgements xI. INTRODUCTION 1(A) Neurochemical Correlates of Positive Reinforcement 4(B) Positive Reinforcement and Drug Self-Administration 8(C) ‘Craving’ and Drug Self-Administration 14(D) Neurochemical Correlates of ‘Craving’ 15(E) Purpose of the Present Studies 20II. METHODS(A) General Methods 21(B) Electrochemical Procedures 25(C) Microdialysis Procedures 32III. EXPERIMENT ONE(A) Introduction 37(B) Methods 38VIV. EXPERIMENT TWO(A) Introduction 44(B) Methods 46V. RESULTS(A) Experiment One: In Vivo Electrochemistry 49(B) Experiment One: In Vivo Microdialysis 63(C) Experiment Two 85VI. DISCUSSION(A) Experiment One 98(B) Experiment Two 104VII. REFERENCES 111viList of FiguresFigure 1: Representative voltammogram 27Figure 2: Illustration of brain coronal sections showing electrode sitesfor experiment one. 50Figure 3: Graph of interval-interfusion intervals for self-administrationof 0.05, 0.10 and 0.20 mg/infusion of amphetamine in theelectrochemistry experiment. 53Figure 4: Changes in behaviour and electrochemical dopamine signalscorresponding to the self-administration of 0.05 mg/infusion ofi.v. amphetamine 55Figure 5: Changes in behaviour and electrochemical dopamine signalscorresponding to the self-administration of 0.10 mg/infusion ofi.v. amphetamine. 57Figure 6: Changes in behaviour and electrochemical dopamine signalscorresponding to the self-administration of 0.20 mg/infusion ofi.v. amphetamine. 59Figure 7: Dose-related differences in duration of change inelectrochemical DA signals corresponding to the selfadministration of 0.05, 0.10 and 0.20 mg/infusions of i.v.amphetamine. 61Figure 8: Illustration of brain coronal sections showing probe sites forexperiment one. 64viiFigure 9: Graph of interval-interfusion intervals for self-administrationof 0.05, 0.10 and 0.20 mg/infusion of amphetamine in themicrodialysis experiment. 67Figure 10: Changes in behaviour and dialysate dopamine concentrationscorresponding to the self-administration of 0.05 mg/infusion ofi.v. amphetamine. 69Figure 11: Changes in behaviour and dialysate dopamine concentrationscorresponding to the self-administration of 0.10 mg/infusion ofi.v. amphetamine. 71Figure 12: Changes in behaviour and dialysate dopamine concentrationscorresponding to the self-administration of 0.20 mg/infusion ofi.v. amphetamine. 73Figure 13: Differences in duration of change in dialysate dopamineconcentrations corresponding to the self-administration of0.05, 0.10 and 0.20 mg/infusion of i.v. amphetamine. 76Figure 14: Changes in behaviour and dialysate DOPAC concentrationscorresponding to the self-administration of 0.05 mg/infusion ofi.v. amphetamine. 79Figure 15: Changes in behaviour and microdialysis DOPACconcentrations corresponding to the self-administration of0.10 mg/infusion of i.v. amphetamine. 81VIIIFigure 16: Changes in behaviour and microdialysis DOPACconcentrations corresponding to the self-administration of0.20 mg/infusion of i.v. amphetamine. 83Figure 17: Illustration of brain coronal sections showing electrode sitesfor experiment two. 87Figure 18: Changes in behaviour and electrochemical dopamine signalscorresponding to an abstinence from drug self-administrationfor rats ci, c2 and c3. 90Figure 19: Changes in behaviour and electrochemical dopamine signalscorresponding to an abstinence from drug self-administrationfor rats c4 and c5. 92Figure 20: Changes in behaviour and electrochemical dopamine signalscorresponding to an abstinence from drug self-administrationfor rat c6. 94Figure 21: Average electrochemical dopamine signal corresponding tovarious time points during an abstinence from drug selfadministration. 96ixList of TablesTable 1: Number of Bar Presses Per Hour Averaged for Six HourBlocks 86xAcknowledgementsI would like to thank my advisor, Dr. Charles D. Blaha, for his guidanceand support over the years. I would also like to thank Dr. Anthony G.Phillips for his help on many aspects of this project.Many thanks to Dr. Ariane Coury, Dennis Fiorino and Soyon Ahn fortheir work on the microdialysis portion of these projects and to FredLePiane for programming the MANX, repairing my equipment, etc, etc...Thanks as well to Penny Lam for helping with the ‘dirty work’, to PaulMackenzie for reading preliminary drafts of my thesis, to Charles Yang forthe use of his computer, to Jeremy Seamans for help with some of myfigures and to everyone else in the lab for helping to make my work hereenjoyable.1INTRODUCTIONDrug abuse is a major social problem. It is estimated that in the UnitedStates alone, 1 to 3 million people are in need of treatment for cocaineaddiction, a figure about 6 times greater than that for heroin addicts (forreview, see: Gawin, 1991). In particular, the use of the psychomotorstimulants, cocaine and the amphetamines, is on the rise with the recentpopularization of the free base form of cocaine, crack, and derivatives ofamphetamine such as ‘speed’ and ‘meth’ (Robinson and Berridge, 1993). Ofspecial interest is the observation that drug users continue to abuse theirsubstance of choice despite severe physical, social or financialconsequences (for reviews, see: Robinson and Berridge, 1993; Gawin,1991; Johanson and Fischman, 1989). These factors suggest that theprocesses contributing to drug abuse are both powerful and complex.Therefore, a detailed understanding of the behavioural and neurobiologicalfactors that contribute to the initiation and long-term use ofpsychostimulants is essential to the success of any program attempting tocontrol abuse of these drugs.Psychologists, and biopsychologists, in particular, can offer a uniqueperspective to drug abuse research. A biopsychological approach to thestudy of drug abuse emphasizes the interactions between learning andprevious experience and the physiological mechanisms which contribute tothe acquisition and maintenance of drug use. An understanding of theseissues will lead to strategies for the development of pharmacologicaltreatments of drug abuse. Theoretically, if one could block or prevent theneural correlates of drug-taking behaviour, then the inherent addictive2properties of the drug could be abolished (for a review of pharmacologicalinterventions of drug taking, see: Kosten and Kosten, 1991).Currently, one of the most widely held theories of drug abuse is thereinforcement model (Beninger, 1983; Wise, 1987; White and Mimer,1992). Reinforcement in this framework does not refer to an internal stateof ‘pleasure’ or ‘hedonia’. Rather, reinforcement is a behavioural anddescriptive term referring to the strengthening of any response due to itsconsequences (strengthening means that the response will occur withgreater probability in the future). The opposite of reinforcement ispunishment: the weakening of a response due to its (undesirable)consequences. Positive and negative reinforcement refer to thestrengthening of a response due to the onset of an (inferred hedonic) event,and the offset of an (inferred aversive) event, respectively (Rescorla, 1988;Robinson and Berridge, 1993).For a stimulus to act as a reinforcer, the operant behaviour associated withthe stimulus must obey certain principles. First, an animal must be able toacquire a new response to obtain the reinforcer. Second, the response-reinforcer contingency must be such that the response immediatelyprecedes the reinforcer. Third, the operant behaviour must be subject toextinction (Pickens and Thomson, 1968). In the context of drug abuse,extinction occurs when a substance that is self-administered fails to retainits reinforcing properties. This can happen when the reinforcer is removed,or when the contingency between a response and its consequences isaltered. In all cases, the operant response fails to have any reinforcingconsequences (Grove and Schuster, 1974). Extinction can be observed in3the laboratory by substituting a reinforcing drug with an inactive agent ordrug vehicle or by reversing the drug-response contingency. Extinction-like responding is characterized by an initial increase in responding (burst)followed by a decline and eventual cessation in operant responding(Pickens and Thomson, 1968; Grove and Schuster, 1974; Yokel and Wise,1975; Roberts, Koob, Kionoff and Fibiger, 1980; Roberts, Loh andVickers, 1989).The majority of biopsychological hypotheses of drug abuse attempt to linkreinforcement with neurochemical events. Most of these hypotheses havein common the premise that psychomotor stimulants are reinforcingbecause of their pharmacological influence on central dopaminergic systems(Yokel and Wise, 1975; Wise, 1978; Beninger, 1983; Carr and White, 1986;Wise and Hoffman, 1992; Caine and Koob, 1993). These hypotheses arealso concerned with two different, and interacting, aspects of drug use:acquisition of drug use and long-term maintenance of drug abuse. Asnoted above, the positive reinforcement theory of drug use states that drugtaking behaviour is acquired because the drug is reinforcing. Thereinforcing aspects of the drug are thought to be due, in part, to its abilityto selectively increase extracellular levels of the neurotransmitter dopamine(DA) in specific forebrain structures. In contrast, maintenance of drugabuse is hypothesized to be due to a state of ‘anhedonia’ associated with anacute depletion of brain extracellular DA during an abstinence (Dackis andGold, 1985; Gawin and Kieber, 1986). This dopamine depletion hypothesismaintains that the anhedonia associated with withdrawal is alleviated by theincrease in DA concentrations that results from the reinitiation of drugtaking.4The present thesis was conducted to examine these two hypotheses ofreinforcement and drug abuse. Specifically, changes in extracellular DAlevels in the forebrain of the rat were measured in vivo during theacquisition and maintenance of intravenous (i.v.) amphetamine self-administration as a direct test of certain predictions of the reinforcementand dopamine depletion hypotheses. Before outlining these hypotheses,several issues will be addressed. The first establishes the role of DA inreinforcement. The second provides evidence that psychomotor stimulantsserve as reinforcers.Neurochemical Correlates of Positive ReinforcementOver the past two decades, a great deal of basic research has been aimed atidentifying the neurochemical substrates of reinforcement. These studieshave led to the hypothesis that both reinforcement and locomotor outputare mediated by mesocorticolimbic dopaminergic systems (Jackson, Andenand Dahlstrom, 1975; Mogenson and Phillips, 1976; Kelly, Seviour andIversen, 1975; Joyce and Koob, 1981). In particular, changes inextracellular DA concentrations in the nucleus accumbens (N.Acc.), alimbic structure and major terminal site of these neurons, are thought to beassociated with reinforcement (for reviews, see: Wise, 1987; Robinson andBerridge, 1993). The N.Acc. is currently considered to be the majorinterface between the limbic system and sensory-motor control systems andtherefore seems to be an ideal locus for the regulation of reinforcedbehaviours (for reviews, see: Mogenson, Jones and Yim, 1980; Beninger,51983; Wiliner, Ahienius, Muscat and Scheel-Kruger, 1991; Mogenson andYang, 1991).Evidence linking DA in the N.Acc to the self-administration of drugs ofabuse comes from studies of the effects of DA receptor antagonists, DAagonists and lesions of the DA system on self-administration behaviour (forreviews, see: Wise and Hoffman, 1992; Koob, 1992). DA receptorblockers are typically given to animals prior to a self-administration sessionon the assumption that a substance which partially blocks thepharmacologically reinforcing properties of a drug of abuse will cause acompensatory increase in responding for the drug (Pickens and Thomson,1968; Yokel and Pickens, 1974; Wilson, Hitomi and Schuster, 1971).Consistent with this notion is the finding that rats pretreated with the DAreceptor blockers pimozide and butaclamol showed accelerated respondingfor i.v. amphetamine (Yokel and Wise, 1975, 1976). These effectsappeared selective, because norepinephrine (NE) receptor antagonistsphentolamine, phenoxybenzamine and l-propranolol were without effect.Similarly, pretreatment of rats with the DA receptor antagonist haloperidol(Roberts, Loh and Vickers, 1989) and other typical neuroleptics (Robertsand Vickers, 1984) has also been shown to increase response rates forcocaine. Collectively, these data have shown that pharmacologicalblockade of central DA receptors attenuates responding for reinforcers aspredicted by a dopaminergic theory of psychostimulant reinforcement.When the reinforcing efficacy of a drug is completely abolished (not justattenuated), extinction-like responding has been typically observed.Extinction-like responding has been reported following systemic6administration of very high doses of DA antagonists (Yokel and Wise,1976; Roberts, Loh and Vickers, 1989) and after selective neurotoxiclesions of the mesocorticolimbic DA system, particularly of the N.Acc. Inthe case of cocaine self-administration, extinction-like responding is seenfollowing 6-hydroxydopamine lesions of the ascending DA system. Thedegree and duration of these behavioural responses corresponded with theextent of DA depletion in the N. Acc. (Roberts, Koob, Kionoff and Fibiger,1980). In contrast, selective destruction of the dorsal or ventral NEbundles were without effect (Roberts, Corcoran and Fibiger, 1977).Decreased responding for cocaine and amphetamine following discretelesions of the N.Acc. has also been reported (Roberts and Koob, 1982;Lyness, Friedle and Moore, 1979). The anatomical specificity of the N.Accin the self-administration of drugs was further supported by the finding thatlesions of other DA-rich areas in the brain, such as the prefrontal cortexand striatum, are without effect on the self-administration of psychomotorstimulants (Martin-Iversen, Szostak and Fibiger, 1986).Recent biochemical and receptor-binding studies have provided a uniqueapproach to the study of the role of N.Acc. DA in the self-administrationof psychomotor stimulants. In one study, genetic strains of ratspredisposed to self-administer drugs of abuse were found to have relativelyhigher brain levels of tyrosine hydroxylase, the rate-limiting enzyme for DAsynthesis (Beitner-Johnson, Guitart and Nestler, 1991). This differencewas found mainly in the N.Acc. but not in the DA-rich regions of thestriatum. As well, Ritz, Lamb, Goldberg and Kuhar (1987) have found apositive correlation between the binding efficacy of cocaine to the DAtransporter in the N.Acc. and the potency of the drug in maintaining self-7administration for cocaine. Overall, these studies lend support to previousbehavioural data pointing to a critical role for DA in the N.Acc. in the self-administration of drugs of abuse.Studies of the unconditioned effects of cocaine and the amphetamines havealso provided support for a dopaminergic hypothesis of drug abuse.Cocaine and amphetamine are potent indirect DA agonists, acting toincrease extracellular levels of DA through synaptic reuptake blockade orvia facilitated-exchange diffusion, respectively (Fischer and Cho, 1979; forreview, see: North, 1992). Amphetamine enters the nerve terminal via aDA uptake carrier and releases DA into the cytosol from newly-synthesizedDA storage vesicles. This, in turn, enhances synaptic concentrations of DAby facilitating (via the uptake carrier) the diffusion of cytosolic DAthrough the nerve terminal (Fischer and Cho, 1979). In humans, bothcompounds are strong euphorics with. hedonic qualities (Chait, 1993;Robinson and Berridge, 1993; Dackis and Gold, 1985) and can increasespontaneous motoric activity (Kelly, Seviour and Iversen, 1975; forreviews, see: Gawin, 1991; Johanson and Fischman, 1989). In vivoneurochemical studies provide further evidence that cocaine andamphetamine enhance DA neurotransmission. For example, systemicamphetamine and cocaine injections dose-dependently increase DAextracellular levels in both the striatum and N.Acc. as measured by in vivomicrodialysis (Segal and Kuczenski, 1992; Segal and Kuczenski, 1992b;Kuczenski and Segal, 1989; Kalivas and Duffy, 1990; Nicolaysen, Pan andJustice, 1988; Coury, Blaha, Atkinson and Phillips, 1992; Di Chiara,Acquas and Carboni, 1992).8Positive Reinforcement and Drug Self-AdministrationThe self-administration of drugs is the prototypical example of drugreinforcement. Evidence that drugs of abuse are positively reinforcing toanimals comes from the demonstration that animals will self-administermany of the drugs that are abused by humans including the amphetamines,narcotic analgesics, tetrahydrocannabinol (THC), cocaine, somebarbiturates, nicotine, opiates, phencyclidine, and ethanol (van Ree,Slangen and deWied, 1978; Brady, 1991; Iwamoto and Martin, 1988).Pickens and Thomson (1968) conducted an elegant study to demonstratethat the use of cocaine obeys the principles of reinforcement. In this study,animals self-administered cocaine only when the response and the drugadministration were contingent. Moreover, the response for drugunderwent rapid extinction when it was replaced by saline or when theresponse-drug contingency was reversed. Grove and Schuster (1974) alsoreported extinction-like responding in monkeys when saline was substitutedfor cocaine during a self-administration session. Consistent with thesefindings, Roberts, Loh and Vickers (1989) found that the ‘breaking point’for responding for cocaine increased when higher doses were available torats. An observed decrease in the ‘breaking point’ following neurolepticpre-treatment is also consistent with the idea that the increase in responserate is due to a DA antagonist-induced decrease in the reinforcing efficacyof the drug. The ‘breaking point’ is considered to be a direct measure ofreinforcing efficacy. By gradually increasing the number of responsesrequired to receive the reinforcer, a point is reached where the animal failsto respond for the drug. This is thought to represent the point at which the9‘price’ the animal has to pay for the reinforcement is no longer ‘worth’ thepayoff. Thus, compounds which can sustain relatively high ‘breakingpoints’ are thought to have relatively high reinforcing value (for review,see: Brady, 1991). Overall, these studies demonstrate that psychomotorstimulants are reinforcing.A variety of factors influence the self-administration of drugs. Two ofthese are the dose of drug available and the schedule of reinforcement.Typically, when allowed access to a given dose, animals will initially ‘loadup’. It is during this phase that peak levels of drug are reached in theanimal’s system. Following this stage, animals typically administer the drugat evenly spaced intervals (maintenance phase) (Yokel and Pickens, 1974;Wilson, Hitomi and Schuster, 1981). Response rates vary with dose. Asthe dose is increased from threshold levels of drug detection, animals willincrease their rate of responding. As the dose continues to increase, apoint will be reached where response rates decrease. This inverted U-shaped function is frequently reported in the drug self-administrationliterature for both amphetamine and cocaine (Pickens and Thomson, 1968;Yokel and Pickens, 1973; Yokel and Pickens, 1974; Wilson, Hitomi,Schuster, 1971; for review, see: Brady, 1991). The finding that thedecrease in rate of responding at higher doses is unrelated to any motoriceffects at these doses has led to the suggestion that the animal may be‘titrating’ the level of drug intake when either the drug dose or schedule ofreinforcement is changed (Pettit and Justice, 1991).Another factor influencing drug self-administration is the schedule ofreinforcement. Animals can be trained to bar press for a drug infusion on10interval or ratio schedules. Interval schedules involve the receipt of drugfollowing the first bar press after an experimenter-determined length oftime. Ratio schedules require that animals respond a certain number oftimes before the drug is administered. Typically, when an animal is trainedon a given schedule, it will administer a constant amount of drug over time.When the response requirement is increased, the animal will adjust itsresponding to maintain a constant amount of drug intake over time(Pickens and Thomson, 1968). For example, if trained on a fixed ratio 1(FR1) schedule, an animal will receive a drug infusion after every response.When the response requirement is changed to FR2 during the session, theanimal will increase its rate of responding (for review, see: Wallace andSinger, 1976).Several hypotheses have been put forth to explain the titration of drugs inself-administering rats (for review, see: Wilson, Hitomi and Schuster,1971). One proposal is that drug titration serves to maintain an optimalblood level of drug. This optimal level corresponds to a concentration atwhich the drug is maximally reinforcing. A similar hypothesis is thatduring to self-administration of a drug, a certain concentration ofextracellular DA, corresponding to an optimal reinforcement threshold, isachieved in brain (for review, see: Wilson, Hitomi and Schuster, 1971).Once this occurs, the animal does not administer drug again untilcorresponding DA levels begin to decline. Reinitiation of selfadministration of drug is associated with a drop in DA levels below athreshold value. A third hypothesis of titration posits that too much drugis aversive and therefore the animal stops administering drug until the11aversive effects diminish. At this point, the animal will again administeranother dose of drug.Most of the experimental evidence supports the hypothesis that animalstitrate drug intake to maintain optimal reinforcement efficacy, above areinforcement threshold, as defined by corresponding blood levels of thedrug and DA extracellular levels in the brain. Evidence for a reinforcementthreshold comes from a study by Risner and Jones (1976), in which ratspretreated with amphetamine, decreased their intake of drug whenpermitted to self-administer amphetamine. The rats titrated their intake sothat the total amount of drug received per hour, including the pretreatmentdose, was the same as the amount previously self-administered without anypretreatment. This effect was not seen following pretreatment with theselective noradrenergic agonist methoxamine, suggesting that the effectwas specific to the DA system.The ‘titration’ hypothesis of responding seems to be more complex than asimple inverse relationship between dose and response rate. Although sucha relationship exists, it does not appear to be linear. Reports indicate thatanimals will self-administer, in total, greater amounts of drug administeredin higher doses, both in the ‘loading’ and ‘maintenance’ phase (Yokel andPickens, 1973). Yokel and Pickens (1974) calculated from knownmetabolic rates that blood levels of both d- and 1-amphetamine isomersincreased with the dose of drug administered. This was confirmed in thesame set of studies by monitoring blood levels of14C-amphetamine duringself-administration. However, dose-dependent increases in blood and bodylevels of drug was evident only during the first 2 hours of self-12administration. During the subsequent maintenance phase, drug levelsremained constant across doses. Experimental evidence also suggests thatanimals self-administer greater amounts of drug at higher doses because itis more reinforcing. Iglauer, Liewellyn and Woods (1976) reported thatwhen given the choice of different doses of cocaine, all normally self-administered, monkeys consistently administered the highest dose available.Support for the hypothesis that higher doses are more reinforcing alsocomes from the previously mentioned study demonstrating that increases in‘breaking points’ were observed at higher doses (Roberts, Loh andVickers,1989). Collectively, the results from these studies support thehypothesis that animals titrate their intake of drugs to achieve and maintainan optimal level of reinforcement. Consistent with this is the finding thatblood levels of drug are also kept at a stable, and supposedly optimal,level. As mentioned above, this optimal level seems to vary with doseonly during the loading phase, after which stable intake is achieved over arange of different doses.The recent advent of in vivo neurochemical techniques has allowed for themonitoring of dynamic changes in brain DA extracellular concentrations infreely-moving animals. These technological advances have been applied toanimals self-administering drugs. Accordingly, one now can test thehypothesis that animals will self-administer cocaine and amphetamine tomaintain an optimal reinforcement threshold which corresponds to anoptimal extracellular brain level of DA. Using in vivo microdialysis, Weiss,Hurd, Ungerstedt, Markson, Plotsky and Koob (1992) monitored DA levelsin the N.Acc. in animals self-administering i.v. cocaine. Initially, anincrease in DA levels was seen as the animals began to self-administer the13drug. Following the loading phase, responding shifted to a more regularinter-response interval and DA levels stabilized around a mean for the restof the self-administration session. Substitution of cocaine with salineproduced a brief extinction-like burst of responding which was correlatedwith the start of a gradual return to baseline DA levels. Pettit and Justice(1989; 1991) investigated the effects of different doses of cocaine onresponse rate and corresponding changes in extracellular DA using in vivomicrodialysis in the N.Acc. These investigators reported a typical inverserelationship between dose and responding. However, this relationship wasnot linear (i.e. greater amounts were administered at the higher doses).This non-linearity was also reflected in brain DA concentrations. Althoughanimals maintained steady levels of extracellular DA during a givensession, the level varied with dose. Increased doses resulted in higheroverall intake and in the maintenance of greater extracellular DAconcentrations at all times during the self-administration session. Theresults of these three studies are consistent with the notion that optimalblood levels of drug are obtained for each dose of cocaine administered byrats and they suggest further that optimal levels of DA exceed areinforcement threshold during a given self-administration session. Inaddition, in contrast to those findings by Yokel and Pickens (1974) withamphetamine, Pettit and Justice (1991) have suggested that the absolutelevel of DA varies with dose, reflecting a change in reinforcement efficacyof different doses of cocaine.14‘Craving’ and Drug Self-AdministrationOne of the most elusive features of drug abuse is its chronic compulsivenature. The reinforcement model has been useful in understanding some ofthe variables involved in drug use, however, it does not account for thetransition from drug use to abuse. In particular, a defining characteristicof drug abuse is relapse. Drug abuse is, in effect, a chronic relapsingdisorder: rehabilitated addicts will often begin to take drugs again monthsafter detoxification (Robinson and Berridge, 1993). A powerful subjectivestate reported to contribute to relapse in humans is ‘craving’ (Pickens andJohanson, 1992). ‘Craving’ is thought to be associated with an intense needor ‘wanting’ for drug that is not motivated by its hedonic properties(Robinson and Berridge, 1993). Although the basis for ‘craving’ is poorlyunderstood, various researchers have put forth different hypotheses for itsmechanisms. One of these is the dopamine depletion hypothesis put forthby Dackis and Gold (1985) who propose that ‘craving’ is associated withthe dissipation of a drug effect or with the abrupt cessation of drug use.This absence of drug is associated with subtle withdrawal symptoms thatare typically opposite to the initial euphoric and stimulant effects of thedrug. The most frequently reported symptoms are decreased energy,hypersomnia, irritability, dysphoria, depressed mood and psychomotorretardation (Dackis and Gold, 1985). In short, abstinence is associatedwith withdrawal and anhedonia. Anhedonia in this context is defined as ‘aninability to derive pleasure from normally pleasurable substances’ (Koob,1992). According to the dopamine depletion hypothesis (Dackis and Gold,1985), the user craves drugs to relieve the anhedonia associated withwithdrawal from drug: drug use during an abstinence therefore acts as a15negative reinforcer. Dackis and Gold (1985) suggest that drug abuse ‘is infact an interplay between positive and