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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 NUCLEUS ACCUMBENS DURING INTRAVENOUS SELF-ADMINISTRATION OF D-AMPHETAMINE BY THE RAT By PATRICIA DI CIANO  B.A. (Hons.), Queen’s University, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in THE FACULTY OF GRADUATE STUDIES Department of Psychology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1993 ©Patricia Di Ciano, 1993  tCciQQ  + 11  1  Hf CD  A  V I  S  UN  In presenting this thesis  in  partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives,  It  is  understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department  of  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  Q  /  Abstract  Current theory suggests that mesolimbic dopamine (DA) in the nucleus accumbens is involved in the self-administration of drugs such as cocaine and amphetamine. The present thesis was conducted to determine some of the behavioural and in vivo dopaminergic correlates of intravenous (i.v.) self-administration of amphetamine in the rat. Experiment one tested the hypothesis that rats will self-administer amphetamine to maintain an optimal extracellular level of DA in the brain that exceeds a critical threshold. In vivo measurements of DA oxidation currents (electrochemical studies) and DA dialysate concentrations (microdialysis study) were used to determine changes in extracellular DA concentrations in rats permitted to self-administer 12 infusions of d-amphetamine a day at a given dose. During all self-administration sessions, distinct ‘loading’ and ‘maintenance’ phases were seen. The loading phase was characterized by rapid responding at the start of the session which slowed during the maintenance phase to a constant level for the duration of the selfadministration session. DA extracellular concentrations in the nucleus accumbens increased at the start of the self-administration session and then levelled off around a stable mean for the duration of the session. An inverse dose-response relationship between rate of responding for amphetamine and the dose of drug administered (0.05, 0.10 or 0.20 mg/infusion) was seen. No dose-related changes in the maximal increase in DA oxidation currents or dialysate concentrations during self administration was evident. Experiment two tested the hypothesis that increased DA levels associated with the reinitiation of responding after an abstinence serve as a negative reinforcer by increasing DA levels that are  III  theoretically depleted during an abstinence (Dackis and Gold, 1985). When permitted continuous access to 0.10 mg/infusion of amphetamine for 48 hours, response rates and in vivo DA oxidation currents were similar to those in experiment one and remained steady during the course of the entire self-administration session. After at least 24 hours, all rats abstained from self-administering amphetamine for a single period of time lasting at least two hours. During this time, a decrease in DA concentrations was seen. Reinitiation of responding for amphetamine did not occur when DA concentrations were at their lowest, but was correlated with an immediate increase in DA concentrations as measured by in vivo electrochemistry. In summary, the findings of experiment one and two suggest that rats titer self-administered i.v. amphetamine to maintain a steady optimal extracellular level of DA in the brain that exceeds a reinforcement threshold.  iv  Table of Contents  Abstract  ii  List of Figures  vi  List of Tables  ix  Acknowledgements  x  I.  II.  III.  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  20  METHODS (A) General Methods  21  (B) Electrochemical Procedures  25  (C) Microdialysis Procedures  32  EXPERIMENT ONE (A) Introduction  37  (B) Methods  38  V  IV.  V.  VI.  VII.  EXPERIMENT TWO (A) Introduction  44  (B) Methods  46  RESULTS (A) Experiment One: In Vivo Electrochemistry  49  (B) Experiment One: In Vivo Microdialysis  63  (C) Experiment Two  85  DISCUSSION (A) Experiment One  98  (B) Experiment Two  104  REFERENCES  111  vi  List of Figures  27  Figure 1:  Representative voltammogram  Figure 2:  Illustration of brain coronal sections showing electrode sites for experiment one.  Figure 3:  50  Graph of interval-interfusion intervals for self-administration of 0.05, 0.10 and 0.20 mg/infusion of amphetamine in the electrochemistry experiment.  Figure 4:  53  Changes in behaviour and electrochemical dopamine signals corresponding to the self-administration of 0.05 mg/infusion of i.v. amphetamine  Figure 5:  55  Changes in behaviour and electrochemical dopamine signals corresponding to the self-administration of 0.10 mg/infusion of i.v. amphetamine.  Figure 6:  57  Changes in behaviour and electrochemical dopamine signals corresponding to the self-administration of 0.20 mg/infusion of i.v. amphetamine.  Figure 7:  59  Dose-related differences in duration of change in electrochemical DA signals corresponding to the self administration of 0.05, 0.10 and 0.20 mg/infusions of i.v. amphetamine.  Figure 8:  61  Illustration of brain coronal sections showing probe sites for experiment one.  64  vii  Figure 9:  Graph of interval-interfusion intervals for self-administration of 0.05, 0.10 and 0.20 mg/infusion of amphetamine in the microdialysis experiment.  Figure 10:  67  Changes in behaviour and dialysate dopamine concentrations corresponding to the self-administration of 0.05 mg/infusion of i.v. amphetamine.  Figure 11:  69  Changes in behaviour and dialysate dopamine concentrations corresponding to the self-administration of 0.10 mg/infusion of i.v. amphetamine.  Figure 12:  71  Changes in behaviour and dialysate dopamine concentrations corresponding to the self-administration of 0.20 mg/infusion of i.v. amphetamine.  Figure 13:  73  Differences in duration of change in dialysate dopamine concentrations corresponding to the self-administration of 0.05, 0.10 and 0.20 mg/infusion of i.v. amphetamine.  Figure 14:  76  Changes in behaviour and dialysate DOPAC concentrations corresponding to the self-administration of 0.05 mg/infusion of i.v. amphetamine.  Figure 15:  79  Changes in behaviour and microdialysis DOPAC concentrations corresponding to the self-administration of 0.10 mg/infusion of i.v. amphetamine.  81  VIII  Figure 16:  Changes in behaviour and microdialysis DOPAC concentrations corresponding to the self-administration of 0.20 mg/infusion of i.v. amphetamine.  Figure 17:  Illustration of brain coronal sections showing electrode sites for experiment two.  Figure 18:  83  87  Changes in behaviour and electrochemical dopamine signals corresponding to an abstinence from drug self-administration for rats ci, c2 and c3.  Figure 19:  90  Changes in behaviour and electrochemical dopamine signals corresponding to an abstinence from drug self-administration for rats c4 and c5.  Figure 20:  92  Changes in behaviour and electrochemical dopamine signals corresponding to an abstinence from drug self-administration for rat c6.  Figure 21:  94  Average electrochemical dopamine signal corresponding to various time points during an abstinence from drug self administration.  96  ix List of Tables  Table 1:  Number of Bar Presses Per Hour Averaged for Six Hour Blocks  86  x  Acknowledgements  I would like to thank my advisor, Dr. Charles D. Blaha, for his guidance and 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 for their work on the microdialysis portion of these projects and to Fred LePiane for programming the MANX, repairing my equipment, etc, etc... Thanks as well to Penny Lam for helping with the ‘dirty work’, to Paul Mackenzie for reading preliminary drafts of my thesis, to Charles Yang for the use of his computer, to Jeremy Seamans for help with some of my figures and to everyone else in the lab for helping to make my work here enjoyable.  1  INTRODUCTION  Drug abuse is a major social problem. It is estimated that in the United States alone, 1 to 3 million people are in need of treatment for cocaine addiction, a figure about 6 times greater than that for heroin addicts (for review, see: Gawin, 1991). In particular, the use of the psychomotor stimulants, cocaine and the amphetamines, is on the rise with the recent popularization of the free base form of cocaine, crack, and derivatives of amphetamine such as ‘speed’ and ‘meth’ (Robinson and Berridge, 1993). Of special interest is the observation that drug users continue to abuse their substance of choice despite severe physical, social or financial consequences (for reviews, see: Robinson and Berridge, 1993; Gawin, 1991; Johanson and Fischman, 1989). These factors suggest that the processes contributing to drug abuse are both powerful and complex. Therefore, a detailed understanding of the behavioural and neurobiological factors that contribute to the initiation and long-term use of psychostimulants is essential to the success of any program attempting to control abuse of these drugs.  Psychologists, and biopsychologists, in particular, can offer a unique perspective to drug abuse research. A biopsychological approach to the study of drug abuse emphasizes the interactions between learning and previous experience and the physiological mechanisms which contribute to the acquisition and maintenance of drug use. An understanding of these issues will lead to strategies for the development of pharmacological treatments of drug abuse. Theoretically, if one could block or prevent the neural correlates of drug-taking behaviour, then the inherent addictive  2  properties of the drug could be abolished (for a review of pharmacological interventions of drug taking, see: Kosten and Kosten, 1991).  Currently, one of the most widely held theories of drug abuse is the reinforcement model (Beninger, 1983; Wise, 1987; White and Mimer, 1992). Reinforcement in this framework does not refer to an internal state of ‘pleasure’ or ‘hedonia’. Rather, reinforcement is a behavioural and descriptive term referring to the strengthening of any response due to its consequences (strengthening means that the response will occur with greater probability in the future). The opposite of reinforcement is punishment: the weakening of a response due to its (undesirable) consequences. Positive and negative reinforcement refer to the strengthening 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 with the stimulus must obey certain principles. First, an animal must be able to acquire a new response to obtain the reinforcer. Second, the responsereinforcer contingency must be such that the response immediately precedes the reinforcer. Third, the operant behaviour must be subject to extinction (Pickens and Thomson, 1968).  In the context of drug abuse,  extinction occurs when a substance that is self-administered fails to retain its reinforcing properties. This can happen when the reinforcer is removed, or when the contingency between a response and its consequences is altered. In all cases, the operant response fails to have any reinforcing consequences (Grove and Schuster, 1974). Extinction can be observed in  3  the laboratory by substituting a reinforcing drug with an inactive agent or drug vehicle or by reversing the drug-response contingency. Extinctionlike 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 and Vickers, 1989).  The majority of biopsychological hypotheses of drug abuse attempt to link reinforcement with neurochemical events. Most of these hypotheses have in common the premise that psychomotor stimulants are reinforcing because 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 are also concerned with two different, and interacting, aspects of drug use: acquisition of drug use and long-term maintenance of drug abuse. As noted above, the positive reinforcement theory of drug use states that drug taking behaviour is acquired because the drug is reinforcing. The reinforcing aspects of the drug are thought to be due, in part, to its ability to selectively increase extracellular levels of the neurotransmitter dopamine (DA) in specific forebrain structures. In contrast, maintenance of drug abuse is hypothesized to be due to a state of ‘anhedonia’ associated with an acute depletion of brain extracellular DA during an abstinence (Dackis and Gold, 1985; Gawin and Kieber, 1986). This dopamine depletion hypothesis maintains that the anhedonia associated with withdrawal is alleviated by the increase in DA concentrations that results from the reinitiation of drug taking.  4  The present thesis was conducted to examine these two hypotheses of reinforcement and drug abuse. Specifically, changes in extracellular DA levels in the forebrain of the rat were measured in vivo during the acquisition and maintenance of intravenous (i.v.) amphetamine selfadministration as a direct test of certain predictions of the reinforcement and dopamine depletion hypotheses. Before outlining these hypotheses, several issues will be addressed. The first establishes the role of DA in reinforcement. The second provides evidence that psychomotor stimulants serve as reinforcers.  Neurochemical Correlates of Positive Reinforcement  Over the past two decades, a great deal of basic research has been aimed at identifying the neurochemical substrates of reinforcement. These studies have led to the hypothesis that both reinforcement and locomotor output are mediated by mesocorticolimbic dopaminergic systems (Jackson, Anden and Dahlstrom, 1975; Mogenson and Phillips, 1976; Kelly, Seviour and Iversen, 1975; Joyce and Koob, 1981). In particular, changes in extracellular DA concentrations in the nucleus accumbens (N.Acc.), a limbic structure and major terminal site of these neurons, are thought to be associated with reinforcement (for reviews, see: Wise, 1987; Robinson and Berridge, 1993). The N.Acc. is currently considered to be the major interface between the limbic system and sensory-motor control systems and therefore seems to be an ideal locus for the regulation of reinforced behaviours (for reviews, see: Mogenson, Jones and Yim, 1980; Beninger,  5  1983; Wiliner, Ahienius, Muscat and Scheel-Kruger, 1991; Mogenson and Yang, 1991).  Evidence linking DA in the N.Acc to the self-administration of drugs of abuse comes from studies of the effects of DA receptor antagonists, DA agonists and lesions of the DA system on self-administration behaviour (for reviews, see: Wise and Hoffman, 1992; Koob, 1992). DA receptor blockers are typically given to animals prior to a self-administration session on the assumption that a substance which partially blocks the pharmacologically reinforcing properties of a drug of abuse will cause a compensatory 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 DA receptor blockers pimozide and butaclamol showed accelerated responding for i.v. amphetamine (Yokel and Wise, 1975, 1976). These effects appeared selective, because norepinephrine (NE) receptor antagonists phentolamine, 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 (Roberts and Vickers, 1984) has also been shown to increase response rates for cocaine. Collectively, these data have shown that pharmacological blockade of central DA receptors attenuates responding for reinforcers as predicted by a dopaminergic theory of psychostimulant reinforcement.  When the reinforcing efficacy of a drug is completely abolished (not just attenuated), extinction-like responding has been typically observed. Extinction-like responding has been reported following systemic  6  administration of very high doses of DA antagonists (Yokel and Wise, 1976; Roberts, Loh and Vickers, 1989) and after selective neurotoxic lesions of the mesocorticolimbic DA system, particularly of the N.Acc. In the case of cocaine self-administration, extinction-like responding is seen following 6-hydroxydopamine lesions of the ascending DA system. The degree and duration of these behavioural responses corresponded with the extent of DA depletion in the N. Acc. (Roberts, Koob, Kionoff and Fibiger, 1980).  In contrast, selective destruction of the dorsal or ventral NE  bundles were without effect (Roberts, Corcoran and Fibiger, 1977). Decreased responding for cocaine and amphetamine following discrete lesions of the N.Acc. has also been reported (Roberts and Koob, 1982; Lyness, Friedle and Moore, 1979). The anatomical specificity of the N.Acc in the self-administration of drugs was further supported by the finding that lesions of other DA-rich areas in the brain, such as the prefrontal cortex and striatum, are without effect on the self-administration of psychomotor stimulants (Martin-Iversen, Szostak and Fibiger, 1986).  Recent biochemical and receptor-binding studies have provided a unique approach to the study of the role of N.Acc. DA in the self-administration of psychomotor stimulants. In one study, genetic strains of rats predisposed to self-administer drugs of abuse were found to have relatively higher brain levels of tyrosine hydroxylase, the rate-limiting enzyme for DA synthesis (Beitner-Johnson, Guitart and Nestler, 1991). This difference was found mainly in the N.Acc. but not in the DA-rich regions of the striatum.  As well, Ritz, Lamb, Goldberg and Kuhar (1987) have found a  positive correlation between the binding efficacy of cocaine to the DA transporter in the N.Acc. and the potency of the drug in maintaining self-  7  administration for cocaine. Overall, these studies lend support to previous behavioural data pointing to a critical role for DA in the N.Acc. in the selfadministration of drugs of abuse.  Studies of the unconditioned effects of cocaine and the amphetamines have also provided support for a dopaminergic hypothesis of drug abuse. Cocaine and amphetamine are potent indirect DA agonists, acting to increase extracellular levels of DA through synaptic reuptake blockade or via facilitated-exchange diffusion, respectively (Fischer and Cho, 1979; for review, see: North, 1992). Amphetamine enters the nerve terminal via a DA uptake carrier and releases DA into the cytosol from newly-synthesized DA storage vesicles. This, in turn, enhances synaptic concentrations of DA by facilitating (via the uptake carrier) the diffusion of cytosolic DA through the nerve terminal (Fischer and Cho, 1979). In humans, both compounds are strong euphorics with. hedonic qualities (Chait, 1993; Robinson and Berridge, 1993; Dackis and Gold, 1985) and can increase spontaneous motoric activity (Kelly, Seviour and Iversen, 1975; for reviews, see: Gawin, 1991; Johanson and Fischman, 1989). In vivo neurochemical studies provide further evidence that cocaine and amphetamine enhance DA neurotransmission. For example, systemic amphetamine and cocaine injections dose-dependently increase DA extracellular levels in both the striatum and N.Acc. as measured by in vivo microdialysis (Segal and Kuczenski, 1992; Segal and Kuczenski, 1992b; Kuczenski and Segal, 1989; Kalivas and Duffy, 1990; Nicolaysen, Pan and Justice, 1988; Coury, Blaha, Atkinson and Phillips, 1992; Di Chiara, Acquas and Carboni, 1992).  8  Positive Reinforcement and Drug Self-Administration  The self-administration of drugs is the prototypical example of drug reinforcement. Evidence that drugs of abuse are positively reinforcing to animals comes from the demonstration that animals will self-administer many of the drugs that are abused by humans including the amphetamines, narcotic analgesics, tetrahydrocannabinol (THC), cocaine, some barbiturates, 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 demonstrate that the use of cocaine obeys the principles of reinforcement. In this study, animals self-administered cocaine only when the response and the drug administration were contingent. Moreover, the response for drug underwent rapid extinction when it was replaced by saline or when the response-drug contingency was reversed. Grove and Schuster (1974) also reported extinction-like responding in monkeys when saline was substituted for cocaine during a self-administration session. Consistent with these findings, Roberts, Loh and Vickers (1989) found that the ‘breaking point’ for responding for cocaine increased when higher doses were available to rats. An observed decrease in the ‘breaking point’ following neuroleptic pre-treatment is also consistent with the idea that the increase in response rate is due to a DA antagonist-induced decrease in the reinforcing efficacy of the drug. The ‘breaking point’ is considered to be a direct measure of reinforcing efficacy. By gradually increasing the number of responses required to receive the reinforcer, a point is reached where the animal fails to respond for the drug. This is thought to represent the point at which the  9  ‘price’ the animal has to pay for the reinforcement is no longer ‘worth’ the payoff. Thus, compounds which can sustain relatively high ‘breaking points’ are thought to have relatively high reinforcing value (for review, see: Brady, 1991). Overall, these studies demonstrate that psychomotor stimulants are reinforcing.  A variety of factors influence the self-administration of drugs. Two of these are the dose of drug available and the schedule of reinforcement. Typically, when allowed access to a given dose, animals will initially ‘load up’. It is during this phase that peak levels of drug are reached in the animal’s system. Following this stage, animals typically administer the drug at evenly spaced intervals (maintenance phase) (Yokel and Pickens, 1974; Wilson, Hitomi and Schuster, 1981). Response rates vary with dose. As the dose is increased from threshold levels of drug detection, animals will increase their rate of responding. As the dose continues to increase, a point will be reached where response rates decrease. This inverted Ushaped function is frequently reported in the drug self-administration literature 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 the decrease in rate of responding at higher doses is unrelated to any motoric effects 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 of reinforcement is changed (Pettit and Justice, 1991).  Another factor influencing drug self-administration is the schedule of reinforcement. Animals can be trained to bar press for a drug infusion on  10  interval or ratio schedules. Interval schedules involve the receipt of drug following the first bar press after an experimenter-determined length of time. Ratio schedules require that animals respond a certain number of times before the drug is administered. Typically, when an animal is trained on a given schedule, it will administer a constant amount of drug over time. When the response requirement is increased, the animal will adjust its responding 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, the animal will increase its rate of responding (for review, see: Wallace and Singer, 1976).  Several hypotheses have been put forth to explain the titration of drugs in self-administering rats (for review, see: Wilson, Hitomi and Schuster, 1971). One proposal is that drug titration serves to maintain an optimal blood level of drug. This optimal level corresponds to a concentration at which the drug is maximally reinforcing. A similar hypothesis is that during to self-administration of a drug, a certain concentration of extracellular DA, corresponding to an optimal reinforcement threshold, is achieved in brain (for review, see: Wilson, Hitomi and Schuster, 1971). Once this occurs, the animal does not administer drug again until corresponding DA levels begin to decline. Reinitiation of self administration of drug is associated with a drop in DA levels below a threshold value. A third hypothesis of titration posits that too much drug is aversive and therefore the animal stops administering drug until the  11  aversive effects diminish. At this point, the animal will again administer another dose of drug.  Most of the experimental evidence supports the hypothesis that animals titrate drug intake to maintain optimal reinforcement efficacy, above a reinforcement threshold, as defined by corresponding blood levels of the drug and DA extracellular levels in the brain. Evidence for a reinforcement threshold comes from a study by Risner and Jones (1976), in which rats pretreated with amphetamine, decreased their intake of drug when permitted to self-administer amphetamine. The rats titrated their intake so that the total amount of drug received per hour, including the pretreatment dose, was the same as the amount previously self-administered without any pretreatment. This effect was not seen following pretreatment with the selective noradrenergic agonist methoxamine, suggesting that the effect was specific to the DA system.  The ‘titration’ hypothesis of responding seems to be more complex than a simple inverse relationship between dose and response rate. Although such a relationship exists, it does not appear to be linear.  Reports indicate that  animals will self-administer, in total, greater amounts of drug administered in higher doses, both in the ‘loading’ and ‘maintenance’ phase (Yokel and Pickens, 1973). Yokel and Pickens (1974) calculated from known metabolic rates that blood levels of both d- and 1-amphetamine isomers increased with the dose of drug administered. This was confirmed in the ine during 14 same set of studies by monitoring blood levels of C-amphetam self-administration. However, dose-dependent increases in blood and body levels of drug was evident only during the first 2 hours of self-  12  administration. During the subsequent maintenance phase, drug levels remained constant across doses. Experimental evidence also suggests that animals self-administer greater amounts of drug at higher doses because it is more reinforcing. Iglauer, Liewellyn and Woods (1976) reported that when given the choice of different doses of cocaine, all normally selfadministered, monkeys consistently administered the highest dose available. Support for the hypothesis that higher doses are more reinforcing also comes from the previously mentioned study demonstrating that increases in ‘breaking points’ were observed at higher doses (Roberts, Loh and Vickers,1989). Collectively, the results from these studies support the hypothesis that animals titrate their intake of drugs to achieve and maintain an optimal level of reinforcement. Consistent with this is the finding that blood levels of drug are also kept at a stable, and supposedly optimal, level.  As mentioned above, this optimal level seems to vary with dose  only during the loading phase, after which stable intake is achieved over a range of different doses.  The recent advent of in vivo neurochemical techniques has allowed for the monitoring of dynamic changes in brain DA extracellular concentrations in freely-moving animals. These technological advances have been applied to animals self-administering drugs. Accordingly, one now can test the hypothesis that animals will self-administer cocaine and amphetamine to maintain an optimal reinforcement threshold which corresponds to an optimal extracellular brain level of DA. Using in vivo microdialysis, Weiss, Hurd, Ungerstedt, Markson, Plotsky and Koob (1992) monitored DA levels in the N.Acc. in animals self-administering i.v. cocaine. Initially, an increase in DA levels was seen as the animals began to self-administer the  13  drug. Following the loading phase, responding shifted to a more regular inter-response interval and DA levels stabilized around a mean for the rest of the self-administration session. Substitution of cocaine with saline produced a brief extinction-like burst of responding which was correlated with the start of a gradual return to baseline DA levels. Pettit and Justice (1989; 1991) investigated the effects of different doses of cocaine on response rate and corresponding changes in extracellular DA using in vivo microdialysis in the N.Acc. These investigators reported a typical inverse relationship between dose and responding. However, this relationship was not linear (i.e. greater amounts were administered at the higher doses). This non-linearity was also reflected in brain DA concentrations. Although animals maintained steady levels of extracellular DA during a given session, the level varied with dose. Increased doses resulted in higher overall intake and in the maintenance of greater extracellular DA concentrations at all times during the self-administration session. The results of these three studies are consistent with the notion that optimal blood levels of drug are obtained for each dose of cocaine administered by rats and they suggest further that optimal levels of DA exceed a reinforcement threshold during a given self-administration session. In addition, in contrast to those findings by Yokel and Pickens (1974) with amphetamine, Pettit and Justice (1991) have suggested that the absolute level of DA varies with dose, reflecting a change in reinforcement efficacy of different doses of cocaine.  