negative reinforcers’.Neurochemical Correlates of CravingGiven the evidence for a role of elevated concentrations of extracellularDA in positive reinforcement (reviewed above), Dackis and Gold (1985)propose that DA may be critically involved in the negative reinforcementproduced by the reinitiation of drug use after an abstinence. Dackis andGold (1985) argue that since an elevation of synaptic DA concentrationsmay be critical to the ‘hedonic’ effects of psychomotor stimulants, it is alsopossible that an acute reduction in extracellular DA concentrations may beassociated with the anhedonia observed during a drug abstinence.According to the dopamine depletion hypothesis (Dackis and Gold, 1985),drug taking in response to ‘craving’ during withdrawal serves as a negativereinforcer by increasing extracellular levels of DA, thereby attenuating thestate of anhedonia. Based on evidence obtained from animal studies on thelong-term effects of cocaine administration, Dackis and Gold (1985)propose that DA levels are decreased during a period of abstinence throughdepletion of intracellular DA stores. Cocaine is a potent reuptake blocker,resulting in increased extracellular levels of DA. Hypothetically, oneconsequence of reuptake blockade is an increase in extracellularmetabolism of DA. Therefore, normal recycling and reuse of released DAis no longer available to the neuron. Citing evidence that increased levelsof tyrosine hydroxylase are found in neurons after the long-term use ofcocaine, Dackis and Gold (1985) hypothesize that, as a consequence of16prolonged cocaine-induced reuptake blockade and increased extracellularDA metabolism, compensatory increases in intracellular synthesis of DAoccur. Eventually an inability of intraneuronal DA synthesis to fullycompensate for the increased use of DA may lead to a DA ‘shunt’, ordepletion of DA in the nerve terminal. A prediction of the dopaminedepletion hypothesis (Dackis and Gold, 1985) is that reinitiation of drugself-administration after an abstinence would be associated with the timeduring an abstinence period at which extracellular DA levels are at theirlowest. Therefore, drug use during an abstinence acts as a negativereinforcer and the dopamine depletion hypothesis would further predictthat extacellular DA levels should again rise immediately after thereinitiation of drug self-administration.The present thesis will test the predictions of the dopamine depletionhypothesis. However, first the literature establishing anhedonia as acorrelate of withdrawal and the evidence establishing an associationbetween decreases in DA and anhedonia will be briefly reviewed.Evidence linking anhedonia with drug abstinence comes from work withintracranial self-stimulation (ICS S). Administration of psychomotorstimulants is known to potentiate responding for ICSS (for a review, see:Koob, 1992b), measured by increased rates of responding and decreasedreward thresholds. Since amphetamine increases brain DA levels, thepotentiation of responding for ICSS is thought to be due to the additiveeffects of the drug and ICSS on DA neurotransmission. During drugabstinence and withdrawal, however, ICSS responding is attenuated: i.e.,response rates are lowered and reward thresholds are increased. (Koob,171992; Kokkinidis and McCarter, 1990). A decrease in response rate isthought to be a type of behavioural depression associated with anhedonia(Dackis and Gold, 1985). As well, Paulson, Camp and Robinson (1991)reported attenuated behavioural responses following amphetamineinjections during withdrawal in rats. Both spontaneous locomotor activityand the locomotor response to a challenge injection of amphetamine weredecreased during withdrawal from amphetamine. These responses wereattenuated to the levels seen in drug-naive control rats given saline.Therefore, these studies demonstrate that drug abstinence and withdrawalare associated with anhedonia and behavioural depression.Evidence from a number of studies support the proposal that decreasedsynaptic DA levels are correlated with temporary cessation of respondingfor drug, specifically, in the associated state of anhedonia. Wise, Spindler,deWit and Gerber (1978) reported that high doses of the DA antagonistpimozide decreased responding for food in a manner similar to that seen inanimals who had their food reward withheld. The behaviour of rats givenpimozide before food access and of those not receiving food were similarto each other, but differed from rats allowed free access to food. Thissuggests that DA may mediate an animal’s experience of hedonic states, andtherefore blockade of synaptic DA transmission may attenuate the positiveeffects of reinforcers. Graeff, Leme and Silva (1965) demonstratedchanges in the amount of spontaneous locomotion after pretreatment withvarious dopaminergic agents. These investigators reported an increase inlocomotion following increases in DA due to administration of the DAprecursor 1-dihydroxyphenylalanine (L-DOPA). These L-DOPA-inducedincreases in spontaneous locomotion were attenuated by depletion of18intracellular DA stores with systemic injections of reserpine. Furthermore,reinstatement of the potentiated locomotor response seen with L-DOPAand MAOI corresponded with the time course of repletion of dopaminergicstores. Since locomotion is thought to be mediated by DA (Kelly, Seviourand Iversen, 1975) and the increase in locomotor response is highlycorrelated with the administration of reinforcers acting on the DA system,locomotor activity is thought to be a predictive measure of reinforcement.Therefore, if one accepts the relationship between locomotion andreinforcement, these findings suggest that behavioural depression, oranhedonia, is correlated with a decrease in synaptic DA concentrations.Direct evidence linking fluctuations in DA levels with drug abstinence orwithdrawal comes from recent in vivo microdialysis studies. Segal andKuczenski (1992; 1992b) found that dopaminergic responses to a drugchallenge were attenuated 48 hours after the end of pretreatment witheither amphetamine or cocaine. As well, Weiss, Hurd, Ungerstedt,Markou, Plotsky and Koob (1992) found direct evidence to support thehypothesis that a significant reduction in DA release corresponded with anabstinence from drug self-administration. Using in vivo microdialysis,Weiss et al. (1992) monitored the in vivo correlates of withdrawalfollowing free access to cocaine. Drug self-administration typically lastedbetween 9.5 and 21.5 hours. During this time, DA concentrations roseinitially with self-administration and remained elevated around a stablemean for the duration of the self-administration session. After a three hourperiod of self-imposed abstinence, saline was substituted for the drug,thereby imposing a withdrawal. Withdrawal was associated with a decreasein DA concentrations below pre drug-taking baseline levels. In support of19postsynaptic changes predicted by the dopamine depletion hypothesis, DAlevels began to decrease, in some animals, before the drug abstinence,despite continued drug administration. When the levels dropped below acertain baseline, responding stopped. However, these findings contradictthe dopamine depletion hypothesis because the dopamine depletionhypothesis predicts that responding should be highest when extracellularDA levels are at their lowest. The study by Weiss et al. (1992) isinconclusive because it does not provide a full test of the dopaminedepletion hypothesis, in particular, the predictions about the reinitiation ofresponding after an abstinence. Future studies must address the specificquestion of whether reinitiation of drug self-administration occurs whenextracellular DA levels are at their lowest and that reinitiation of drug useresults in an immediate increase in these levels and a return to normalpatterns of responding. Positive results would support a major tenet of thedopamine depletion hypothesis, namely that drug administration during awithdrawal serves as a negative reinforcer and that rats reinitiate drug selfadministration when DA synaptic concentrations are depleted.20Purpose of the Present StudiesTwo experiments were planned. In the first, the effects of different dosesof amphetamine on self-administration behaviour and on DA concentrationsin the N.Acc. were measured with in vivo electrochemistry and with in vivomicrodialysis. This study tested the hypothesis that animals self-administeramphetamine to maintain an optimal level of DA release, above a so-calledreinforcement threshold.The second study provided long-term measurement of DA in the N.Acc.during 24 to 48 hour sessions in which rats often cease to respond for i.v.amphetamine of their own accord. Therefore, it permitted a description ofsome of the changes in behaviour and in vivo DA corresponding to atemporary drug abstinence following self-administration of amphetamine asmeasured with in vivo electrochemistry. These studies therefore provideda direct test of some of the predictions of the dopamine depletionhypothesis.21METHODSGeneral MethodsApparatus for operant responding for food reinforcementRats were trained to bar press for food in ‘Plexiglas’ chambers (25 cm X30cm X 30cm) surrounded by a sound and light-proof black wooden box.One side of the Plexiglas chamber was made of stainless-steel with one 28VD.C. 170 nA light bulb (Spectro), an operant lever and pellet dispenserfixed to one wall of these chambers. The floor of the chamber was linedwith corn-cob bedding (Sanicel) and covered with a metal grid. Duringfood training a ‘session on’ light was present. The lights, operant lever,and pellet dispenser (with food hopper) were interfaced to a computersystem (MANX) (Gilbert and Rice, 1979).Training for foodPrior to surgery, rats were trained to bar press for 45 mg pellets (Noyes).Criterion was set at 150 bar presses in a 30 mm session, on an FR-2schedule, for 3 out of 4 days. During training, all rats were givenapproximately 12 gms of rat chow (Purina) per day in their home cage.Water was available ad libitum.IV catheter preparationCatheters were made from custom-made 22 gauge cannulae with elongatedends (5 mm) (Plastic Products Inc.). The bottom end of each cannula wasbent to a right angle and Silastic tubing (0.012 I.D.; 0.025 O.D.; Dow22Corning) was secured to the bottom end of the cannula. To secure thecatheter to the jugular vein, a small quantity of silicone gel (Home Seal)was fixed to the Silastic approximately 4 cm from the junction of theSilastic tubing and cannula (for 350-450 gm rats). For sterilizationpurposes, 70% ethanol was flushed through the catheters and the catheterswere then stored in 70% ethanol until use.IV catheter surgeryAll instruments were cold sterilized immediately prior to surgery with0.15% alkylbenzyldimethylammonium chloride (ADC; EMI Industries) for20 mm, followed by 70% ethanol for at least 5 mm. The jugular catheter,suture strings and wound clips were all sterilized with 70% ethanol for 30minutes. Rats were anaesthetized with xylazine (9 mg/kg i.p.; Rompun)and ketamine hydrochloride (100 mg/kg i.p.; MTC Pharmaceuticals) andsupplemented with ketamine (- 20 mg) when needed. All rats wereimplanted with a single jugular catheter aimed at the left vena cava. Thecatheter was then tied and glued (LePage superglue) to the vein and thefree end of the cannula was passed subcutaneously to the top of the headand then cemented in place with dental acrylic cement (followingstereotaxic procedures). All catheterized rats were given garamycin (8 mgi.m.) and ampicillin (50 mg i.m.) prior to surgery and twice daily for oneday after surgery. Immediately following the catheterization, rats wereprepared for stereotaxic surgery and implantation of either electrochemicalelectrodes or microdialysis probes.23Catheter maintenanceAfter surgery, catheters were flushed twice daily with streptokinasekabikinase (0.2mg, i.v.; KABI) if clots were detected. Catheters wereflushed with 10 unit heparin (dissolved in sterile 0.9% physiological saline)during the experimental protocol, both prior to being connected to the testapparatus, and after being removed from the apparatus. Before flushing,catheters were swabbed with (0.85%) sodium chlorite (clidox; Pharmacal).Syringes for passing liquids through the catheters were stored in, and filledwith, 70% ethanol when not in use.Drugsd-Amphetamine sulphate was obtained from Smith Klein Beecham. Thedrug was mixed fresh daily in a concentration of 1 mg amphetaminesulphate per 1 ml of 1 unit heparin (dissolved in 0.9% sterile physiologicalsaline). One unit and 10 unit heparin solutions were made freshapproximately every three weeks. Heparin was purchased as aconcentrated solution and was diluted in 0.9% sterile physiological saline.Streptokinase was made fresh as needed (2 mg/i ml/1 unit heparinsolution). Clidox was made fresh every month. All antibiotics andanaesthetics were purchased as sterile solutions.I. V. injection procedureAll drugs were dissolved in a sterile heparin solution (1 unit/mI heparindissolved in 0.9% sterile physiological saline) and passed through a 0.22 i.m sterilizing filter (Lida Manufacturing Company) before use. All druginfusions were 100 tl in volume and given at a rate of 20 p.1/s.24I. V. self-administration procedureThe syringe pumps used to deliver infusions were interfaced to a computer(MANX) (Gilbert and Rice, 1979). The computer recorded the latency (inseconds) between successive infusions. Following each second bar-press,the MANX (Gilbert and Rice, 1979) delivered an infusion of drug to therat. This computer was also interfaced to the electrochemical detector(GMA Technology) so that each infusion was recorded and displayedgraphically as a tic mark as a function of time on the electrochemicalreadings. Therefore, a graph of the change in the electrochemical signaland the time at which drug infusions occurred were automatically plottedas a function of time on the same graph. Since drug infusions were plottedon this graph to the nearest minute, more accuracy in determining the timeof each infusion was obtained from the hard copy of inter-infusion latenciesobtained from the MANX system (Gilbert and Rice, 1979) (see page 31).HistologyRats were sacrificed with an overdose of