14  ‘Craving’ and Drug Self-Administration  One of the most elusive features of drug abuse is its chronic compulsive nature. The reinforcement model has been useful in understanding some of the variables involved in drug use, however, it does not account for the transition from drug use to abuse. In particular, a defining characteristic of drug abuse is relapse. Drug abuse is, in effect, a chronic relapsing disorder: rehabilitated addicts will often begin to take drugs again months after detoxification (Robinson and Berridge, 1993). A powerful subjective state reported to contribute to relapse in humans is ‘craving’ (Pickens and Johanson, 1992). ‘Craving’ is thought to be associated with an intense need or ‘wanting’ for drug that is not motivated by its hedonic properties (Robinson and Berridge, 1993). Although the basis for ‘craving’ is poorly understood, various researchers have put forth different hypotheses for its mechanisms. One of these is the dopamine depletion hypothesis put forth by Dackis and Gold (1985) who propose that ‘craving’ is associated with the dissipation of a drug effect or with the abrupt cessation of drug use. This absence of drug is associated with subtle withdrawal symptoms that are typically opposite to the initial euphoric and stimulant effects of the drug. The most frequently reported symptoms are decreased energy, hypersomnia, irritability, dysphoria, depressed mood and psychomotor retardation (Dackis and Gold, 1985). In short, abstinence is associated with withdrawal and anhedonia. Anhedonia in this context is defined as ‘an inability 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 with withdrawal from drug: drug use during an abstinence therefore acts as a  15  negative reinforcer. Dackis and Gold (1985) suggest that drug abuse ‘is in fact an interplay between positive and negative reinforcers’.  Neurochemical Correlates of Craving  Given the evidence for a role of elevated concentrations of extracellular DA in positive reinforcement (reviewed above), Dackis and Gold (1985) propose that DA may be critically involved in the negative reinforcement produced by the reinitiation of drug use after an abstinence. Dackis and Gold (1985) argue that since an elevation of synaptic DA concentrations may be critical to the ‘hedonic’ effects of psychomotor stimulants, it is also possible that an acute reduction in extracellular DA concentrations may be associated 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 negative reinforcer by increasing extracellular levels of DA, thereby attenuating the state of anhedonia. Based on evidence obtained from animal studies on the long-term effects of cocaine administration, Dackis and Gold (1985) propose that DA levels are decreased during a period of abstinence through depletion of intracellular DA stores. Cocaine is a potent reuptake blocker, resulting in increased extracellular levels of DA. Hypothetically, one consequence of reuptake blockade is an increase in extracellular metabolism of DA. Therefore, normal recycling and reuse of released DA is no longer available to the neuron. Citing evidence that increased levels of tyrosine hydroxylase are found in neurons after the long-term use of cocaine, Dackis and Gold (1985) hypothesize that, as a consequence of  16  prolonged cocaine-induced reuptake blockade and increased extracellular DA metabolism, compensatory increases in intracellular synthesis of DA occur. Eventually an inability of intraneuronal DA synthesis to fully compensate for the increased use of DA may lead to a DA ‘shunt’, or depletion of DA in the nerve terminal. A prediction of the dopamine depletion hypothesis (Dackis and Gold, 1985) is that reinitiation of drug self-administration after an abstinence would be associated with the time during an abstinence period at which extracellular DA levels are at their lowest. Therefore, drug use during an abstinence acts as a negative reinforcer and the dopamine depletion hypothesis would further predict that extacellular DA levels should again rise immediately after the reinitiation of drug self-administration.  The present thesis will test the predictions of the dopamine depletion hypothesis. However, first the literature establishing anhedonia as a correlate of withdrawal and the evidence establishing an association between decreases in DA and anhedonia will be briefly reviewed.  Evidence linking anhedonia with drug abstinence comes from work with intracranial self-stimulation (ICS S). Administration of psychomotor stimulants is known to potentiate responding for ICSS (for a review, see: Koob, 1992b), measured by increased rates of responding and decreased reward thresholds. Since amphetamine increases brain DA levels, the potentiation of responding for ICSS is thought to be due to the additive effects of the drug and ICSS on DA neurotransmission. During drug abstinence and withdrawal, however, ICSS responding is attenuated: i.e., response rates are lowered and reward thresholds are increased. (Koob,  17  1992; Kokkinidis and McCarter, 1990). A decrease in response rate is thought 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 amphetamine injections during withdrawal in rats. Both spontaneous locomotor activity and the locomotor response to a challenge injection of amphetamine were decreased during withdrawal from amphetamine. These responses were attenuated to the levels seen in drug-naive control rats given saline. Therefore, these studies demonstrate that drug abstinence and withdrawal are associated with anhedonia and behavioural depression.  Evidence from a number of studies support the proposal that decreased synaptic DA levels are correlated with temporary cessation of responding for drug, specifically, in the associated state of anhedonia. Wise, Spindler, deWit and Gerber (1978) reported that high doses of the DA antagonist pimozide decreased responding for food in a manner similar to that seen in animals who had their food reward withheld. The behaviour of rats given pimozide before food access and of those not receiving food were similar to each other, but differed from rats allowed free access to food. This suggests that DA may mediate an animal’s experience of hedonic states, and therefore blockade of synaptic DA transmission may attenuate the positive effects of reinforcers. Graeff, Leme and Silva (1965) demonstrated changes in the amount of spontaneous locomotion after pretreatment with various dopaminergic agents. These investigators reported an increase in locomotion following increases in DA due to administration of the DA precursor 1-dihydroxyphenylalanine (L-DOPA). These L-DOPA-induced increases in spontaneous locomotion were attenuated by depletion of  18  intracellular DA stores with systemic injections of reserpine. Furthermore, reinstatement of the potentiated locomotor response seen with L-DOPA and MAOI corresponded with the time course of repletion of dopaminergic stores. Since locomotion is thought to be mediated by DA (Kelly, Seviour and Iversen, 1975) and the increase in locomotor response is highly correlated 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 and reinforcement, these findings suggest that behavioural depression, or anhedonia, is correlated with a decrease in synaptic DA concentrations.  Direct evidence linking fluctuations in DA levels with drug abstinence or withdrawal comes from recent in vivo microdialysis studies. Segal and Kuczenski (1992; 1992b) found that dopaminergic responses to a drug challenge were attenuated 48 hours after the end of pretreatment with either amphetamine or cocaine. As well, Weiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992) found direct evidence to support the hypothesis that a significant reduction in DA release corresponded with an abstinence from drug self-administration. Using in vivo microdialysis, Weiss et al. (1992) monitored the in vivo correlates of withdrawal following free access to cocaine. Drug self-administration typically lasted between 9.5 and 21.5 hours. During this time, DA concentrations rose initially with self-administration and remained elevated around a stable mean for the duration of the self-administration session. After a three hour period of self-imposed abstinence, saline was substituted for the drug, thereby imposing a withdrawal. Withdrawal was associated with a decrease in DA concentrations below pre drug-taking baseline levels. In support of  19  postsynaptic changes predicted by the dopamine depletion hypothesis, DA levels began to decrease, in some animals, before the drug abstinence, despite continued drug administration. When the levels dropped below a certain baseline, responding stopped. However, these findings contradict the dopamine depletion hypothesis because the dopamine depletion hypothesis predicts that responding should be highest when extracellular DA levels are at their lowest. The study by Weiss et al. (1992) is inconclusive because it does not provide a full test of the dopamine depletion hypothesis, in particular, the predictions about the reinitiation of responding after an abstinence. Future studies must address the specific question of whether reinitiation of drug self-administration occurs when extracellular DA levels are at their lowest and that reinitiation of drug use results in an immediate increase in these levels and a return to normal patterns of responding. Positive results would support a major tenet of the dopamine depletion hypothesis, namely that drug administration during a withdrawal serves as a negative reinforcer and that rats reinitiate drug self administration when DA synaptic concentrations are depleted.  20  Purpose of the Present Studies  Two experiments were planned. In the first, the effects of different doses of amphetamine on self-administration behaviour and on DA concentrations in the N.Acc. were measured with in vivo electrochemistry and with in vivo microdialysis. This study tested the hypothesis that animals self-administer amphetamine to maintain an optimal level of DA release, above a so-called reinforcement 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 of some of the changes in behaviour and in vivo DA corresponding to a temporary drug abstinence following self-administration of amphetamine as measured with in vivo electrochemistry. These studies therefore provided a direct test of some of the predictions of the dopamine depletion hypothesis.  21  METHODS  General Methods  Apparatus for operant responding for food reinforcement Rats were trained to bar press for food in ‘Plexiglas’ chambers (25 cm X 30cm 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 28V D.C. 170 nA light bulb (Spectro), an operant lever and pellet dispenser fixed to one wall of these chambers. The floor of the chamber was lined with corn-cob bedding (Sanicel) and covered with a metal grid. During food training a ‘session on’ light was present. The lights, operant lever, and pellet dispenser (with food hopper) were interfaced to a computer system (MANX) (Gilbert and Rice, 1979).  Training for food Prior 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-2  schedule, for 3 out of 4 days. During training, all rats were given approximately 12 gms of rat chow (Purina) per day in their home cage. Water was available ad libitum.  IV catheter preparation Catheters were made from custom-made 22 gauge cannulae with elongated ends (5 mm) (Plastic Products Inc.). The bottom end of each cannula was bent to a right angle and Silastic tubing (0.012 I.D.; 0.025 O.D.; Dow  22  Corning) was secured to the bottom end of the cannula. To secure the catheter to the jugular vein, a small quantity of silicone gel (Home Seal) was fixed to the Silastic approximately 4 cm from the junction of the Silastic tubing and cannula (for 350-450 gm rats). For sterilization purposes, 70% ethanol was flushed through the catheters and the catheters were then stored in 70% ethanol until use.  IV catheter surgery All instruments were cold sterilized immediately prior to surgery with 0.15% alkylbenzyldimethylammonium chloride (ADC; EMI Industries) for 20 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 30 minutes. Rats were anaesthetized with xylazine (9 mg/kg i.p.; Rompun) and ketamine hydrochloride (100 mg/kg i.p.; MTC Pharmaceuticals) and supplemented with ketamine  (-  20 mg) when needed. All rats were  implanted with a single jugular catheter aimed at the left vena cava. The catheter was then tied and glued (LePage superglue) to the vein and the free end of the cannula was passed subcutaneously to the top of the head and then cemented in place with dental acrylic cement (following stereotaxic procedures). All catheterized rats were given garamycin (8 mg i.m.) and ampicillin (50 mg i.m.) prior to surgery and twice daily for one day after surgery. Immediately following the catheterization, rats were prepared for stereotaxic surgery and implantation of either electrochemical electrodes or microdialysis probes.  23  Catheter maintenance After surgery, catheters were flushed twice daily with streptokinase kabikinase (0.2mg, i.v.; KABI) if clots were detected. Catheters were flushed with 10 unit heparin (dissolved in sterile 0.9% physiological saline) during the experimental protocol, both prior to being connected to the test apparatus, 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 filled with, 70% ethanol when not in use.  Drugs d-Amphetamine sulphate was obtained from Smith Klein Beecham. The drug was mixed fresh daily in a concentration of 1 mg amphetamine sulphate per 1 ml of 1 unit heparin (dissolved in 0.9% sterile physiological saline). One unit and 10 unit heparin solutions were made fresh approximately every three weeks. Heparin was purchased as a concentrated solution and was diluted in 0.9% sterile physiological saline. Streptokinase was made fresh as needed (2 mg/i ml/1 unit heparin solution). Clidox was made fresh every month. All antibiotics and anaesthetics were purchased as sterile solutions.  I. V. injection procedure All drugs were dissolved in a sterile heparin solution (1 unit/mI heparin dissolved in 0.9% sterile physiological saline) and passed through a 0.22 m sterilizing filter (Lida Manufacturing Company) before use. All drug infusions were 100 tl in volume and given at a rate of 20 p.1/s.  i.  24  I. V. self-administration procedure The syringe pumps used to deliver infusions were interfaced to a computer (MANX) (Gilbert and Rice, 1979). The computer recorded the latency (in seconds) between successive infusions. Following each second bar-press, the MANX (Gilbert and Rice, 1979) delivered an infusion of drug to the rat. This computer was also interfaced to the electrochemical detector (GMA Technology) so that each infusion was recorded and displayed graphically as a tic mark as a function of time on the electrochemical readings. Therefore, a graph of the change in the electrochemical signal and the time at which drug infusions occurred were automatically plotted as a function of time on the same graph. Since drug infusions were plotted on this graph to the nearest minute, more accuracy in determining the time of each infusion was obtained from the hard copy of inter-infusion latencies obtained from the MANX system (Gilbert and Rice, 1979) (see page 31).  Histology Rats were sacrificed with an overdose of ketamine (i.p., or i.v. if the catheters were still patent at the time of sacrifice). Brains were then removed and stored in buffered 4% neutral formalin for at least 24 hours before sectioning. Brains were sectioned into 50 im slices and every third slice through the N.Acc. was saved and mounted on glass microscope slides. Brains were stained with cresyl violet and placements were verified under a light microscope.  25  Electrochemical Procedures  In vivo electrochemistry can be used to measure the steady state levels (concentration) and changes in the levels of oxidizable species at the tip of a recording electrode placed in a brain area of interest (Stamford, 1986; Lane, Blaha and Han, 1987). When an electroactive species is oxidized at the surface of a recording electrode in brain by application of a voltage via an auxiliary (or counter) electrode, it loses electrons. The current produced by the flow of these electrons through the recording electrode can be quantified. The amount of oxidation current detected at the recording electrode is proportional to the amount of species being oxidized at the electrode tip. A reference electrode placed in an arbitrary area of the cerebral cortex provides a ‘reference’ ground potential from which to apply either positive or negative voltages to the brain. Individual electroactive compounds in the brain typically oxidize at different applied voltages. Thus, by application of appropriate voltages, many oxidizable species in brain extracellular fluid can be quantified (Adams and Marsden, 1982).  Voltamme try The application of a voltammetric sweep is an electrochemical technique used to measure oxidation peak currents of several electroactive species and to evaluate the response characteristics of a recording electrode implanted in brain. To obtain a voltammogram, a range of potentials are applied in the form of an ascending voltage ramp (typically 10 to 100 mV/sec every 5 to 10 minutes). A plot of the resulting oxidation current with respect to the applied voltage sweep yields a plot of ‘oxidation waves’,  26  or voltammogram. The peak height of these waves are proportional to the concentration of species in solution at the tip of the recording electrode.  Semidifferentiation of the voltammetric current is a standard signal processing procedure which provides more clearly defined peak oxidation waves. In brain tissue, DA oxidizes at a graphite paste recording electrode at a potential of approximately +100 mV when scanned at 10 mV/sec (Figure 1) (Lane, Blaha and Han, 1987; Blaha and Jung, 1991; Blaha and Lane, 1983).  Chronoamperometry Repetitive square-wave pulse amperometry, or chronoamperometry, is an electrochemical technique in which 1-second duration potential pulses corresponding to the voltammetric peak oxidation potential of a species of interest is applied to a recording electrode (Adams and Marsden, 1982). The pulse is applied at an inter-sample interval of at least 30 seconds per electrode. Each applied potential pulse results in a single current sample that is directly proportional to the concentration of species in solution at the electrode tip. By plotting these concentrations (oxidation currents) with respect to time, a temporal profile of the change in extracellular concentrations of the electroactive species can be generated.  Apparatus During daily sessions, rats were placed in 32cm X 32cm X 41cm Plexiglas chambers. Fixed to the outside of this box was a grounded Faraday cage designed to screen external 60 Hz electrical noise. The interior of the box contained a stainless-steel lever with a white cue light (28 V 170 nA;  27  Figure 1: Representative voltammogram recorded at a chronicallyimplanted stearate-graphite paste electrode in the nucleus accumbens of an awake, freely-moving rat. Oxidation current is plotted against the applied voltage (ramp ratelO mV/sec). Peaks 1-3 correspond to dopamine, serotonin and metabolites of DA, respectively. Dopamine peak oxidation typically occurs between approximately 150 and 200 millivolts.  -  -o x 0  C  0  C  0  D  0  C  c  0  50  100 150 200 250 300 350 400 450 500  Voltage Applied vs. Ag/AgCI (mV)  -150 -100 -50  00  29  Spectro) directly above it. The bottom of the cage was lined with corn-cob bedding (Sanicel) and covered with a metal grid. Care was taken to ensure that the inside of the box and the rat were not grounded. The testing chamber and surrounding Faraday cage were placed within a sound-proof and light-proof black wooden chamber. An electrochemical recording lead extended from the rat’s head to a mercury-filled commutator and liquid swivel mounted to the top of the Plexiglas box. Shielded co-axial cables extended from the commutator and swivel ensemble up through the wooden box to an electrometer device (E-Chempro, GMA Technologies, Inc.) mounted on top of the wooden chamber.  I.V. tubing extended from the  animal to the liquid swivel and through the wooden chamber to a Harvard apparatus pump (Sage Apparatus, pump model 341) mounted on top of the wooden box. This pump and the electrometer device were then interfaced to a computer control system (MANX) (Gilbert and Rice, 1979).  Electrode preparation A three-electrode electrochemical recording system was used in all experiments. Recording electrodes were made from Teflon-coated stainless-steel wire (0.008° bare, 0.011” coated; Leico Industries).  The  Teflon was pulled away from the tip of the stainless-steel to create a small well of approximately 0.5 mm in depth. The well was then packed with stearate-modified graphite paste (Blaha and Lane, 1983; Blaha and Jung, 1991) (graphite powder, liquid paraffin oil and stearic acid, in a 3:2:0.2 wt./wt. mixture). The electrode tip was then surfaced on smooth glass to obtain a slightly convex graphite paste surface when viewed under a light microscope (mag. X 625) (Blaha and Jung, 1991). The auxiliary electrode consisted of a stainless-steel wire (0.008” bare, 0.011” coated; Leico  30  Industries), covered with Teflon, except for -4mm from the end of the electrode. The third type of electrode, the reference electrode, was made from Teflon-coated silver wire (0.003” bare, 0.0045” coated; Leico Industries). The Teflon was stripped away -‘1-2 mm from the end of the tip of the wire. The exposed end of the wire was chlorided in an electrolysis bath (Blaha and Lane, 1983; Stamford, 1986). The free ends of all three types of electrodes were soldered to miniature gold Amphenol pins.  Surgery Recording electrodes were aimed bilaterally at the N.Acc. (+1.2mm anterior to bregma, +/-1.2mm lateral to the midline and -6.5mm ventral to cortical dura; incisor bar -3.3mm; Paxinos and Watson, 1986).  Care was  taken to remove all blood and dura prior to lowering the electrodes into brain. A single reference electrode was implanted —‘2-3 mm posterior to bregma and lateral to the midline into the cortex of the right hemisphere. The electrode was lowered until the exposed chiorided tip was completely implanted in cortex. Four skull screws were then fixed in place to the skull. The exposed length of the auxiliary electrode was wrapped around one of the anterior skull screws. This screw was lowered its entire length into brain (-‘1 mm). The gold pins from each electrode were seated into a threaded cylindrical mini-socket holder (Science Technology Centre, Carleton University) and mounted, along with the catheter inlet, to the top of the rats’ head with dental acrylic cement.  Electrochemical recording procedures One day following surgery, recording electrodes were electrochemically conditioned overnight by applying voltammetric sweeps to each electrode  31  every hour. The day after being conditioned overnight, experimental testing began. During testing, voltammograms were obtained before each test session to determine the status of the electrode and the appropriate parameters for the applied potential pulse (chronoamperometry). All daily voltammograms were ramped from -150 mV to 450 mV at a rate of 10 mV/see; a sweep was taken from each electrode every 5 minutes and approximately 3 to 4 sweeps were collected for each electrode.  To determine daily chronoamperometric pulse parameters, the value at the trough 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 final value (voltages were typically applied from -150 to +200 mV). If the trough of the DA oxidation wave occurred at more than +275 mV, the electrodes were considered no longer patent and the rat was discontinued. The rat was also discontinued if the chronoamperometric recording was poor. An electrochemical signal was considered inadequate if large amounts of electrical noise or frequent current ‘spikes’ greater then approximately 30 nanoamperes were present.  Data collection Behavioural 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-infusion time for each infusion each day and was collected daily as a hard copy from the MANX (Gilbert and Rice, 1979) computer. Electrochemical data was interfaced to a Hewlett-Packard, IBM-PC compatible, 386DX, 20MHz  32  computer. Custom-made batch files (GMA Computer Technologies, Inc.) designed for Lotus 1-2-3 version 2.2 (Lotus) converted the daily data for each rat into Lotus worksheet graph files.  Microdialysis Procedures  In vivo microdialysis is the simultaneous sampling of a variety of extracellular molecules by perfusion of a physiological buffer through a probe inserted into a brain area of interest (Church, Justice and Neill, 1987). The active portion of the probe consists of a semi-permeable membrane. Artificial extracellular fluid (perfusate) (Moghaddam and Bunney, 1989) is passed continuously through the probe via an inlet and the molecules surrounding the probe travel down their concentration gradient across the semi-permeable membrane and into the probe. In this manner, a representative sample of the molecules in the brain extracellular compartment can be collected as dialysate from an outlet tubing of the probe. Once collected, this sample is analysed using standard analytical methods. In the present study, electroactive substances such as DA and its metabolites, 3,4-dihdroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as the serotonergic metabolite 5-hydroxyindole-3-acetic acid (5-HIAA), were separated and quantified using high pressure liquid chromatography with an electrochemical detector (HPLC-ED).  DOPAC is a presynaptic metabolite of DA formed principally in the intracellular compartment and is thought to reflect the rate of DA metabolism intracellularly. The ratio of DOPAC/DA levels in tissue has been used previously as an index of dopaminergic activity (Moore, Chieuh  33  and Zeldes, 1977; Westerink, 1979). In the case of uptake inhibitors such as amphetamine, extracellular levels of DOPAC decrease following their administration (Kuczenski and Segal, 1989). In this study, both DA and DOPAC will be measured to demonstrate the known actions of amphetamine and to provide confirmation of our in vivo DA results.  Apparatus The microdialysis chamber was a clear Plexiglas box (26.5 cm X 24cm X 29cm) placed within a sound and light-proof white chamber. The bottom of 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 parallel with the i.v. line that extended through a dual liquid swivel (Instech, Inc.) to the i.v. (Sage Instruments, model 341A) and dialysis (Harvard Apparatus, model 22) pumps.  Probe preparation The microdialysis probes were constructed from a semi-permeable hollow fibre (340 p.m O.D., 65,000 MW cutoff, Filtral 12, Hospal) with a 2mm exposed fibre length. A 55 cm length of PE 10 inlet tubing and a 55 cm length of fused silica outlet tubing (75 p.m I.D. X 150 p.m O.D) were sealed to the membrane with epoxy (Devcon 2-ton). All joints and the dialysis fibre tip were sealed with epoxy.  Surgery Two 23 gauge permanent guide cannulae with dummy probes were lowered bilaterally into brain above the target site (+1.8 mm AP; 1.0 mm ML;+I-1.0 mm DV; -3.3 mm incisor bar; Paxinos and Watson, 1986). The cannulae  34  and i.v. catheter were fixed to the skull with dental acrylic cement, anchored by 4 skull screws.  Probe insertion To minimize the effects of tissue damage on concentrations of extracellular DA, rats were perfused for approximately 16 hours prior to collection of samples. Probes were inserted into brain the day before testing and were aimed at the N.Acc. (+1.8 mm AP; 1.0 mm ML; +1-7.0 mm DV; -3.3 mm incisor bar; Paxinos and Watson, 1986) and fixed to the permanent guide cannula with brass holders (Fiorino, Coury, Fibiger and Phillips, 1993). During this time, perfusate was flushed through the probe at a rate of 1.5 tl/min.  