ketamine (i.p., or i.v. if thecatheters were still patent at the time of sacrifice). Brains were thenremoved and stored in buffered 4% neutral formalin for at least 24 hoursbefore sectioning. Brains were sectioned into 50 im slices and every thirdslice through the N.Acc. was saved and mounted on glass microscopeslides. Brains were stained with cresyl violet and placements were verifiedunder a light microscope.25Electrochemical ProceduresIn vivo electrochemistry can be used to measure the steady state levels(concentration) and changes in the levels of oxidizable species at the tip ofa recording electrode placed in a brain area of interest (Stamford, 1986;Lane, Blaha and Han, 1987). When an electroactive species is oxidized atthe surface of a recording electrode in brain by application of a voltage viaan auxiliary (or counter) electrode, it loses electrons. The currentproduced by the flow of these electrons through the recording electrodecan be quantified. The amount of oxidation current detected at therecording electrode is proportional to the amount of species being oxidizedat the electrode tip. A reference electrode placed in an arbitrary area ofthe cerebral cortex provides a ‘reference’ ground potential from which toapply either positive or negative voltages to the brain. Individualelectroactive compounds in the brain typically oxidize at different appliedvoltages. Thus, by application of appropriate voltages, many oxidizablespecies in brain extracellular fluid can be quantified (Adams and Marsden,1982).Voltamme tryThe application of a voltammetric sweep is an electrochemical techniqueused to measure oxidation peak currents of several electroactive speciesand to evaluate the response characteristics of a recording electrodeimplanted in brain. To obtain a voltammogram, a range of potentials areapplied in the form of an ascending voltage ramp (typically 10 to 100mV/sec every 5 to 10 minutes). A plot of the resulting oxidation currentwith respect to the applied voltage sweep yields a plot of ‘oxidation waves’,26or voltammogram. The peak height of these waves are proportional to theconcentration of species in solution at the tip of the recording electrode.Semidifferentiation of the voltammetric current is a standard signalprocessing procedure which provides more clearly defined peak oxidationwaves. In brain tissue, DA oxidizes at a graphite paste recording electrodeat a potential of approximately +100 mV when scanned at 10 mV/sec(Figure 1) (Lane, Blaha and Han, 1987; Blaha and Jung, 1991; Blaha andLane, 1983).ChronoamperometryRepetitive square-wave pulse amperometry, or chronoamperometry, is anelectrochemical technique in which 1-second duration potential pulsescorresponding to the voltammetric peak oxidation potential of a species ofinterest is applied to a recording electrode (Adams and Marsden, 1982).The pulse is applied at an inter-sample interval of at least 30 seconds perelectrode. Each applied potential pulse results in a single current samplethat is directly proportional to the concentration of species in solution atthe electrode tip. By plotting these concentrations (oxidation currents)with respect to time, a temporal profile of the change in extracellularconcentrations of the electroactive species can be generated.ApparatusDuring daily sessions, rats were placed in 32cm X 32cm X 41cm Plexiglaschambers. Fixed to the outside of this box was a grounded Faraday cagedesigned to screen external 60 Hz electrical noise. The interior of the boxcontained a stainless-steel lever with a white cue light (28 V 170 nA;27Figure 1: Representative voltammogram recorded at a chronically-implanted stearate-graphite paste electrode in the nucleus accumbensof an awake, freely-moving rat. Oxidation current is plotted againstthe applied voltage (ramp ratelO mV/sec). Peaks 1-3 correspondto dopamine, serotonin and metabolites of DA, respectively.Dopamine peak oxidation typically occurs between approximately -150 and 200 millivolts.c C 0 D 0 C 0 C -o x 0-150-100-50050100150200250300350400450500VoltageAppliedvs.Ag/AgCI(mV)0029Spectro) directly above it. The bottom of the cage was lined with corn-cobbedding (Sanicel) and covered with a metal grid. Care was taken to ensurethat the inside of the box and the rat were not grounded. The testingchamber and surrounding Faraday cage were placed within a sound-proofand light-proof black wooden chamber. An electrochemical recording leadextended from the rat’s head to a mercury-filled commutator and liquidswivel mounted to the top of the Plexiglas box. Shielded co-axial cablesextended from the commutator and swivel ensemble up through the woodenbox to an electrometer device (E-Chempro, GMA Technologies, Inc.)mounted on top of the wooden chamber. I.V. tubing extended from theanimal to the liquid swivel and through the wooden chamber to a Harvardapparatus pump (Sage Apparatus, pump model 341) mounted on top of thewooden box. This pump and the electrometer device were then interfacedto a computer control system (MANX) (Gilbert and Rice, 1979).Electrode preparationA three-electrode electrochemical recording system was used in allexperiments. Recording electrodes were made from Teflon-coatedstainless-steel wire (0.008° bare, 0.011” coated; Leico Industries). TheTeflon was pulled away from the tip of the stainless-steel to create a smallwell of approximately 0.5 mm in depth. The well was then packed withstearate-modified graphite paste (Blaha and Lane, 1983; Blaha and Jung,1991) (graphite powder, liquid paraffin oil and stearic acid, in a 3:2:0.2wt./wt. mixture). The electrode tip was then surfaced on smooth glass toobtain a slightly convex graphite paste surface when viewed under a lightmicroscope (mag. X 625) (Blaha and Jung, 1991). The auxiliary electrodeconsisted of a stainless-steel wire (0.008” bare, 0.011” coated; Leico30Industries), covered with Teflon, except for -4mm from the end of theelectrode. The third type of electrode, the reference electrode, was madefrom Teflon-coated silver wire (0.003” bare, 0.0045” coated; LeicoIndustries). The Teflon was stripped away -‘1-2 mm from the end of the tipof the wire. The exposed end of the wire was chlorided in an electrolysisbath (Blaha and Lane, 1983; Stamford, 1986). The free ends of all threetypes of electrodes were soldered to miniature gold Amphenol pins.SurgeryRecording electrodes were aimed bilaterally at the N.Acc. (+1.2mmanterior to bregma, +/-1.2mm lateral to the midline and -6.5mm ventral tocortical dura; incisor bar -3.3mm; Paxinos and Watson, 1986). Care wastaken to remove all blood and dura prior to lowering the electrodes intobrain. A single reference electrode was implanted —‘2-3 mm posterior tobregma and lateral to the midline into the cortex of the right hemisphere.The electrode was lowered until the exposed chiorided tip was completelyimplanted in cortex. Four skull screws were then fixed in place to theskull. The exposed length of the auxiliary electrode was wrapped aroundone of the anterior skull screws. This screw was lowered its entire lengthinto brain (-‘1 mm). The gold pins from each electrode were seated into athreaded cylindrical mini-socket holder (Science Technology Centre,Carleton University) and mounted, along with the catheter inlet, to the topof the rats’ head with dental acrylic cement.Electrochemical recording proceduresOne day following surgery, recording electrodes were electrochemicallyconditioned overnight by applying voltammetric sweeps to each electrode31every hour. The day after being conditioned overnight, experimentaltesting began. During testing, voltammograms were obtained before eachtest session to determine the status of the electrode and the appropriateparameters for the applied potential pulse (chronoamperometry). All dailyvoltammograms were ramped from -150 mV to 450 mV at a rate of 10mV/see; a sweep was taken from each electrode every 5 minutes andapproximately 3 to 4 sweeps were collected for each electrode.To determine daily chronoamperometric pulse parameters, the value at thetrough of the DA oxidation wave served as the final pulse potential value.The initial pulse potential was set to a value 350 mV lower than the finalvalue (voltages were typically applied from -150 to +200 mV). If thetrough of the DA oxidation wave occurred at more than +275 mV, theelectrodes were considered no longer patent and the rat was discontinued.The rat was also discontinued if the chronoamperometric recording waspoor. An electrochemical signal was considered inadequate if largeamounts of electrical noise or frequent current ‘spikes’ greater thenapproximately 30 nanoamperes were present.Data collectionBehavioural and electrochemical data were collected separately.Behavioural data was recorded via a MANX (Gilbert and Rice, 1979)computer interface system which controlled the delivery of drug and food,as well as light cues. The behavioural data was given as the inter-infusiontime for each infusion each day and was collected daily as a hard copy fromthe MANX (Gilbert and Rice, 1979) computer. Electrochemical data wasinterfaced to a Hewlett-Packard, IBM-PC compatible, 386DX, 20MHz32computer. Custom-made batch files (GMA Computer Technologies, Inc.)designed for Lotus 1-2-3 version 2.2 (Lotus) converted the daily data foreach rat into Lotus worksheet graph files.Microdialysis ProceduresIn vivo microdialysis is the simultaneous sampling of a variety ofextracellular molecules by perfusion of a physiological buffer through aprobe inserted into a brain area of interest (Church, Justice and Neill,1987). The active portion of the probe consists of a semi-permeablemembrane. Artificial extracellular fluid (perfusate) (Moghaddam andBunney, 1989) is passed continuously through the probe via an inlet andthe molecules surrounding the probe travel down their concentrationgradient across the semi-permeable membrane and into the probe. In thismanner, a representative sample of the molecules in the brain extracellularcompartment can be collected as dialysate from an outlet tubing of theprobe. Once collected, this sample is analysed using standard analyticalmethods. In the present study, electroactive substances such as DA and itsmetabolites, 3,4-dihdroxyphenylacetic acid (DOPAC) and homovanillic acid(HVA), as well as the serotonergic metabolite 5-hydroxyindole-3-aceticacid (5-HIAA), were separated and quantified using high pressure liquidchromatography with an electrochemical detector (HPLC-ED).DOPAC is a presynaptic metabolite of DA formed principally in theintracellular compartment and is thought to reflect the rate of DAmetabolism intracellularly. The ratio of DOPAC/DA levels in tissue hasbeen used previously as an index of dopaminergic activity (Moore, Chieuh33and Zeldes, 1977; Westerink, 1979). In the case of uptake inhibitors suchas amphetamine, extracellular levels of DOPAC decrease following theiradministration (Kuczenski and Segal, 1989). In this study, both DA andDOPAC will be measured to demonstrate the known actions ofamphetamine and to provide confirmation of our in vivo DA results.ApparatusThe microdialysis chamber was a clear Plexiglas box (26.5 cm X 24cm X29cm) placed within a sound and light-proof white chamber. The bottomof the box was lined with corn-cob bedding and covered with a metal grid.The dialysis probe was connected to an outlet line which ran in parallelwith the i.v. line that extended through a dual liquid swivel (Instech, Inc.)to the i.v. (Sage Instruments, model 341A) and dialysis (HarvardApparatus, model 22) pumps.Probe preparationThe microdialysis probes were constructed from a semi-permeable hollowfibre (340 p.m O.D., 65,000 MW cutoff, Filtral 12, Hospal) with a 2mmexposed fibre length. A 55 cm length of PE 10 inlet tubing and a 55 cmlength of fused silica outlet tubing (75 p.m I.D. X 150 p.m O.D) were sealedto the membrane with epoxy (Devcon 2-ton). All joints and the dialysisfibre tip were sealed with epoxy.SurgeryTwo 23 gauge permanent guide cannulae with dummy probes were loweredbilaterally into brain above the target site (+1.8 mm AP; 1.0 mm ML;+I-1.0mm DV; -3.3 mm incisor bar; Paxinos and Watson, 1986). The cannulae34and i.v. catheter were fixed to the skull with dental acrylic cement,anchored by 4 skull screws.Probe insertionTo minimize the effects of tissue damage on concentrations of extracellularDA, rats were perfused for approximately 16 hours prior to collection ofsamples. Probes were inserted into brain the day before testing and wereaimed at the N.Acc. (+1.8 mm AP; 1.0 mm ML; +1-7.0 mm DV; -3.3 mmincisor bar; Paxinos and Watson, 1986) and fixed to the permanent guidecannula with brass holders (Fiorino, Coury, Fibiger and Phillips, 1993).During this time, perfusate was flushed through the probe at a rate of1.5 tl/min.Dialysate and perfusateDuring behavioural testing, collected dialysates were assayed for DA andmetabolites using HPLC-ED. The perfusion medium consisted of 0.01 Mphosphate buffer (pH=7.4) containing (mM) NA 147, K 3.0, Ca 1.3and Mg++1.0. A probe perfusion flow rate of 1.5 p.1/mm was used for allsamples collected.HPL C-EDThe compounds of interest in each dialysate sample were separated on areverse-phase chromatographic column (Beckman ultrasphere ODS 5p.m, 15cm length, 2.0 mm I.D.) using a 0.1M sodium acetate buffer (pH=3.5, 5%methanol). The glassy carbon working electrode was set at +0.65 V. Theapparatus consisted of a Waters 501 HPLC pump, a Waters U6K injector,and a Waters 460 electrochemical detector. Waters Maxima software was35used for calculation of chromatographic peaks corresponding to eachmeasured compound (DA, DOPAC, 5-HIAA, HVA).Both in vivo electrochemical and microdialysis techniques areimprovements over previous ex vivo methods of neurochemicalmeasurement. Traditional ex vivo methods relied on analysis of postmortem tissue, making data collection difficult and confounded by handlingeffects and by enzymatic-induced changes in neurotransmitter andmetabolite concentrations following death. Both electrochemistry andmicrodialysis techniques circumvent the reliance on post-mortem tissue.These procedures allow neurochemical species of interest to be monitoredin the extracellular fluid of intact tissue in awake or anaesthetized rats.Both techniques have