Dialysate and perfusate During behavioural testing, collected dialysates were assayed for DA and metabolites using HPLC-ED. The perfusion medium consisted of 0.01 M phosphate buffer (pH=7.4) containing (mM) NA 147, K 3.0, Ca 1.3 and Mg++1.0. A probe perfusion flow rate of 1.5 p.1/mm  was used for all  samples collected.  HPL C-ED The compounds of interest in each dialysate sample were separated on a reverse-phase chromatographic column (Beckman ultrasphere ODS 5p.m, 15 cm 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. The apparatus consisted of a Waters 501 HPLC pump, a Waters U6K injector, and a Waters 460 electrochemical detector. Waters Maxima software was  35  used for calculation of chromatographic peaks corresponding to each measured compound (DA, DOPAC, 5-HIAA, HVA).  Both in vivo electrochemical and microdialysis techniques are improvements over previous ex vivo methods of neurochemical measurement. Traditional ex vivo methods relied on analysis of post mortem tissue, making data collection difficult and confounded by handling effects and by enzymatic-induced changes in neurotransmitter and metabolite concentrations following death. Both electrochemistry and microdialysis techniques circumvent the reliance on post-mortem tissue. These procedures allow neurochemical species of interest to be monitored in 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 of time and over many consecutive days. This permits the use of powerful within-subjects designs. Conversely, dialysis is a one-trial measurement limiting analysis to the use of between-subjects designs. Although electrochemistry allows finer time discrimination (30 sec sampling as opposed to a 15 mm  sampling with dialysis), microdialysis permits a wider  spectrum of neurochemical species to be monitored, including precursors and metabolites. Another factor to be considered is the relative sizes of the electrochemical electrodes and microdialysis probes. Electrodes have smaller 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, and therefore significantly greater sampling area (100-200 X) than electrochemical electrodes, which have a planar active surface (area  =  1.8  36  X  4 1o  . Therefore, electrochemical electrodes allow for a more cm ) 2  localized monitoring of the sites in the brain. An integration of both in vivo electrochemical and microdialysis techniques in any study can provide the benefit of both temporal resolution and a wealth of information about the dynamics of neurotransmitters and their metabolites and also offer cross-validation of methodological results.  37  EXPERIMENT ONE  Introduction  The first experiment was designed to measure changes in DA levels in the N.Acc. during self-administration of different doses of amphetamine (0.05, 0.10 and 0.20 mg/infusion). Using both in vivo electrochemical and microdialysis techniques, DA levels in the freely-moving rat were monitored during i.v. self-administration of 12 infusions of a given dose of amphetamine. The effects of different doses of amphetamine on both responding for drug and in vivo DA concentrations were monitored. It was hypothesized that an inverse relationship between dose and response rate would be observed. It also was hypothesized that, consistent with a reinforcement threshold concept, distinct ‘loading’ and ‘maintenance’ phases would be seen, with the former correlated with maximal rises in DA concentration, and the latter with the maintenance of a steady level of DA efflux around a stable mean. Consistent with findings by Yokel and Pickens (1974), it was also hypothesized that rats would maintain a steady level of extracellular DA across all doses. This study represents the first of its kind to monitor DA efflux during the self-administration of different doses of amphetamine by rats. As well, this study is important because it directly compares in vivo electrochemistry and microdialysis results during motivated behaviour. Although past studies have validated in vivo electrochemistry with microdialysis pharmacologically, this study provides convincing evidence that electrochemistry can be used to monitor DA efflux in behavioural studies.  38  Methods  Electrochemistry subjects Subjects were 30 male Long-Evans rats (3 50-450 gms at the time of surgery) from Charles River Canada. Rats were housed individually in Sanicel-lined cages and were given food and water ad libitum, except during food training. The temperature in the colony was controlled at 21°C and the humidity was constant at 40%. Rats were kept on a twelve hour light-dark cycle (0700h-1900h; lights on at 0700h) and were always tested during the light phase.  Electrochemistry  -  behavioural method  Behavioural testing began three days after surgery.  Animals had pre  exposure to the testing chamber during the overnight period of electrode conditioning. On test days, rats were attached to the i.v. catheter line and the electrochemical recording device.  The chamber was dark during the  sampling of voltammograms, and during a further 30 minute chronoamperometric baseline period. Food and water were not available at any point during testing. After chronoamperometric baselines were established, rats were given a priming injection that was of the same dose as all other injections received that day. Following this, rats were given an infusion 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 second time-out during which the house light was turned off and the operant lever was inactive. The session was terminated when the rats had received 12  39  self-administered infusions (a total of 13 infusions including the priming dose). At the end of the session the house light and bar were inactivated. The rats remained in the test chamber until the chronoamperometric signal returned to pre-infusion baseline levels. Testing sessions were typically 79 hours in length. Rats were tested daily for approximately eight days. On the 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 either received 0.05 mg dose (low) or 0.20 mg dose (high) of amphetamine per infusion. 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 high dose not previously experienced. The order in which the rats received the high and low dose was counterbalanced.  Rats were eliminated from the experiment if they failed to bar press for drug during the first self-administration session, if their catheter failed at any point during the experiment, or if their electrochemical signal was poor. All rats with intact catheters and drug lines learned to bar press during the first session of drug self-administration.  Statistical analyses  -  behaviour  A two-way repeated measures ANOVA of dose x inter-infusion interval (INI) was conducted. For all analyses, comparisons were considered significant if p<.05 (alpha.05).  Statistical analyses  -  dopamine  A two-way dose x time repeated measures ANOVA of the pre-drug baseline and first three hours of the drug effect was conducted. The purpose of this  40  analysis was to determine the maximal DA oxidation currents attained during a session (peak height). Prior to the analysis of electrochemical data, a linear transformation on the raw data was performed for each rat. A constant was subtracted from each data point in the electrochemical record so that the oxidation current corresponding to the start of the session time was at zero current. The transformed data from each rat on each day were then averaged over successive 15 minute time intervals (bins). These mean current values were analysed by a two-way repeated measures dose x time ANOVA. If a significant dose effect was found, interaction comparisons on the main effect of dose were conducted between all doses. This analysis considered the days on which rats received the high and low doses (test doses), and the medium (training) dose on the day immediately prior to receiving the first test dose. A DunnetCs post hoc analysis was conducted to determine when elevations in DA oxidation currents were significantly different from pre-drug baseline. For the Dunnett’s test, mean DA oxidation current values corresp.onding to every 15 minute time bin after the start of the self-administration session was compared to a single control DA oxidation mean current value representing the pre-drug baseline. All Dunnett’s tests were computed manually. However, the MSresidual for these analyses were computed separately with Systat® (Systat, Inc.).  For the analysis of the duration of drug effect on the elevation in the DA oxidation signal, an estimate of the value for the duration was taken as the time point where a line manually drawn through a hard copy of the pre drug baseline intersected with the electrochemical signal. If there was no intersection point, the maximum duration of drug effect on the DA  41  oxidation signals was taken as the value where the post-drug electrochemical signal became parallel with this line. A one-way repeated analysis of variance was conducted to test for a significant effect of dose on the duration of elevation in the DA oxidation signal. If a significant dose effect was revealed, interaction comparisons on the dose effect were conducted between all doses.  Statistical analyses  -  assumption of within-treatment homogeneity  Repeated measures analyses are extremely sensitive to violations of homogeneity of within-treatment variances. Any violation of this assumption can lead to erroneous rejection of the null hypothesis (type I error). When doing repeated measures analyses, most statistical packages provide correction factors for violations of the assumption of homogeneity, such as the Geisser-Greenhouse adjusted F value and the Hyund-Feldt adjusted degrees of freedom. However, caution must be used when selecting a correction factor. The former overcorrects and the latter has limited applicability. For the present analyses, we followed the suggestion of Keppel (1982) and used the Hyund-Feldt adjusted degrees of freedom for all repeated measures analyses when the Hyund-Feldt  8  was below 0.75  (denoted PHF in the text). For all other repeated-measures designs, the Geisser-Greenhouse correction was used (denoted PGG in text).  Microdialysis subjects Subjects were 17 male Long-Evans rats (3 50-450 gms) from Charles River Canada. All housing conditions were identical to those described in the electrochemical study in experiment one.  42  Microdialysis  -  behavioural testing  Three days after implantation of the dialysis guide cannula, amphetamine self-administration was begun. Rats were allowed to self-administer a total of 12 infusions of 0.10 mg of amphetamine in daily sessions. After at least three days of stable responding at this dose, microdialysis probes were inserted. Approximately sixteen hours after probes were inserted, rats were 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 either the high (0.20 mg) or the low (0.05 mg) dose. All rats were dialysed once on each side of the brain. All sessions were the same as those for the electrochemical recording phase of this experiment. Animals were not included if they did not learn to bar press for drug or if they did not selfadminister amphetamine within the first hour of a dialysis test day.  Statistical analyses  -  behaviour  See electrochemical experiment.  Statistical analyses  -  dopamine  Analyses of dialysate DA concentration were conducted as a function of dose and time. A two-way between-subjects dose x time ANOVA was conducted on the 15 minute samples of DA concentration (nanomolar; nM) in the dialysate. All other analyses of peak heights were the same as those discussed for the electrochemistry data. For the analysis of the duration of drug effect on the change in dialysate concentrations, the duration of the change in dialysate DA concentration was taken as the time when DA concentrations returned to pre-drug baseline concentrations. A one-way ANOVA of duration of elevated DA concentrations as a function of dose  43  was conducted. Dunnett’s tests were computed as in the electrochemistry described above.  Statistical analyses  -  DOPAC  All analyses of dialysate DOPAC concentrations were the same as those described above for DA. Since decreases in DOPAC concentrations seen after amphetamine treatment typically lasted longer than those for DA, the full time-course of DOPAC concentration changes was not monitored. Therefore, an analysis of the total duration of effect of amphetamine on DOPAC dialysate concentrations was not conducted.  44  EXPERIMENT TWO  Introduction  The second experiment tested the effects of prolonged amphetamine selfadministration on both response rate and in vivo DA oxidation currents in the N.Acc. in an explicit test of some of the predictions of the dopamine depletion hypothesis (Dackis and Gold, 1985). It was hypothesized that, consistent with the dopamine depletion hypothesis, periods of cessation of amphetamine self-administration would be associated with a decrease in DA concentrations. It was also hypothesized, consistent with the findings of Weiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992), that DA levels would drop to, or below, pre-amphetamine baseline levels, before and during the drug abstinence periods. The present experiment expanded on the findings of Weiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992) by allowing rats to reinitiate responding for amphetamine after an abstinence. Therefore, this study was able to monitor the behavioural and neurochemical correlates of both the initiation and termination of a selfimposed period of abstinence in self-administration of amphetamine. Consistent with the suggestion of the dopamine depletion hypothesis that DA may serve as a negative reinforcer, it was hypothesized that DA concentrations would be at their lowest during the abstinence when rats reinitiated drug self-administration and DA concentrations would increase again after reinitiation of drug self-administration. It is noteworthy that this is the first study to monitor the in vivo correlates of a reinitiation of self-administration of amphetamine after an abstinence period, thus allowing for an explicit test of the dopamine depletion hypothesis. The use  45  of in vivo electrochemical techniques permit, not only a within-subjects design, but also, for the first time, the evaluation of neurochemical changes in individual animals over a prolonged period of time.  46  Methods  Subjects Subjects were 9 male Long-Evans rats (3 50-450 gms) from Charles River Canada. Housing conditions were the same as for the other studies.  