strengths and limitations of their own.Electrochemistry allows measurements to be taken for prolonged periods oftime and over many consecutive days. This permits the use of powerfulwithin-subjects designs. Conversely, dialysis is a one-trial measurementlimiting analysis to the use of between-subjects designs. Althoughelectrochemistry allows finer time discrimination (30 sec sampling asopposed to a 15 mm sampling with dialysis), microdialysis permits a widerspectrum of neurochemical species to be monitored, including precursorsand metabolites. Another factor to be considered is the relative sizes ofthe electrochemical electrodes and microdialysis probes. Electrodes havesmaller diameters (outer diameter200 p.m) than microdialysis probes(outer diameter 300 p.m) (Blaha, Coury, Phillips and Fibiger, 1990).Microdialysis probes also have a longer active surface, 2-4 mm, andtherefore significantly greater sampling area (100-200 X) thanelectrochemical electrodes, which have a planar active surface (area = 1.836X 1o4 cm2). Therefore, electrochemical electrodes allow for a morelocalized monitoring of the sites in the brain. An integration of both invivo electrochemical and microdialysis techniques in any study can providethe benefit of both temporal resolution and a wealth of information aboutthe dynamics of neurotransmitters and their metabolites and also offercross-validation of methodological results.37EXPERIMENT ONEIntroductionThe first experiment was designed to measure changes in DA levels in theN.Acc. during self-administration of different doses of amphetamine (0.05,0.10 and 0.20 mg/infusion). Using both in vivo electrochemical andmicrodialysis techniques, DA levels in the freely-moving rat weremonitored during i.v. self-administration of 12 infusions of a given dose ofamphetamine. The effects of different doses of amphetamine on bothresponding for drug and in vivo DA concentrations were monitored. It washypothesized that an inverse relationship between dose and response ratewould be observed. It also was hypothesized that, consistent with areinforcement threshold concept, distinct ‘loading’ and ‘maintenance’phases would be seen, with the former correlated with maximal rises in DAconcentration, and the latter with the maintenance of a steady level of DAefflux around a stable mean. Consistent with findings by Yokel andPickens (1974), it was also hypothesized that rats would maintain a steadylevel of extracellular DA across all doses. This study represents the firstof its kind to monitor DA efflux during the self-administration of differentdoses of amphetamine by rats. As well, this study is important because itdirectly compares in vivo electrochemistry and microdialysis results duringmotivated behaviour. Although past studies have validated in vivoelectrochemistry with microdialysis pharmacologically, this study providesconvincing evidence that electrochemistry can be used to monitor DAefflux in behavioural studies.38MethodsElectrochemistry subjectsSubjects were 30 male Long-Evans rats (3 50-450 gms at the time ofsurgery) from Charles River Canada. Rats were housed individually inSanicel-lined cages and were given food and water ad libitum, exceptduring food training. The temperature in the colony was controlled at21°C and the humidity was constant at 40%. Rats were kept on a twelvehour light-dark cycle (0700h-1900h; lights on at 0700h) and were alwaystested during the light phase.Electrochemistry - behavioural methodBehavioural testing began three days after surgery. Animals had preexposure to the testing chamber during the overnight period of electrodeconditioning. On test days, rats were attached to the i.v. catheter line andthe electrochemical recording device. The chamber was dark during thesampling of voltammograms, and during a further 30 minutechronoamperometric baseline period. Food and water were not available atany point during testing. After chronoamperometric baselines wereestablished, rats were given a priming injection that was of the same doseas all other injections received that day. Following this, rats were given aninfusion of amphetamine following every second bar press (FR2 schedule).During the time in which drug was available, the house lights remained on(‘drug available’ cue). Following the prime and every subsequent infusion,the house light flashed for 5 seconds. This was followed by a 30 secondtime-out during which the house light was turned off and the operant leverwas inactive. The session was terminated when the rats had received 1239self-administered infusions (a total of 13 infusions including the primingdose). At the end of the session the house light and bar were inactivated.The rats remained in the test chamber until the chronoamperometric signalreturned to pre-infusion baseline levels. Testing sessions were typically 7-9 hours in length. Rats were tested daily for approximately eight days. Onthe first 4 days, rats were tested with 0.10 mg per infusion of amphetamine(the training dose and also medium dose). On the fifth day, rats eitherreceived 0.05 mg dose (low) or 0.20 mg dose (high) of amphetamine perinfusion. On the next two days the rats again received the training/medium(0.10 mg/infusion) dose. On the last day, rats received the low or highdose not previously experienced. The order in which the rats received thehigh and low dose was counterbalanced.Rats were eliminated from the experiment if they failed to bar press fordrug during the first self-administration session, if their catheter failed atany point during the experiment, or if their electrochemical signal waspoor. All rats with intact catheters and drug lines learned to bar pressduring the first session of drug self-administration.Statistical analyses - behaviourA two-way repeated measures ANOVA of dose x inter-infusion interval(INI) was conducted. For all analyses, comparisons were consideredsignificant if p<.05 (alpha.05).Statistical analyses - dopamineA two-way dose x time repeated measures ANOVA of the pre-drug baselineand first three hours of the drug effect was conducted. The purpose of this40analysis was to determine the maximal DA oxidation currents attainedduring a session (peak height). Prior to the analysis of electrochemicaldata, a linear transformation on the raw data was performed for each rat.A constant was subtracted from each data point in the electrochemicalrecord so that the oxidation current corresponding to the start of thesession time was at zero current. The transformed data from each rat oneach day were then averaged over successive 15 minute time intervals(bins). These mean current values were analysed by a two-way repeatedmeasures dose x time ANOVA. If a significant dose effect was found,interaction comparisons on the main effect of dose were conducted betweenall doses. This analysis considered the days on which rats received thehigh and low doses (test doses), and the medium (training) dose on the dayimmediately prior to receiving the first test dose. A DunnetCs post hocanalysis was conducted to determine when elevations in DA oxidationcurrents were significantly different from pre-drug baseline. For theDunnett’s test, mean DA oxidation current values corresp.onding to every15 minute time bin after the start of the self-administration session wascompared to a single control DA oxidation mean current value representingthe pre-drug baseline. All Dunnett’s tests were computed manually.However, the MSresidual for these analyses were computed separately withSystat® (Systat, Inc.).For the analysis of the duration of drug effect on the elevation in the DAoxidation signal, an estimate of the value for the duration was taken as thetime point where a line manually drawn through a hard copy of the predrug baseline intersected with the electrochemical signal. If there was nointersection point, the maximum duration of drug effect on the DA41oxidation signals was taken as the value where the post-drugelectrochemical signal became parallel with this line. A one-way repeatedanalysis of variance was conducted to test for a significant effect of doseon the duration of elevation in the DA oxidation signal. If a significantdose effect was revealed, interaction comparisons on the dose effect wereconducted between all doses.Statistical analyses - assumption of within-treatment homogeneityRepeated measures analyses are extremely sensitive to violations ofhomogeneity of within-treatment variances. Any violation of thisassumption can lead to erroneous rejection of the null hypothesis (type Ierror). When doing repeated measures analyses, most statistical packagesprovide correction factors for violations of the assumption of homogeneity,such as the Geisser-Greenhouse adjusted F value and the Hyund-Feldtadjusted degrees of freedom. However, caution must be used whenselecting a correction factor. The former overcorrects and the latter haslimited applicability. For the present analyses, we followed the suggestionof Keppel (1982) and used the Hyund-Feldt adjusted degrees of freedomfor all repeated measures analyses when the Hyund-Feldt 8 was below 0.75(denoted PHF in the text). For all other repeated-measures designs, theGeisser-Greenhouse correction was used (denoted PGG in text).Microdialysis subjectsSubjects were 17 male Long-Evans rats (3 50-450 gms) from Charles RiverCanada. All housing conditions were identical to those described in theelectrochemical study in experiment one.42Microdialysis - behavioural testingThree days after implantation of the dialysis guide cannula, amphetamineself-administration was begun. Rats were allowed to self-administer a totalof 12 infusions of 0.10 mg of amphetamine in daily sessions. After at leastthree days of stable responding at this dose, microdialysis probes wereinserted. Approximately sixteen hours after probes were inserted, ratswere dialysed. On the first day of dialysis, rats were given the medium(0.10 mg) dose of amphetamine. On the second day, rats were given eitherthe high (0.20 mg) or the low (0.05 mg) dose. All rats were dialysed onceon each side of the brain. All sessions were the same as those for theelectrochemical recording phase of this experiment. Animals were notincluded if they did not learn to bar press for drug or if they did not self-administer amphetamine within the first hour of a dialysis test day.Statistical analyses - behaviourSee electrochemical experiment.Statistical analyses - dopamineAnalyses of dialysate DA concentration were conducted as a function ofdose and time. A two-way between-subjects dose x time ANOVA wasconducted on the 15 minute samples of DA concentration (nanomolar; nM)in the dialysate. All other analyses of peak heights were the same as thosediscussed for the electrochemistry data. For the analysis of the duration ofdrug effect on the change in dialysate concentrations, the duration of thechange in dialysate DA concentration was taken as the time when DAconcentrations returned to pre-drug baseline concentrations. A one-wayANOVA of duration of elevated DA concentrations as a function of dose43was conducted. Dunnett’s tests were computed as in the electrochemistrydescribed above.Statistical analyses - DOPACAll analyses of dialysate DOPAC concentrations were the same as thosedescribed above for DA. Since decreases in DOPAC concentrations seenafter amphetamine treatment typically lasted longer than those for DA, thefull time-course of DOPAC concentration changes was not monitored.Therefore, an analysis of the total duration of effect of amphetamine onDOPAC dialysate concentrations was not conducted.44EXPERIMENT TWOIntroductionThe second experiment tested the effects of prolonged amphetamine self-administration on both response rate and in vivo DA oxidation currents inthe N.Acc. in an explicit test of some of the predictions of the dopaminedepletion hypothesis (Dackis and Gold, 1985). It was hypothesized that,consistent with the dopamine depletion hypothesis, periods of cessation ofamphetamine self-administration would be associated with a decrease in DAconcentrations. It was also hypothesized, consistent with the findings ofWeiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992), that DAlevels would drop to, or below, pre-amphetamine baseline levels, beforeand during the drug abstinence periods. The present experiment expandedon the findings of Weiss, Hurd, Ungerstedt, Markou, Plotsky and Koob(1992) by allowing rats to reinitiate responding for amphetamine after anabstinence. Therefore, this study was able to monitor the behavioural andneurochemical correlates of both the initiation and termination of a self-imposed period of abstinence in self-administration of amphetamine.Consistent with the suggestion of the dopamine depletion hypothesis thatDA may serve as a negative reinforcer, it was hypothesized that DAconcentrations would be at their lowest during the abstinence when ratsreinitiated drug self-administration and DA concentrations would increaseagain after reinitiation of drug self-administration. It is noteworthy thatthis is the first study to monitor the in vivo correlates of a reinitiation ofself-administration of amphetamine after an abstinence period, thusallowing for an explicit test of the dopamine depletion hypothesis. The use45of in vivo electrochemical techniques permit, not only a within-subjectsdesign, but also, for the first time, the evaluation of neurochemical changesin individual animals over a prolonged period of time.46MethodsSubjectsSubjects were 9 male Long-Evans rats (3 50-450 gms) from Charles RiverCanada. Housing conditions were the same as for the other studies.Behavioural testingIn this protocol, rats experienced a total of three drug self-administrationsessions. The first involved 12 infusions and a prime of amphetamine on anFR-2 response schedule (same as experiment one), the second lasted for 24hours and allowed free access to amphetamine on an FR-2 schedule, andthe final session allowed the animal free access to drug for 48 hours, alsoon an FR-2 schedule. The self-administration sessions were similar to thedose-response sessions of the first experiment with a few exceptions: 1) thedose of amphetamine was held constant at 0.10 mg per infusion, 2) ratswere not limited to 12 infusions during the 24 and 48 hour sessions, buthad access to amphetamine at all times and 3) rats were given food andwater overnight in the experimental chamber. After both the 24 and 48hour sessions, voltammograms were recorded to assess the condition andpatency of each electrode. The purpose of the first two sessions was toestablish reliable responding for amphetamine. Therefore, only the resultsfrom the abstinence periods during the third session are presented here.47Statistical analyses- behaviourFor each rat, the inter-infusion latencies were computed as number of barpresses per hour for each rat. A one-way repeated measures analysis of barpresses as a function of time was conducted.Statistical analyses - dopamineFor the test session in which rats were permitted 48 hours of continuousaccess to amphetamine, both the changes in DA oxidation current over the48 hours and during the drug abstinence periods were analysed. For theformer, the transformed (see experiment one, electrochemical study)chronoamperometric data from each rat was averaged over one hour timeperiods and a one-way repeated measures ANOVA was conducted. ADunnett’s post hoc test compared the average current values of eachconsecutive one hour time period to a single mean pre-drug baselinecurrent value (same procedure as experiment one, electrochemistry study).See note on assumption of homogeneity of within-treatment variance inexperiment one.An abstinence period was defined as the first pause in responding for drugthat lasted for more than 2 hours and that was initiated at least 20 hoursinto the 48 hour continuous access session. To analyse the change in DAoxidation current during the abstinence periods, six data points per ratwere considered: 1) the mean current values corresponding to the 5minutes immediately before and 2) after the last self-administered druginfusion before the abstinence period; 3) the mean current valuescorresponding to the 5 minute averages before and 4) after the timecorresponding to the midpoint between the last self-administered druginfusion before the abstinence period and the first self-administered druginfusion after the abstinence period; 5) the mean values corresponding tothe 5 minutes immediately before and 6) after the first infusion after theabstinence. A one-way analysis of variance followed by Newman-Keulspost hoc analysis was conducted on these six data points.4849RESULTSExperiment One: In Vivo ElectrochemistrySubjectsOf the 30 rats used in this experiment, six rats were excluded from analysesbecause of broken catheters, 15 were excluded because they had poorelectrochemical records, two were excluded because they received a drugthat was not amphetamine during at least one session and one rat wasexcluded because the behavioural testing protocol was not correct.Therefore, of the 30 rats used in the experiment, only six met the criterionfor inclusion in the analyses.HistologyHistological examination of the six rats showed that four had electrodesplaced bilaterally in the N.Acc. The remaining two rats had one of the twoelectrodes placed in the N.Acc. Only data considered from correctlyplaced electrodes were considered in the overall analysis. For subjectswith two electrodes placed in the N.Acc., the chronoamperometric recordwith the least amount of electrical noise was used (Figure 2).BehaviourAll rats learned to bar press (FR-2 schedule) for amphetamine on the firstday of drug exposure. At all doses, response rates were initially rapid andthen slowed to a constant rate for the duration of the self-administrationsession. The average and standard error of the mean (+/-SEM) for the50Figure 2: Representative sections showing the placement (solid circles) ofelectrochemical electrodes in the nucleus accumbens used in theanalyses of experiment one (redrawn from the atlas of Paxinos andWatson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations:nucleus accumbens (N.Acc.); anterior commissure (ac); corpuscallosum (cc); caudate putamen (Cpu).51AP+2.2mmAP +1.7mmAP+1.2mmAP+O.7mmI I I I I I I3210123Left Right52inter-infusion interval (INI) between each of the 12 self-administered druginfusions was 500 +1- 215 seconds for the 0.05 mg/infusion dose, 1336 +1-304 seconds for the 0.10 mg/infusion dose and 3030 +1- 723 seconds forthe 0.20 mg/infusion dose (Figure 3). A two-way repeated measuresanalysis of dose x INI confirmed that higher doses produced significantlylonger INIs [F(2,10)27, p<.O5] (For clarity, the behavioural data foreach group of rats were shown as the mean +/- SEMs number of barpresses over 15 minute time intervals in Figures 3, 4 and 5).Change in Dopamine Oxidation CurrentTransformed chronoamperometric records were averaged and the +/-SEMswere determined for the 0.05 mg dose, 0.10 mg dose and 0.20 mg dose ofamphetamine (Figures 4, 5 and 6). Figures 4, 5 and 6 illustrate that the DAoxidation current increased above pre-drug baseline following the self-administration of each dose of amphetamine tested. Within one hour of thestart of the session, DA oxidation currents plateaued at —6 nA for all threedoses. A two-way dose x time repeated measures ANOVA revealed thatthe difference in peak DA oxidation current between these doses was notsignificant [F(2,10)0.83, PGG>.05]. However, the total duration of thechange in the electrochemical signal increased as a function of dose ofamphetamine self-administered: larger doses resulted in longer plateaus. Aone-way repeated measures ANOVA revealed a significant dose-relatedchange in the total duration of amphetamine-induced increases in DAoxidation currents [F(2,10)32.43, PGG<•°5l• As shown in Figure 7,interaction comparisons revealed a significant dose-dependent increase induration of the change in DA oxidation current between all doses (the lowcompared to the medium dose: [F(1,5)14.47, PHF<.°5]the low compared53Figure 3: Plot of the average inter-infusion latencies and +1- SEMs for the12 i.v. self-administered infusions of 0.05 mg/infusion (dark solidline), 0.10 mg/infusion (light solid line) and 0.20 mg/infusion (darkstippled line) of amphetamine in the electrochemistry study.54C’)1c’J011-0)-Co-U)U)--L()--Cr)CbJ-I1I ICr) CsJ(0001 x ceo) ewi55Figure 4: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, rightaxis) and corresponding change in mean (+1- SEM) dopamineoxidation currents in the nucleus accumbens (top, left axis) of ratsself-administering twelve infusions of amphetamine (0.05mg/infusion). Dark and pale solid lines are the means and +/- SEMsfor the electrochemical data, respectively. Open symbols (boxes) arethe means and +/-SEMs for the electrochemical data averaged over15-minute time bins.ChangeinDopamineOxidationCurrent(nA)oDU!gjjsesSeJdiogO)O)CD IIIIII-1I-1I-I************3CD*0F’)CA)C)10)CDCD0*0013D-hC0DII0)II*-A9c57Figure 5: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, rightaxis) and corresponding change in mean (+1- SEM) dopamineoxidation currents in the nucleus accumbens (top, left axis) of ratsself-administering twelve infusions of amphetamine (0.10mg/infusion). Dark and pale solid lines are the means and +1- SEMsfor the electrochemical data, respectively. Open symbols (boxes) arethe means and +/-SEMs for the electrochemical data averaged over15-minute time bins.ChangeinDopamineOxidationCurrent(nA)-Cog/sesseJdJDQ[0-0)00IIIIIIIFFI-IH****3(DF-’1EICi)00)00(0-**********II0)Cl II* -c,AD01 8c59Figure 6: Bar graph of mean (+1- SEM) rate of bar pressing (bottom, rightaxis) and corresponding change in mean (+I SEM) dopamineoxidation currents in the nucleus accumbens (top, left axis) of ratsself-administering twelve infusions of amphetamine (0.20mg/infusion). Dark and pale solid lines are the means and +/- SEMsfor the electrochemical data, respectively. Open symbols (boxes) arethe means and +/-SEMs for the electrochemical data averaged over15-minute time bins.ChangeinDopamineOxidationCurrent(nA)I)O)OO************-3CDzHHHHHHH—IIHHHHC)C,,0)CoCo*********pF’)3Cl)0DII0)IIQ.CD*AC,,Uw\Jg[/sesseidJD0961Figure 7: Bar graph illustrating mean (+1- SEM) duration of effect of self-administered amphetamine on change in dopamine oxidation currentsin the nucleus accumbens as a function of dose (0.05, 0.10 and 0.20mg/infusion). The value for these effects corresponded to the timepoint where a line drawn manually through the pre-drug baselineintersected with the electro chemical signal on a hard copy of thedata. If there was no intersection point, the maximum duration ofeffect was taken as the time value where the post-drugelectrochemical signal became parallel with this best-fit line.ci0C’,CDCDD-hCC’,0pN)0DIIDIIDurationofDrugEffect(Hrs)N)0000bC),pCHIIZ963to the high dose: [F(1,5)58.71, PHF<.O5]and the medium compared tothe high dose: [F(1,5)=19.36, PHF<.05]).Dunnett’s post hoc tests revealed that for the 0.05 mg dose, theelectrochemical signal first increased significantly above baseline at 45minutes after the start of the session and remained significantly elevatedfor four hours (Figure 4). The DA oxidation signal for both the 0.10 mgand 0.20 mg doses was first increased significantly above baseline at onehour after the start of the session and remained significantly above baselinefor 5 and 8 hours, respectively (Figures 5 and 6).Experiment One: In Vivo MicrodialysisSubjectsOf the 17 rats used in the present experiment, one was excluded fromanalyses because it did not bar press for amphetamine during the first hourof the self-administration session, one was excluded because its catheterbroke and two were excluded because they received a drug that was notamphetamine during at least one session. Therefore, 13 rats met thecriterion for inclusion in the present analyses.HistologyProbe placements (n=13) were verified histologically and found to be in theN.Acc. Data were included from four rats receiving amphetamine in the0.10 mg dose, four rats in the 0.20 mg dose, and five rats in the 0.05 mgdose (Figure 8).64Figure 8: Representative sections showing the placement (vertical lines)of microdialysis probes in the nucleus accumbens used in theanalyses of experiment one (redrawn from the atlas of Paxinos andWatson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations:nucleus accumbens (N.Acc.); anterior commissure (ac); corpuscallosum (cc); caudate nucleus (CPu).65AP +2.2mmJH.p:I AP+1.7mm0 accCPuAP+1.2mmccCPuAP+O.7mmI I I I I I I321012366BehaviourAll rats learned to bar press (FR-2 schedule) for amphetamine on the firstday of drug exposure. At all doses, response rates were initially rapid andthen slowed to a constant rate for the duration of the self-administrationsession. The average and +1- SEM of the inter-infusion interval (INI)between each of the 12 self-administered drug infusions was 400 +1- 78seconds for a dose of 0.05 mg/infusion, 729 +/- 181 seconds for a dose of0.10 mg/infusion and 1266 +/- 359 for the high dose of 0.20 mg/infusion(Figure 9). A two-way between-subjects dose x INI ANOVA confirmedthat higher doses produced longer INIs [F(2,10)=8.77, p<.O5J. (Forclarity, behavioural data were not represented as INI, but rather as theaverages and +1- SEMs for the number of bar presses over 15 minute timeintervals in Figures 10, 11 and 12).Dopamine ConcentrationsDA concentrations during each self-administration session was averagedacross all rats for each dose (0.05, 0.10 and 0.20 mg/infusion) and +1-SEMs were obtained. As shown in Figures 10, 11 and 12 theconcentrations of DA (nM) increased above the pre-drug baseline followingthe self-administration of each dose of amphetamine tested. Within onehalf hour of the start of the session, DA concentrations plateaued atapproximately the same peak values for all doses (2.5, 2.0 and 3.0 nM for0.05, 0.10 and 0.20 mg/infusion, respectively).67Figure 9: Plot of the average inter-infusion latencies and +1- SEMs for the12 i.v. self-administered infusions of 0.05 mg/infusion (dark solidline), 0.10 mg/infusion (light solid line) and 0.20 mg/infusion (darkstippled line) of amphetamine in the microdialysis study.-oCDCl)C,)CDTime(SecX1000)r’)C)3CDI)C,)C)10)0)00-r%)C,)8969Figure 10: Bar graph of mean (+I SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+I SEM) dopaminedialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.05mg/infusion). The dark symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDopamineConcentration(nM)0ro—IIII.FI--—I*—I—1**I-__II*FH—114*II=1i/3i/CDH•HIH•H/IC)/0 I-•H/•0Cii /1•/•(71-D*IICl)—.00)DII0.CDU!J9L/SeSSeJdJDOL71Figure 11: Bar graph of mean (+1- SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+/ SEM) dopaminedialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.10mg/infusion). The dark symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDopamineConcentration(nM)-—III•UUU_____F—_HNUH*IHH—U—*H----1*H5•4/F—/II=1r) 3CD/HUH/IU -‘wICI)I/ U•IF-U-H___[UI0 \3c13.1UD—I’*DCoADIIU!LAJL/SeSSeJdJDZL73Figure 12: Bar graph of mean (+/ SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+1- SEM) dopaminedialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.20mg/infusion). The dark symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDopamineConcentration(nM)CI’)—IIII.0*I/1111*H—•—-H*HH——-H*/H—B---H*HH-•-H*=!H—çH*HCD1*0 H-H1\)0./3 F•H I—I /I—.