Behavioural testing In this protocol, rats experienced a total of three drug self-administration sessions. The first involved 12 infusions and a prime of amphetamine on an FR-2 response schedule (same as experiment one), the second lasted for 24 hours and allowed free access to amphetamine on an FR-2 schedule, and the final session allowed the animal free access to drug for 48 hours, also on an FR-2 schedule. The self-administration sessions were similar to the dose-response sessions of the first experiment with a few exceptions: 1) the dose of amphetamine was held constant at 0.10 mg per infusion, 2) rats were not limited to 12 infusions during the 24 and 48 hour sessions, but had access to amphetamine at all times and 3) rats were given food and water overnight in the experimental chamber. After both the 24 and 48 hour sessions, voltammograms were recorded to assess the condition and patency of each electrode. The purpose of the first two sessions was to establish reliable responding for amphetamine. Therefore, only the results from the abstinence periods during the third session are presented here.  47  Statistical analyses  -  behaviour  For each rat, the inter-infusion latencies were computed as number of bar presses per hour for each rat. A one-way repeated measures analysis of bar presses as a function of time was conducted.  Statistical analyses  -  dopamine  For the test session in which rats were permitted 48 hours of continuous access to amphetamine, both the changes in DA oxidation current over the 48 hours and during the drug abstinence periods were analysed. For the former, the transformed (see experiment one, electrochemical study) chronoamperometric data from each rat was averaged over one hour time periods and a one-way repeated measures ANOVA was conducted. A Dunnett’s post hoc test compared the average current values of each consecutive one hour time period to a single mean pre-drug baseline current value (same procedure as experiment one, electrochemistry study). See note on assumption of homogeneity of within-treatment variance in experiment one.  An abstinence period was defined as the first pause in responding for drug that lasted for more than 2 hours and that was initiated at least 20 hours into the 48 hour continuous access session. To analyse the change in DA oxidation current during the abstinence periods, six data points per rat were considered: 1) the mean current values corresponding to the 5 minutes immediately before and 2) after the last self-administered drug infusion before the abstinence period; 3) the mean current values corresponding to the 5 minute averages before and 4) after the time corresponding to the midpoint between the last self-administered drug  48  infusion before the abstinence period and the first self-administered drug infusion after the abstinence period; 5) the mean values corresponding to the 5 minutes immediately before and 6) after the first infusion after the abstinence. A one-way analysis of variance followed by Newman-Keuls post hoc analysis was conducted on these six data points.  49  RESULTS  Experiment One: In Vivo Electrochemistry  Subjects  Of the 30 rats used in this experiment, six rats were excluded from analyses because of broken catheters, 15 were excluded because they had poor electrochemical records, two were excluded because they received a drug that was not amphetamine during at least one session and one rat was excluded because the behavioural testing protocol was not correct. Therefore, of the 30 rats used in the experiment, only six met the criterion for inclusion in the analyses.  Histology  Histological examination of the six rats showed that four had electrodes placed bilaterally in the N.Acc. The remaining two rats had one of the two electrodes placed in the N.Acc. Only data considered from correctly placed electrodes were considered in the overall analysis. For subjects with two electrodes placed in the N.Acc., the chronoamperometric record with the least amount of electrical noise was used (Figure 2).  Behaviour  All rats learned to bar press (FR-2 schedule) for amphetamine on the first day of drug exposure. At all doses, response rates were initially rapid and then slowed to a constant rate for the duration of the self-administration session. The average and standard error of the mean (+/-SEM) for the  50  Figure 2: Representative sections showing the placement (solid circles) of electrochemical electrodes in the nucleus accumbens used in the analyses of experiment one (redrawn from the atlas of Paxinos and Watson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations: nucleus accumbens (N.Acc.); anterior commissure (ac); corpus callosum (cc); caudate putamen (Cpu).  51  AP+2.2mm  AP +1.7mm  AP+1.2mm  AP+O.7mm Left  I  I  I  I  I  I  I  3210123  Right  52  inter-infusion interval (INI) between each of the 12 self-administered drug infusions was 500 +1- 215 seconds for the 0.05 mg/infusion dose, 1336 +1304 seconds for the 0.10 mg/infusion dose and 3030 +1- 723 seconds for the 0.20 mg/infusion dose (Figure 3). A two-way repeated measures analysis of dose x INI confirmed that higher doses produced significantly longer INIs [F(2,10)27,  . p<.O ] 5  (For clarity, the behavioural data for  each group of rats were shown as the mean +/- SEMs number of bar presses over 15 minute time intervals in Figures 3, 4 and 5).  Change in Dopamine Oxidation Current Transformed chronoamperometric records were averaged and the +/-SEMs were determined for the 0.05 mg dose, 0.10 mg dose and 0.20 mg dose of amphetamine (Figures 4, 5 and 6). Figures 4, 5 and 6 illustrate that the DA oxidation current increased above pre-drug baseline following the selfadministration of each dose of amphetamine tested. Within one hour of the start of the session, DA oxidation currents plateaued at —6 nA for all three doses. A two-way dose x time repeated measures ANOVA revealed that the difference in peak DA oxidation current between these doses was not significant [F(2,10)0.83, PGG>. ]. However, the total duration of the 05 change in the electrochemical signal increased as a function of dose of amphetamine self-administered: larger doses resulted in longer plateaus. A one-way repeated measures ANOVA revealed a significant dose-related change in the total duration of amphetamine-induced increases in DA oxidation currents [F(2,10)32.43, PGG<•° l• As shown in Figure 7, 5 interaction comparisons revealed a significant dose-dependent increase in duration of the change in DA oxidation current between all doses (the low compared to the medium dose: [F(1,5)14.47, PHF<.° ] the low compared 5  53  Figure 3: Plot of the average inter-infusion latencies and +1- SEMs for the 12 i.v. self-administered infusions of 0.05 mg/infusion (dark solid line), 0.10 mg/infusion (light solid line) and 0.20 mg/infusion (dark stippled line) of amphetamine in the electrochemistry study.  54  C’) 1  c’J 1  0 1  -0)  -Co -  U) U) -  -L()  -  -Cr) CbJ  -  I  I  Cr)  CsJ  I  (0001  x ceo) ewi  1  55  Figure 4: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, right  axis) and corresponding change in mean (+1- SEM) dopamine oxidation currents in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.05 mg/infusion). Dark and pale solid lines are the means and +/- SEMs for the electrochemical data, respectively. Open symbols (boxes) are the means and +/-SEMs for the electrochemical data averaged over 15-minute time bins.  3  CD  Change in Dopamine Oxidation Current (nA) O)O)CD I  I  I  I  I  0 I-1 I-1  * * * * *  I-I  F’)  * * * *  CA)  * * * * *  C)1 0)  0 01  3  CD  D  CD  -h  C  D II  0)  0 I  * -  A  0  I  oD U!  g jjsesSeJd iog  9c  57  Figure 5: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+1- SEM) dopamine oxidation currents in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.10 mg/infusion). Dark and pale solid lines are the means and +1- SEMs for the electrochemical data, respectively. Open symbols (boxes) are the means and +/-SEMs for the electrochemical data averaged over 15-minute time bins.  3  (D  Ci)  Change in Dopamine Oxidation Current (nA) Q I  [0 I  I  I I F  0)00 I  I  * * * * * * * * * * * * * *  F I-I H  F-’ 1EI  0 0)  00  (0-  II 0) I -  Cl  *  -c,  A D 01  I Co  g/sesseJd JD 8c  59  Figure 6: Bar graph of mean (+1- SEM) rate of bar pressing (bottom, right  axis) and corresponding change in mean (+I SEM) dopamine oxidation currents in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.20 mg/infusion). Dark and pale solid lines are the means and +/- SEMs for the electrochemical data, respectively. Open symbols (boxes) are the means and +/-SEMs for the electrochemical data averaged over 15-minute time bins.  Change in Dopamine Oxidation Current (nA) I)  O)OO  H * *  H H H H H H  * * * * * *  —  *  C)  * * *  -  3  CD  z  C,,  *  II H H H H  * * * * * * * *  0)  p  F’)  Co  3  Co  *  II 0) I  Q  .  C,,  D  A  Cl)  0  I  CD  Uw\J  g[/sesseid  JD  09  61  Figure 7: Bar graph illustrating mean (+1- SEM) duration of effect of selfadministered amphetamine on change in dopamine oxidation currents in the nucleus accumbens as a function of dose (0.05, 0.10 and 0.20 mg/infusion). The value for these effects corresponded to the time point where a line drawn manually through the pre-drug baseline intersected with the electro chemical signal on a hard copy of the data. If there was no intersection point, the maximum duration of effect was taken as the time value where the post-drug electrochemical signal became parallel with this best-fit line.  Duration of Drug Effect (Hrs) N)  00  0  D II  0  b C),  ci 0 C’, CD CD  D II  p C  D -h C C’,  0  H  p  N) 0  II  Z9  63  to the high dose: [F(1,5)58.71, PHF<.O ] and the medium compared to 5 the high dose: [F(1,5)=19.36, PHF<. ]). 05  Dunnett’s post hoc tests revealed that for the 0.05 mg dose, the electrochemical signal first increased significantly above baseline at 45 minutes after the start of the session and remained significantly elevated for four hours (Figure 4). The DA oxidation signal for both the 0.10 mg and 0.20 mg doses was first increased significantly above baseline at one hour after the start of the session and remained significantly above baseline for 5 and 8 hours, respectively (Figures 5 and 6).  Experiment One: In Vivo Microdialysis  Subjects Of the 17 rats used in the present experiment, one was excluded from analyses because it did not bar press for amphetamine during the first hour of the self-administration session, one was excluded because its catheter broke and two were excluded because they received a drug that was not amphetamine during at least one session. Therefore, 13 rats met the criterion for inclusion in the present analyses.  Histology Probe placements (n=13) were verified histologically and found to be in the N.Acc. Data were included from four rats receiving amphetamine in the 0.10 mg dose, four rats in the 0.20 mg dose, and five rats in the 0.05 mg dose (Figure 8).  64  Figure 8: Representative sections showing the placement (vertical lines) of microdialysis probes in the nucleus accumbens used in the analyses of experiment one (redrawn from the atlas of Paxinos and Watson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations: nucleus accumbens (N.Acc.); anterior commissure (ac); corpus callosum (cc); caudate nucleus (CPu).  65  AP +2.2mm  JH .p:I  0  AP+1.7mm  a  cc  CPu AP+1.2mm cc  CPu  AP+O.7mm I  I  I  I  I  I  I  3210123  66  Behaviour All rats learned to bar press (FR-2 schedule) for amphetamine on the first day of drug exposure. At all doses, response rates were initially rapid and then slowed to a constant rate for the duration of the self-administration  session. The average and +1- SEM of the inter-infusion interval (INI) between each of the 12 self-administered drug infusions was 400 +1- 78 seconds for a dose of 0.05 mg/infusion, 729 +/- 181 seconds for a dose of 0.10 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 confirmed that higher doses produced longer INIs [F(2,10)=8.77, p<.O5J. (For clarity, behavioural data were not represented as INI, but rather as the averages and +1- SEMs for the number of bar presses over 15 minute time intervals in Figures 10, 11 and 12).  Dopamine Concentrations DA concentrations during each self-administration session was averaged across all rats for each dose (0.05, 0.10 and 0.20 mg/infusion) and +1SEMs were obtained. As shown in Figures 10, 11 and 12 the concentrations of DA (nM) increased above the pre-drug baseline following the self-administration of each dose of amphetamine tested. Within one half hour of the start of the session, DA concentrations plateaued at approximately the same peak values for all doses (2.5, 2.0 and 3.0 nM for 0.05, 0.10 and 0.20 mg/infusion, respectively).  67  Figure 9: Plot of the average inter-infusion latencies and +1- SEMs for the 12 i.v. self-administered infusions of 0.05 mg/infusion (dark solid line), 0.10 mg/infusion (light solid line) and 0.20 mg/infusion (dark stippled line) of amphetamine in the microdialysis study.  r’)  Time (Sec X 1000) C)  C)1  0)  3  CD  I) C,)  0)  -o CD Cl) C,)  00 CD -  r%) C,)  89  69  Figure 10: Bar graph of mean (+I SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+I SEM) dopamine dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.05 mg/infusion). The dark symbols represent dialysate samples collected at 15 minute intervals.  Change in Dopamine Concentration (nM) 0 —  I  I  ro I  I  .  F  I--—  —  I  I-  —  I  *  1*  * I  I  F  *  H—114*  I  =1  I  i  3  /  i  CD I  /  H•H I  H•H /  C)  0  / I-•H /  •  0 Cii  /  1  • /  •  D  (71-  II  Cl) —.  0)  *  0  D  I  I  0.CD  U!J 9L/SeSSeJd JD OL  71  Figure 11: Bar graph of mean (+1- SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+/ SEM) dopamine dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.10 mg/infusion). The dark symbols represent dialysate samples collected at 15 minute intervals.  