•/—1•1*/1D \0)!.1’ He-H.jii00/II0-i’Ccu,tgjjsesseJJDgt’L75The total duration of amphetamine-induced increases in DA concentrationsincreased as a function of dose. A one-way between-subjects ANOVA ofthe duration of amphetamine-induced increases in DA concentrationsconfirmed a significant dose effect [F(2,1O)18.11, p<.O5]. As shown inFigure 13, interaction comparisons on the total duration of change in DAconcentrations revealed a significant difference between the low and highdoses [F(1,7)37.14, p<.O5], the high and medium doses [F(1,6)=27.35,p<.O5], but not the low and medium doses [F(1,7)=1.19, p>.O5].To analyse the peak height of the DA concentrations, a two-way dose xtime mixed ANOVA of the three doses and twenty consecutive 1 5 minutesamples (4 baseline and then the first 4 hours of the self-administrationsession) was conducted. The analysis confirmed that there was no effect ofdose on the peak height of DA dialysate concentrations [F(2,O)=3.53,p>.O5] (Figures 10, 11 and 12).Dunnett’s post hoc tests revealed that for all doses, the DA concentrationwas first significantly elevated above baseline levels at 30 minutes after thestart of the self-administration sessions for amphetamine. For the 0.05 mgdose, the elevation remained significant for one and a half hours (Figure10). For the 0.10 mg and 0.20 mg doses, the DA concentration remainedelevated for one and three hours, respectively (Figures 11 and 12).76Figure 13: Bar graph showing the mean (+1- SEM) total duration of effectof self-administered amphetamine on change in dopamineconcentrations in the nucleus accumbens as a function of dose (0.05,0.10 and O2O mg/infusion). Values for these effects correspond tothe time point where post-drug dialysate concentrations of dopaminereturned to pre-drug levels.0C,)(D(0D-hCl)0:3DurationofDrugEffect(Hrs)0)Co:3II0010p000:3II:3IIU78DOPA C ConcentrationsDOPAC concentrations corresponding to the self-administration ofamphetamine as determined by microdialysis were averaged across all ratsfor each dose, and SEMs were obtained. As shown in Figures 14, 15 and16, the DOPAC concentrations decreased for all doses and remained belowbaseline levels during the entire self-administration session. A two-waydose x time between-within ANOVA did not reveal a significant main effectof dose [F(2,0)0.77, p>.O5].DunnetCs test revealed that for the 0.05 mg/infusion and 0.10 mg/infusiondoses, DOPAC levels were first significantly decreased by 45 minutes and30 minutes, respectively. For both doses, DOPAC concentrations remainedsignificantly decreased for the entire duration of the session. For the 0.10mg/infusion dose, a high degree of between-subjects variability was seen(Figures 14 and 16) and a Dunnett’s test failed to find a significant changefrom baseline at any time during the drug session (Figure 15).79Figure 14: Bar graph of mean (+/ SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+1- SEM) DOPACdialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.05mg/infusion). The open symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDOPACConcentration(X100nM)o0)CDIIIIIIIII•II•I\•/0-___I__________•I—.4*—.4*-—/ F—-H*•/F*F—RH*\F—R---H*I_=:!F)I•—H* IIF—•—-i*3I CDI**•*I1*/*I•I**0.II-•cI713CDC71D*DciiA0).II0-CDugL/SeSSeJdJD0881Figure 15: Bar graph of mean (+I SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+1- SEM) DOPACdialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.10mg/infusion). The open symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDOPACConcentration(X100nM)l’s)0)CDIIIIIIIII•-HHIH•4h’[1/\H/=131/CD•—‘4.-,I.I3C,’—I.’*Cl)AIIUI[AJgi/sesSeidiD1883Figure 16: Bar graph of mean (+1- SEM) rate of bar pressing (bottom,right axis) and corresponding change in mean (+1- SEM) DOPACdialysate concentrations in the nucleus accumbens (top, left axis) ofrats self-administering twelve infusions of amphetamine (0.20mg/infusion). The open symbols represent dialysate samplescollected at 15 minute intervals.ChangeinDOPACConcentration(X100nM)1)0)CD—IIIIIIIIHHHHI/*/F--*F-*H_*HF•-*I—•-H*HI H•H*Fl.*H (1-H*I—s-H*CA)--1*l-_*#_*III —H*I\)1h*H-R-H*[I/-—-*3 II He—H*II—-l*—.(31-H*HH**IF•-l*-A•*U—-a)•*-0 I01 —*IIUf\J91/sesSeJdJDt885Experiment TwoAll rats acquired self-administration behaviour on the first day of drugexposure: both the response rates and DA efflux were similar to thoseobserved on the first day of experiment one. Only the abstinence periodsfrom the third session are presented here.SubjectsOf the nine rats used in the present experiment, one was excluded from theanalyses because it had a poor electrochemical signal, one was excludedbecause a power failure caused self-administration to be terminated mid-session and one was excluded because it overdosed on amphetamine.Therefore, six rats met the criterion for inclusion in the presentexperiment.HistologyHistological examination of the six rats tested in this study showed thatfour had electrodes placed bilaterally in the N.Acc. The remaining two ratshad one of the two electrodes placed in the N.Acc. Only data fromcorrectly placed electrodes were considered in the overall analysis. Forsubjects with two electrodes placed in the N.Acc., the electrochemicalrecord with the least amount of electrical noise was used (Figure 17).BehaviourBar press rates were averaged (+/_ SEM) for six hour blocks, as seen inTable 1. A one-way repeated measures ANOVA confirmed that the hourly86Table 1.Number of Bar Presses Per Hour Averaged (+1- SEM) for Six Hour BlocksTime Interval (Hrs) Average +1- SEM1-6 3.00 0.567-12 3.80 0.7713-18 2.97 0.2919-24 2.69 0.1125-30 2.61 0.6831-36 2.08 0.4137-42 2.92 0.5643-48 3.28 0.5187Figure 17: Representative sections showing the placement (solid circles)of electrochemical electrodes in the nucleus accumbens used in theanalyses of experiment two (redrawn from the atlas of Paxinos andWatson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations:nucleus accumbens (N.Acc.); anterior commissure (ac); corpuscallosum (cc); caudate putamen (CPu).88AP+2.2mmAP +1.7mmAP+1.2mmAPi-O.7mmI I I I I I RigIitLeft I I I I I I32 1012389rate of bar pressing for the groups did not differ significantly with time[F(47,235)=9.79, PHF>05i.Abstinence Periods-ElectrochemistryEach rat displayed at least one abstinence period during the 48 hour testsession (Figures 18, 19 and 20). Each abstinence period was characterizedby a complete cessation of bar pressing and concurrent decreases in theelectrochemical signal corresponding to extracellular DA. The end of theabstinence period corresponded to the reinitiation of a regular pattern ofresponding and a subsequent rise in DA oxidation current levels. Theapproximate duration of the abstinence periods for rats ci, c2, c3. c4. c5and c6 were 3.5, 2.0, 3.8, 4.5, 4.2 and 15 hours, respectively.As shown in Figure 21, one-way repeated measures ANOVA of the changesin DA oxidation current during the abstinence period confirmed that therewas a significant effect between the different time intervals that constitutedthe different phases of the abstinence period [F(5,25)=6.15, PHF<•°l• ANeuman-Keuls analysis revealed that the first and second of these meantime values, corresponding to the five minutes before and five minutes afterthe initiation of the abstinence period, were significantly different from allother time values, but they were not different from each other.90Figure 18: Examples of behavioural correlates (tic mark = one druginfusion) for individual subjects prior to, during, and following thedrug abstinence period. Corresponding time-course of changes indopamine oxidation current is indicated by the dark continuous line.The horizontal line represents the estimate of a best-fit line drawnmanually, on a hard copy of the data, through one hour of the predrug abstinence baseline. Data are from rats ci, c2 and c3. Numbershown in parentheses for rat ci indicate the number of bar presses atthat time.91I 4 Nanoampsratcirat c2rat c3ThJH U(13) (14)JLJ____J_J______5 6-1 0 1 2 3 4Time (Hrs)92Figure 19: Examples of behavioural correlates (tic mark = one druginfusion) for individual subjects prior to, during, and following thedrug abstinence period. Corresponding time-course of changes indopamine oxidation currents is indicated by the dark continuous line.The horizontal line represents the estimate of a best-fit line drawnmanually, on a hard copy of the data, through one hour of the predrug abstinence baseline. Data are from rats c4 and c5.93I 4 Nanoampsrat c4rat c5I AJ1 0 1 2 3 4 5 6Time (Hrs)94Figure 20: Examples of behavioural correlates (tic mark = one druginfusion) for individual subjects prior to, during, and following thedrug abstinence period. Corresponding time-course of changes indopamine oxidation currents is indicated by the dark continuous line.The horizontal line represents the estimate of a best-fit line drawnmanually, on a hard copy of the data, through one hour of the predrug abstinence baseline. Data are from rat c6.95I 4 Nanoampsrat c6—1 1 3 5 7 9 11 13 15 17Time (Hrs)vII96Figure 21: Group mean (+/ SEM) changes in dopamine oxidation currentin the nucleus accumbens corresponding to the time periods pre, postand during the abstinence period. A=mean current from the fiveminute period prior to last self-administered infusion of amphetamine(0.10 mg/infusion) before abstinence. Bmean current from the fiveminute period following the last infusion before abstinence. C=meancurrent from the five minute period prior to the midpoint in timebetween the last infusion before abstinence and the first infusionafter abstinence. D=mean current from the five minute periodfollowing the midpoint between the last infusion before abstinenceand the first infusion after abstinence. E=mean current from the fiveminute period prior to the first infusion after abstinence. Fmeancurrent from the five minute period following the first infusion afterabstinence. The vertical stippled lines represent the last infusionprior to the drug abstinence and the first drug infusion after the drugabstinence, respectively.F’)01’)3CD-QCD0a-DopamineOxidationCurrent(nA)DCDwaQcn.CDDCDm-c3____-noCi)*-+AbC)’***DII0)L698DISCUSSIONExperiment OneThe behavioural findings from experiment one demonstrated that rats willself-administer increasingly larger doses of amphetamine with longer inter-infusion latencies. All doses tested produced two phases of responding.One of these, the loading phase, was characterized by rapid responding atthe start of the self-administration session. A second maintenance phasefollowed the loading phase and was characterized by a decrease in responserates to a stable level maintained throughout the remaining duration of self-administration. Both DA oxidation currents and dialysate DAconcentrations corresponding to extracellular DA efflux in the N.Acc.initially increased during the loading phase of self-administration and thenplateaued during the maintenance phase remaining constant for the durationof self-administration. Maximum increases in DA efflux attained during asession did not vary with dose, despite dose-related changes in the rate ofself-administration. As well, the maximal increase in DA levels remainedconstant within rats across days for the same dose. However, eachincrement in the dose of amphetamine self-administered increased thelength of time that DA levels remained elevated between infusions, therebyincreasing the duration of the sessio,n required to self-administer 12infusions of the drug. Correspondingly, a dose-related increase in the totalduration of change in DA concentration from pre-drug baseline was found.Although no dose-dependent changes in the maximal height of DA effluxattained during self-administration sessions were seen in either the99electrochemistry or microdialysis studies of experiment one, statisticalanalyses revealed differences between the two sets of findings. Forexample, in the in vivo electrochemistry study, a longer period of time wasrequired for the chronoamperometric signal to differ significantly from predrug baseline levels. These changes in chronoamperometric signal alsoremained elevated for a longer time than for rats in the in vivomicrodialysis study. As well, the total duration of amphetamine-inducedincreases in DA oxidation currents was significantly different between alldoses tested in the electrochemistry study. However, in the microdialysisstudy, there was no difference in the total duration of amphetamine-induced effect on DA dialysate concentrations between the medium dose(0.10 mg/infusion) and the low dose (0.05 mg/infusion). In addition,statistical analyses revealed some unexpected changes in DOPAC dialysatelevels during amphetamine self-administration. As expected, DOPAC levelsdropped during self-administration (Kuczenski and Segal, 1989; Kuczenski,Segal and Aizenstein, 1991) and this change corresponded in time to therise in extracellular DA levels. However, there was a large amount ofbetween-subjects variability in the time-dependent decreases in DOPACconcentrations during self-administration ofthe middle (0.10 mg/infusion)dose. Not surprisingly, statistical analyses failed to reveal a significanttime-dependent decrease in DOPAC concentrations during the selfadministration of this dose.Some of the discrepancies between the neurochemical findings of theelectrochemistry and microdialysis studies are clearly related to the resultsobtained with the medium dose (0.10 mg/infusion) in the microdialysisexperiment. One possible explanation for the discrepancy in the findings100between the two studies would be that one study (electrochemistry)employed a within-subjects design and the other (microdialysis) a between-subjects design. As such, the between subjects design would not accountfor the individual variability in basal DA levels or metabolism (Kuczenski,Segal and Aizenstein, 1991). As well, the high variability in the resultsfrom the microdialysis experiment could reflect the small and unevennumber of subjects used in this study (for 0.05 mg/infusion, n=5; for 0.10mg/infusion, n=4; for 0.20 mg/infusion, n=4). Increasing the. number ofsubjects in all groups in the microdialysis study may lead to greateragreement between the results of the electrochemistry and microdialysisstudies.It is important to note that discrepancies between the chronoamperometricand dialysate temporal profiles of drug-induced changes in DA efflux havebeen reported in the past (Blaha, Coury, Phillips and Fibiger, 1990). Whenmeasured with microdialysis, drug-induced changes in DA efflux arefrequently seen to be shorter in duration relative to thechronoamperometric findings reported for similar studies. Microdialysis isbased on the premise that particles in solution will travel down theirconcentration gradient. Therefore, when obtaining estimates ofextracellular DA concentrations, a brain area is dialysed with aphysiological perfusate devoid of DA. DA in the extracellular fluid willtherefore cross into the dialysis probe down its concentration gradient. Ithas recently been shown that dialysing brain tissue results in a significantperturbation (decrease) in basal extracellular DA (Blaha, 1992). Sincemost of the DA extracted from the interstitial space during dialysis wouldnormally be taken back up into the nerve terminal and metabolized or101recycled, microdialysis could in effect decrease