Change in Dopamine Concentration (nM) -  —  I  I  I  •  U  U U F—  _  HNUH  IH  *  H—U—  *  *  H----1  H  5•4 /  F— /  I  =1 r) 3  CD  I -‘  /  HUH / UI  w  CI)  I  /  U  •  I  F-U-H  0  [UI \  3 3.1 1 c  U  D  —I’  *  D Co  A  D  I  I  U!LAJ L/SeSSeJd JD ZL  73  Figure 12: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+1- SEM) dopamine dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.20 mg/infusion). The dark symbols represent dialysate samples collected at 15 minute intervals.  Change in Dopamine Concentration (nM) C —  I  I  I’) I  I  .  0 * 1*  /111 I H—•—-H H——-H  H  /  H—B---H  H =!  H-•-H  * * * *  H—çH*  H  CD  1*  0 1\) 0  H-H  .  3  F•H /  I  —I / I  —.  • /  —1•1  /  1  D  \  0)  He-H  !.  .jii  0  *  1’  0  /  I  I  0 -i’Cc  u,tgjjsesseJJDg  t’L  75  The total duration of amphetamine-induced increases in DA concentrations increased as a function of dose. A one-way between-subjects ANOVA of the duration of amphetamine-induced increases in DA concentrations confirmed a significant dose effect [F(2,1O)18.11,  p<.O5].  As shown in  Figure 13, interaction comparisons on the total duration of change in DA concentrations revealed a significant difference between the low and high doses [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 x time mixed ANOVA of the three doses and twenty consecutive 1 5 minute samples (4 baseline and then the first 4 hours of the self-administration session) was conducted. The analysis confirmed that there was no effect of dose 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 concentration was first significantly elevated above baseline levels at 30 minutes after the start of the self-administration sessions for amphetamine. For the 0.05 mg dose, the elevation remained significant for one and a half hours (Figure 10). For the 0.10 mg and 0.20 mg doses, the DA concentration remained elevated for one and three hours, respectively (Figures 11 and 12).  76  Figure 13: Bar graph showing the mean (+1- SEM) total duration of effect of self-administered amphetamine on change in dopamine concentrations in the nucleus accumbens as a function of dose (0.05, 0.10 and O2O mg/infusion). Values for these effects correspond to the time point where post-drug dialysate concentrations of dopamine returned to pre-drug levels.  Duration of Drug Effect (Hrs) 0)  Co  0  :3 II  01 0  0 C,) (D  p (0  :3 II  0  D  -h Cl)  0 :3  0  :3 II  0  U  78  DOPA C Concentrations DOPAC concentrations corresponding to the self-administration of amphetamine as determined by microdialysis were averaged across all rats for each dose, and SEMs were obtained. As shown in Figures 14, 15 and 16, the DOPAC concentrations decreased for all doses and remained below baseline levels during the entire self-administration session. A two-way dose x time between-within ANOVA did not reveal a significant main effect of dose [F(2,0)0.77,  p>.O5].  DunnetCs test revealed that for the 0.05 mg/infusion and 0.10 mg/infusion doses, DOPAC levels were first significantly decreased by 45 minutes and 30 minutes, respectively. For both doses, DOPAC concentrations remained significantly decreased for the entire duration of the session. For the 0.10 mg/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 change from baseline at any time during the drug session (Figure 15).  79  Figure 14: Bar graph of mean (+/ SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+1- SEM) DOPAC dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.05 mg/infusion). The open symbols represent dialysate samples collected at 15 minute intervals.  =:!  ___  Change in DOPAC Concentration (X100 nM) o I  I  I  I  I  I  0) I  I  /H F—/  CD  I  \ • /  I  I  F  \  •  *  * *  * *  •—H  I  I  F—•—-i  I  I  I  •  •  I  0-  —.4 —.4  -— •  F—RH  * * *  F—R---H  I I  F)  3  * * *  I  CD •  1*  I  /  •  I .II-  * * *  I  •  0 cI71  3  CD C71  D  Dc ii  0).  *  A  I  I  0 -CD  u gL/SeSSeJd  JD  08  81  Figure 15: Bar graph of mean (+I SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+1- SEM) DOPAC dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.10 mg/infusion). The open symbols represent dialysate samples collected at 15 minute intervals.  =1  3  Change in DOPAC Concentration (X100 nM) l’s) I  I  I  I  I  I  0) I  I  CD I  -H  •  H IH [1  h’ 4 • / \  H  /  /  1  CD  •—  I  ‘4.-,  .  I  3 C,’ —I.’  Cl)  I  *  A  I  gi/sesSeid  UI[AJ  iD  18  83  Figure 16: Bar graph of mean (+1- SEM) rate of bar pressing (bottom, right axis) and corresponding change in mean (+1- SEM) DOPAC dialysate concentrations in the nucleus accumbens (top, left axis) of rats self-administering twelve infusions of amphetamine (0.20 mg/infusion). The open symbols represent dialysate samples collected at 15 minute intervals.  Change in DOPAC Concentration (X100 nM) 1) —  I  I  I  I  I  I  0) I  CD I  HH HH /  I  H  H  *  *  H•H  *  I—•-H  *  F•-  *  H_  *  F-  *  / F--  I  Fl  H  (1  CA)  *  -H  *  .  *  --1  *  I—s-H l-_  * *  #_  I  I  —H  1h  I\)  * * *  H-R-H /  [I  3  *  -—-  I  I  *  He—H I  (31  *  HH  *  -H  *  I—-l  I  a)  *  •  *  F•-l  —.  * -  A  U  *  •  I —  —-  -  0  01  *  I  I  91/sesSeJd JD  Uf\J  t8  85  Experiment Two  All rats acquired self-administration behaviour on the first day of drug exposure: both the response rates and DA efflux were similar to those observed on the first day of experiment one. Only the abstinence periods from the third session are presented here.  Subjects Of the nine rats used in the present experiment, one was excluded from the analyses because it had a poor electrochemical signal, one was excluded because a power failure caused self-administration to be terminated midsession and one was excluded because it overdosed on amphetamine. Therefore, six rats met the criterion for inclusion in the present experiment.  Histology Histological examination of the six rats tested in this study showed that four had electrodes placed bilaterally in the N.Acc. The remaining two rats had one of the two electrodes placed in the N.Acc. Only data from correctly placed electrodes were considered in the overall analysis.  For  subjects with two electrodes placed in the N.Acc., the electrochemical record with the least amount of electrical noise was used (Figure 17).  Behaviour Bar press rates were averaged (+/_ SEM) for six hour blocks, as seen in Table 1. A one-way repeated measures ANOVA confirmed that the hourly  86  Table 1. Number of Bar Presses Per Hour Averaged (+1- SEM) for Six Hour Blocks  Time Interval (Hrs)  Average  +1- SEM  1-6  3.00  0.56  7-12  3.80  0.77  13-18  2.97  0.29  19-24  2.69  0.11  25-30  2.61  0.68  31-36  2.08  0.41  37-42  2.92  0.56  43-48  3.28  0.51  87  Figure 17: Representative sections showing the placement (solid circles) of electrochemical electrodes in the nucleus accumbens used in the analyses of experiment two (redrawn from the atlas of Paxinos and Watson, 1986; +0.7 to +2.2 mm anterior to bregma). Abbreviations: nucleus accumbens (N.Acc.); anterior commissure (ac); corpus callosum (cc); caudate putamen (CPu).  88  AP+2.2mm  AP +1.7mm  AP+1.2mm  APi-O.7mm Left  I  I I  32  I I  I I  I  I I  I I  10123  RigIit  89  rate of bar pressing for the groups did not differ significantly with time [F(47,235)=9.79, PHF> i. 05  Abstinence Periods-Electrochemistry Each rat displayed at least one abstinence period during the 48 hour test session (Figures 18, 19 and 20). Each abstinence period was characterized by a complete cessation of bar pressing and concurrent decreases in the electrochemical signal corresponding to extracellular DA. The end of the abstinence period corresponded to the reinitiation of a regular pattern of responding and a subsequent rise in DA oxidation current levels. The approximate duration of the abstinence periods for rats ci, c2, c3. c4. c5 and 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 changes in DA oxidation current during the abstinence period confirmed that there was a significant effect between the different time intervals that constituted the different phases of the abstinence period [F(5,25)=6.15, PHF<•° l• A 5 Neuman-Keuls analysis revealed that the first and second of these mean time values, corresponding to the five minutes before and five minutes after the initiation of the abstinence period, were significantly different from all other time values, but they were not different from each other.  90  Figure 18: Examples of behavioural correlates (tic mark  =  one drug  infusion) for individual subjects prior to, during, and following the drug abstinence period. Corresponding time-course of changes in dopamine oxidation current is indicated by the dark continuous line. The horizontal line represents the estimate of a best-fit line drawn manually, on a hard copy of the data, through one hour of the pre drug abstinence baseline. Data are from rats ci, c2 and c3. Number shown in parentheses for rat ci indicate the number of bar presses at that time.  91  I  4 Nanoamps  Th  U  ratci  JH  rat c2  JLJ____  rat c3  J_J______  (14)  (13)  -1  0  1  2  3  Time (Hrs)  4  5  6  92  Figure 19: Examples of behavioural correlates (tic mark  =  one drug  infusion) for individual subjects prior to, during, and following the drug abstinence period. Corresponding time-course of changes in dopamine oxidation currents is indicated by the dark continuous line. The horizontal line represents the estimate of a best-fit line drawn manually, on a hard copy of the data, through one hour of the pre drug abstinence baseline. Data are from rats c4 and c5.  93  I  4 Nanoamps  rat c4  I  rat c5  J 1  A  0  1  2  3  Time (Hrs)  4  5  6  94  Figure 20: Examples of behavioural correlates (tic mark  =  one drug  infusion) for individual subjects prior to, during, and following the drug abstinence period. Corresponding time-course of changes in dopamine oxidation currents is indicated by the dark continuous line. The horizontal line represents the estimate of a best-fit line drawn manually, on a hard copy of the data, through one hour of the pre drug abstinence baseline. Data are from rat c6.  95  I  4 Nanoamps  rat c6  vI  I —1  1  3  5  7  Time (Hrs)  9  11  13  15  17  96  Figure 21: Group mean (+/ SEM) changes in dopamine oxidation current in the nucleus accumbens corresponding to the time periods pre, post and during the abstinence period. A=mean current from the five minute period prior to last self-administered infusion of amphetamine (0.10 mg/infusion) before abstinence. Bmean current from the five minute period following the last infusion before abstinence. C=mean current from the five minute period prior to the midpoint in time between the last infusion before abstinence and the first infusion after abstinence. D=mean current from the five minute period following the midpoint between the last infusion before abstinence and the first infusion after abstinence. E=mean current from the five minute period prior to the first infusion after abstinence. Fmean current from the five minute period following the first infusion after abstinence. The vertical stippled lines represent the last infusion prior to the drug abstinence and the first drug infusion after the drug abstinence, respectively.  3  CD -Q CD 0  Dopamine Oxidation Current (nA) F’)  0  1’)  D CD  w a Qcn.  *  CD D  a-  *  CD  m  * -c3  -no  Ci) -+  *  D  II 0)  A  b C)’  L6  98  DISCUSSION  Experiment One  The behavioural findings from experiment one demonstrated that rats will self-administer increasingly larger doses of amphetamine with longer interinfusion latencies. All doses tested produced two phases of responding. One of these, the loading phase, was characterized by rapid responding at the start of the self-administration session. A second maintenance phase followed the loading phase and was characterized by a decrease in response rates to a stable level maintained throughout the remaining duration of selfadministration. Both DA oxidation currents and dialysate DA concentrations corresponding to extracellular DA efflux in the N.Acc. initially increased during the loading phase of self-administration and then plateaued during the maintenance phase remaining constant for the duration of self-administration. Maximum increases in DA efflux attained during a session did not vary with dose, despite dose-related changes in the rate of self-administration. As well, the maximal increase in DA levels remained constant within rats across days for the same dose. However, each increment in the dose of amphetamine self-administered increased the length of time that DA levels remained elevated between infusions, thereby increasing the duration of the sessio,n required to self-administer 12 infusions of the drug. Correspondingly, a dose-related increase in the total duration of change in DA concentration from pre-drug baseline was found.  Although no dose-dependent changes in the maximal height of DA efflux attained during self-administration sessions were seen in either the  99  electrochemistry or microdialysis studies of experiment one, statistical analyses revealed differences between the two sets of findings. For example, in the in vivo electrochemistry study, a longer period of time was required for the chronoamperometric signal to differ significantly from pre drug baseline levels. These changes in chronoamperometric signal also remained elevated for a longer time than for rats in the in vivo microdialysis study. As well, the total duration of amphetamine-induced increases in DA oxidation currents was significantly different between all doses tested in the electrochemistry study. However, in the microdialysis study, there was no difference in the total duration of amphetamineinduced 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 dialysate levels during amphetamine self-administration. As expected, DOPAC levels dropped during self-administration (Kuczenski and Segal, 1989; Kuczenski, Segal and Aizenstein, 1991) and this change corresponded in time to the rise in extracellular DA levels. However, there was a large amount of between-subjects variability in the time-dependent decreases in DOPAC concentrations during self-administration ofthe middle (0.10 mg/infusion) dose. Not surprisingly, statistical analyses failed to reveal a significant time-dependent decrease in DOPAC concentrations during the self administration of this dose.  