intracellular stores of DAavailable for synaptic release. This would result in a blunted time-courseresponse to drug. Thus, it is possible that differences in the temporalprofile of drug-induced changes in DA measured with electrochemistry andmicrodialysis in experiment one may be due to the tendency ofmicrodialysis to extract DA, thereby decreasing basal levels.Despite these technical differences between the two studies in experimentone, the findings of both studies revealed that maximal levels of DAattained during the self-administration of various doses of amphetamineremained constant across doses. The findings from experiment one aretherefore consistent with the notion of a reinforcement threshold definedby blood levels of drug and brain levels of DA. The reinforcementthreshold refers to the minimum level of drug in the blood and DA efflux inthe brain required to maintain self-administration of a drug. Althoughthere is no suggestion in the literature as to what the absolute ‘value’ of thereinforcement threshold would be, the exact determination of such a valuemay not be as critical to the validity of the DA reinforcement thresholdhypothesis as the demonstration that a constant relative change in DAlevels is achieved with different drug doses during a self-administrationsession. That is, evidence of the maintenance of steady levels ofextracellular DA efflux in brain and blood levels of drug by a predictablepattern of titration of drug intake, is entirely consistent with a DAreinforcement threshold. Implicit in this interpretation of a reinforcementthreshold is the assumption that the titered level of DA in the brain anddrug in the blood is sufficient to exceed the reinforcement threshold. Thefindings of experiment one that a constant efflux of DA was seen both102within sessions for a given animal and across different doses is consistentwith the notion of such a reinforcement threshold. These findings supportthose of Yokel and Pickens (1974), who found that rats will titer the self-administration of different doses of amphetamine to maintain a steadyblood level of drug both between and within doses and across a self-administration session. As well, the finding that rats decreased their rateof responding for higher doses supports the hypothesis that animals titeramphetamine to maintain an optimal level of drug in their blood and DAefflux in the brain and is consistent with the dose-related behaviouralchanges seen with amphetamine that have been reported in the past (Brady,1991; Yokel and Pickens, 1974).The results of the present study are also consistent with the findings ofPettit and Justice (1991) who showed that rats titer their intake of cocaineat a given dose, to maintain a stable level of DA efflux during a self-administration session. However, cocaine and amphetamine do appear todiffer in one important respect, as Pettit and Justice (1991) observed thatrats self-administered greater amounts of higher doses of cocaine whenmade available for injection. The maximal level DA efflux maintainedduring a self-administration session also increased with higher doses. Thedose-dependent increase in DA efflux reported by Pettit and Justice (1991)is difficult to reconcile with the present results. It is possible thatamphetamine, being both a reuptake inhibitor and releaser of DA, cansupport greater absolute increases in DA efflux than cocaine, which acts onDA only as a reuptake blocker. At the very least, these cocaine data areconsistent with the hypothesis that rats titer drug intake to maintain a103steady level of drug in the blood and DA in the brain, which exceeds areinforcement threshold corresponding to a specific amount of DA efflux.104Experiment TwoExperiment two demonstrates that rats will self-administer amphetamine(0.10 mg/infusion) for prolonged periods of time and that during this time,rats will spontaneously cease to respond for drug for a period of timeexceeding two hours. These abstinence periods were accompanied by adrop in DA levels to below pre-drug baseline concentrations in four rats(ci, c3, c4, c6). The DA oxidation currents for one rat dropped tobaseline (c5) and remained above baseline for the other subject (c2). Aswell, in two rats (c2 and c5), the decrease in DA concentrations precededdrug abstinence. For the other four rats, the decrease in DA levelsoccurred when the rats stopped self-administering. As well, half of the ratsin this group (ci, c3 and c4) reinitiated drug self-administering when DAlevels were approximately at their lowest level and the other halfreinitiated responding after DA levels began to rise spontaneously. Bydefinition, at the end of the abstinence period, normal responding occurredin all cases. It is important to note that it was not clear from the presentdata whether an increase in the DA signal occurred before or after thereinitiation of responding. Nevertheless, it is evident that the DA systemresponded to the reinitiation of self-administration of amphetamine and thatthe abstinence period ended at a time when the DA system was responsiveto the pharmacological actions of the drug.The neurochemical findings from this experiment support those reported byWeiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992). In theirstudy, extracellular DA efflux initially increased with the onset of selfadministration and then stabilized around a steady mean value for the105duration of the self-administration session. For most rats, DAconcentrations decreased to values lower than pre-drug baseline levelsduring drug abstinence. Together, these data are also consistent with thedopamine depletion hypothesis which predicts that drug abstinence shouldbe associated with a decrease in extracellular DA concentrations in specificregions of the brain.As described in the Introduction, the dopamine depletion hypothesis(Dackis and Gold, 1985) predicts that a decrease in extracellular DA levelsduring drug abstinence, occurs as a result of depletion of intracellular DAstores after prolonged stimulation of dopaminergic neurons. It is alsohypothesized that this depletion of dopaminergic stores causes a state ofanhedonia (an inability to derive pleasure from normally pleasurable events(Koob, 1992b)). Subsequent responding for a drug under thesecircumstances serves as a negative reinforcer because the drug causes anelevation of extracellular DA levels (Dackis and Gold, 1985). With respectto the present experiment, the dopamine depletion hypothesis wouldpredict that the reinitiation of self-administration of drug during anabstinence should occur at the time of maximal depletion of DA stores andthat a reinitiation of self-administration would lead to a rise in extracellularDA levels. These predictions are partially confirmed by the data fromexperiment two as DA oxidation currents were seen to increase afterreinitiation of amphetamine self-administration. However, no clearevidence was found to support the prediction that reinitiation of selfadministration would occur when DA levels were at their lowest values.Even though rats self-administered amphetamine when DA efflux was low,106some rats reinitiated self-administration only after a spontaneous rise inextracellular DA levels.The data from experiment two can be interpreted in light of thereinforcement threshold hypothesis. These findings are consistent with theidea that a minimal increase in DA efflux must be maintained to supportdrug self-administration. The fact that rats still continued to self-administer amphetamine when extracellular DA levels had declined to levelsnear pre-drug baseline values, suggests that the reinforcement threshold isquite low. Perhaps the best evidence for a reinforcement threshold comesfrom experiment two in which rats ceased to self-administer amphetaminewhen the extracellular levels of DA dropped below a certain value. Thefact that self-administration of amphetamine was reinitiated when the DAsystem again became responsive is also consistent with this concept. Otherevidence indicates that drug self-administration is correlated with anincrease in extracellular DA efflux (reviewed in Robinson and Berridge,1993). A true test of the DA hypothesis of drug reinforcement would be todemonstrate that abstinence from drug self-administration corresponds to atime when the DA system is unable to exceed a reinforcement thresholdand therefore support self-administration. Experimenter-administeredinfusions of amphetamine and selective DA agents at specific times duringan abstinence period could be used to ascertain the time course of thisrefractory period of the mesolimbic DA system and thereby determine ifrats will only reinitiate responding for amphetamine when the DA systemcan exceed a nominal reinforcement threshold.107It is possible that certain unconditioned cues and behavioural tasks caninfluence DA efflux. Among these are locomotor activity, lights andcircadian rhythmicity. Since the N.Acc. is a putative limbic-motor outputregion, it is possible that changes in DA efflux seen during self-administration are simply an artifact of increased locomotor output. Thisis a particular problem for experiment two. The increase in DA efflux seenprior to reinitiation of self-administration may be due to the locomotionand arousal associated with events prior to the reinitiation of bar pressing.However, there is little evidence to support this possibility. Louilot, LeMoal and Simon (1986) reported that social interactions between rats canincrease DA efflux in the N.Acc. as measured by in vivo voltammetry.These researchers also demonstrated that these DA neurons are most activeduring those hostile social interactions which render the rats immobile. Aswell, Damsma, Pfaus, Wenkstern, Phillips and Fibiger (1992) monitoredchanges in DA concentration as measured by in vivo microdialysis duringforced activity on a treadmill. These studies showed no notable change inDA efflux in the N.Acc. during such locomotor activity. As well, evidencedissociating conditioned locomotion and DA efflux supports the claim thatlocomotor output does not influence N.Acc. DA efflux (Brown and Fibiger,1992). The possibility that locomotion and arousal may contribute to theDA signal prior to self-administration at the end of an abstinence can betested in future studies by monitoring the amount of locomotion andgeneral activity of the rat during the period immediately prior to thereinitiation of self-administration after an abstinence.The present study used light cues and required rats to remain in the testchamber for prolonged periods of time. Appropriate controls are important108to establish that the increase in DA seen with the self-administration ofamphetamine does not reflect the unconditioned effects of lights orcircadian rhythmicity. Preliminary findings with control subjects (n=2),receiving yoked-saline paired with the same CS+ as the rats self-administering amphetamine, have shown no increases in DA oxidationcurrent associated with circadian rhythms, feeding or the onset or offset oflight cues. Nevertheless, additional control subjects are needed to confirmthat the DA efflux in the present study are free of these confounds and aredue to the unconditioned effects of self-administered amphetamine.It is possible that the rats in experiments and two at times inadvertently‘bumped’ the operant lever. This would result in a greater amount of druginfusions than the rat had ‘intended’. The possibility that such a ‘bump rate’is low can be tested by video-taping the rats during a self-administrationsession or by placing a rat in the operant chamber with only saline availableas a reinforcer. Although such studies have not been conducted inexperiments one and two, drug-naive rats in other experiments, withoutprior operant training for food, that were placed in the operant chamber fordrug self-administration training rarely press the lever prior to acquisitionof the operant task.In summary, the findings of both experiments in this study support thehypothesis that DA is an essential correlate of drug-reinforcement. Thefindings that rats will titer their self-administration of amphetamine tomaintain stable levels of extracellular DA across doses, is consistent withthe concept of a reinforcement threshold. As well, the findings fromexperiment two demonstrate that patterns of amphetamine self-109administration correspond to periods when DA neurons in the N.Acc. areresponsive to the drug. Although the absolute change in DA effluxrequired to maintain self-administration may not be critical, it is clear thata rise in DA levels above a nominal reinforcement threshold is a necessarycondition to support drug self-administration. Combined, the results fromboth experiments provide strong support for the positive reinforcementmodel of drug self-administration (Wise, 1987). Future hypotheses about‘craving’ and drug abstinence will need to consider that a change in DAefflux is a critical component to the acquisition of both drug use and themaintenance of drug abuse.An alternative hypothesis, which takes into consideration the phenomena of‘craving’, has recently been proposed by Robinson and Berridge (1993).Like Dackis and Gold (1985) with their dopamine depletion hypothesis,Robinson and Berridge (1993) attribute a central role to DA in the processof ‘craving’. The incentive-sensitization theory of drug addiction(Robinson and Berridge, 1993) maintains that activity in the mesolimbicDA system is critically involved in processes by which incentive salience isattributed to the drug and to conditioned stimuli paired with theunconditioned effects of drugs of abuse. Furthermore, the DA systembecomes sensitized as a result of repeated exposure to drugs of abuse. Bysensitization of the DA system, the salience of conditioned stimuliassociated with drug taking is enhanced. Robinson and Berridge (1993)propose that the sensitization of the incentive salience of the conditionedstimulus, underlies the process of ‘craving’ in the absence of drug.Therefore, this theory, unlike the dopamine depletion hypothesis, accountsfor both the long-term nature of ‘craving’ and ‘craving’ in the absence of110any systemic drugs or withdrawal symptoms. 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