Some of the discrepancies between the neurochemical findings of the electrochemistry and microdialysis studies are clearly related to the results obtained with the medium dose (0.10 mg/infusion) in the microdialysis experiment. One possible explanation for the discrepancy in the findings  100  between the two studies would be that one study (electrochemistry) employed a within-subjects design and the other (microdialysis) a betweensubjects design. As such, the between subjects design would not account for the individual variability in basal DA levels or metabolism (Kuczenski, Segal and Aizenstein, 1991). As well, the high variability in the results from the microdialysis experiment could reflect the small and uneven number of subjects used in this study (for 0.05 mg/infusion, n=5; for 0.10 mg/infusion, n=4; for 0.20 mg/infusion, n=4). Increasing the. number of subjects in all groups in the microdialysis study may lead to greater agreement between the results of the electrochemistry and microdialysis studies.  It is important to note that discrepancies between the chronoamperometric and dialysate temporal profiles of drug-induced changes in DA efflux have been reported in the past (Blaha, Coury, Phillips and Fibiger, 1990). When measured with microdialysis, drug-induced changes in DA efflux are frequently seen to be shorter in duration relative to the chronoamperometric findings reported for similar studies. Microdialysis is based on the premise that particles in solution will travel down their concentration gradient. Therefore, when obtaining estimates of extracellular DA concentrations, a brain area is dialysed with a physiological perfusate devoid of DA. DA in the extracellular fluid will therefore cross into the dialysis probe down its concentration gradient. It has recently been shown that dialysing brain tissue results in a significant perturbation (decrease) in basal extracellular DA (Blaha, 1992). Since most of the DA extracted from the interstitial space during dialysis would normally be taken back up into the nerve terminal and metabolized or  101  recycled, microdialysis could in effect decrease intracellular stores of DA available for synaptic release. This would result in a blunted time-course response to drug. Thus, it is possible that differences in the temporal profile of drug-induced changes in DA measured with electrochemistry and microdialysis in experiment one may be due to the tendency of microdialysis to extract DA, thereby decreasing basal levels.  Despite these technical differences between the two studies in experiment one, the findings of both studies revealed that maximal levels of DA attained during the self-administration of various doses of amphetamine remained constant across doses. The findings from experiment one are therefore consistent with the notion of a reinforcement threshold defined by blood levels of drug and brain levels of DA. The reinforcement threshold refers to the minimum level of drug in the blood and DA efflux in the brain required to maintain self-administration of a drug. Although there is no suggestion in the literature as to what the absolute ‘value’ of the reinforcement threshold would be, the exact determination of such a value may not be as critical to the validity of the DA reinforcement threshold hypothesis as the demonstration that a constant relative change in DA levels is achieved with different drug doses during a self-administration session. That is, evidence of the maintenance of steady levels of extracellular DA efflux in brain and blood levels of drug by a predictable pattern of titration of drug intake, is entirely consistent with a DA reinforcement threshold. Implicit in this interpretation of a reinforcement threshold is the assumption that the titered level of DA in the brain and drug in the blood is sufficient to exceed the reinforcement threshold. The findings of experiment one that a constant efflux of DA was seen both  102  within sessions for a given animal and across different doses is consistent with the notion of such a reinforcement threshold. These findings support those of Yokel and Pickens (1974), who found that rats will titer the selfadministration of different doses of amphetamine to maintain a steady blood level of drug both between and within doses and across a selfadministration session. As well, the finding that rats decreased their rate of responding for higher doses supports the hypothesis that animals titer amphetamine to maintain an optimal level of drug in their blood and DA efflux in the brain and is consistent with the dose-related behavioural changes 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 of Pettit and Justice (1991) who showed that rats titer their intake of cocaine at a given dose, to maintain a stable level of DA efflux during a selfadministration session. However, cocaine and amphetamine do appear to differ in one important respect, as Pettit and Justice (1991) observed that rats self-administered greater amounts of higher doses of cocaine when made available for injection. The maximal level DA efflux maintained during a self-administration session also increased with higher doses. The dose-dependent increase in DA efflux reported by Pettit and Justice (1991) is difficult to reconcile with the present results. It is possible that amphetamine, being both a reuptake inhibitor and releaser of DA, can support greater absolute increases in DA efflux than cocaine, which acts on DA only as a reuptake blocker. At the very least, these cocaine data are consistent with the hypothesis that rats titer drug intake to maintain a  103  steady level of drug in the blood and DA in the brain, which exceeds a reinforcement threshold corresponding to a specific amount of DA efflux.  104  Experiment Two  Experiment 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 time exceeding two hours. These abstinence periods were accompanied by a drop in DA levels to below pre-drug baseline concentrations in four rats (ci, c3, c4, c6). The DA oxidation currents for one rat dropped to baseline (c5) and remained above baseline for the other subject (c2). As well, in two rats (c2 and c5), the decrease in DA concentrations preceded drug abstinence. For the other four rats, the decrease in DA levels occurred when the rats stopped self-administering. As well, half of the rats in this group (ci, c3 and c4) reinitiated drug self-administering when DA levels were approximately at their lowest level and the other half reinitiated responding after DA levels began to rise spontaneously. By definition, at the end of the abstinence period, normal responding occurred in all cases. It is important to note that it was not clear from the present data whether an increase in the DA signal occurred before or after the reinitiation of responding. Nevertheless, it is evident that the DA system responded to the reinitiation of self-administration of amphetamine and that the abstinence period ended at a time when the DA system was responsive to the pharmacological actions of the drug.  The neurochemical findings from this experiment support those reported by Weiss, Hurd, Ungerstedt, Markou, Plotsky and Koob (1992). In their study, extracellular DA efflux initially increased with the onset of self administration and then stabilized around a steady mean value for the  105  duration of the self-administration session. For most rats, DA concentrations decreased to values lower than pre-drug baseline levels during drug abstinence. Together, these data are also consistent with the dopamine depletion hypothesis which predicts that drug abstinence should be associated with a decrease in extracellular DA concentrations in specific regions of the brain.  As described in the Introduction, the dopamine depletion hypothesis (Dackis and Gold, 1985) predicts that a decrease in extracellular DA levels during drug abstinence, occurs as a result of depletion of intracellular DA stores after prolonged stimulation of dopaminergic neurons. It is also hypothesized that this depletion of dopaminergic stores causes a state of anhedonia (an inability to derive pleasure from normally pleasurable events (Koob, 1992b)). Subsequent responding for a drug under these circumstances serves as a negative reinforcer because the drug causes an elevation of extracellular DA levels (Dackis and Gold, 1985). With respect to the present experiment, the dopamine depletion hypothesis would predict that the reinitiation of self-administration of drug during an abstinence should occur at the time of maximal depletion of DA stores and that a reinitiation of self-administration would lead to a rise in extracellular DA levels. These predictions are partially confirmed by the data from experiment two as DA oxidation currents were seen to increase after reinitiation of amphetamine self-administration. However, no clear evidence was found to support the prediction that reinitiation of self administration would occur when DA levels were at their lowest values. Even though rats self-administered amphetamine when DA efflux was low,  106  some rats reinitiated self-administration only after a spontaneous rise in extracellular DA levels.  The data from experiment two can be interpreted in light of the reinforcement threshold hypothesis. These findings are consistent with the idea that a minimal increase in DA efflux must be maintained to support drug self-administration. The fact that rats still continued to selfadminister amphetamine when extracellular DA levels had declined to levels near pre-drug baseline values, suggests that the reinforcement threshold is quite low. Perhaps the best evidence for a reinforcement threshold comes from experiment two in which rats ceased to self-administer amphetamine when the extracellular levels of DA dropped below a certain value. The fact that self-administration of amphetamine was reinitiated when the DA system again became responsive is also consistent with this concept. Other evidence indicates that drug self-administration is correlated with an increase in extracellular DA efflux (reviewed in Robinson and Berridge, 1993). A true test of the DA hypothesis of drug reinforcement would be to demonstrate that abstinence from drug self-administration corresponds to a time when the DA system is unable to exceed a reinforcement threshold and therefore support self-administration. Experimenter-administered infusions of amphetamine and selective DA agents at specific times during an abstinence period could be used to ascertain the time course of this refractory period of the mesolimbic DA system and thereby determine if rats will only reinitiate responding for amphetamine when the DA system can exceed a nominal reinforcement threshold.  107  It is possible that certain unconditioned cues and behavioural tasks can influence DA efflux. Among these are locomotor activity, lights and circadian rhythmicity. Since the N.Acc. is a putative limbic-motor output region, it is possible that changes in DA efflux seen during selfadministration are simply an artifact of increased locomotor output. This is a particular problem for experiment two. The increase in DA efflux seen prior to reinitiation of self-administration may be due to the locomotion and arousal associated with events prior to the reinitiation of bar pressing. However, there is little evidence to support this possibility. Louilot, Le Moal and Simon (1986) reported that social interactions between rats can increase DA efflux in the N.Acc. as measured by in vivo voltammetry. These researchers also demonstrated that these DA neurons are most active during those hostile social interactions which render the rats immobile. As well, Damsma, Pfaus, Wenkstern, Phillips and Fibiger (1992) monitored changes in DA concentration as measured by in vivo microdialysis during forced activity on a treadmill. These studies showed no notable change in DA efflux in the N.Acc. during such locomotor activity. As well, evidence dissociating conditioned locomotion and DA efflux supports the claim that locomotor output does not influence N.Acc. DA efflux (Brown and Fibiger, 1992). The possibility that locomotion and arousal may contribute to the DA signal prior to self-administration at the end of an abstinence can be tested in future studies by monitoring the amount of locomotion and general activity of the rat during the period immediately prior to the reinitiation of self-administration after an abstinence.  The present study used light cues and required rats to remain in the test chamber for prolonged periods of time. Appropriate controls are important  108  to establish that the increase in DA seen with the self-administration of amphetamine does not reflect the unconditioned effects of lights or circadian rhythmicity. Preliminary findings with control subjects (n=2), receiving yoked-saline paired with the same CS+ as the rats selfadministering amphetamine, have shown no increases in DA oxidation current associated with circadian rhythms, feeding or the onset or offset of light cues. Nevertheless, additional control subjects are needed to confirm that the DA efflux in the present study are free of these confounds and are due 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 drug infusions 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-administration session or by placing a rat in the operant chamber with only saline available as a reinforcer. Although such studies have not been conducted in experiments one and two, drug-naive rats in other experiments, without prior operant training for food, that were placed in the operant chamber for drug self-administration training rarely press the lever prior to acquisition of the operant task.  In summary, the findings of both experiments in this study support the hypothesis that DA is an essential correlate of drug-reinforcement. The findings that rats will titer their self-administration of amphetamine to maintain stable levels of extracellular DA across doses, is consistent with the concept of a reinforcement threshold. As well, the findings from experiment two demonstrate that patterns of amphetamine self-  109  administration correspond to periods when DA neurons in the N.Acc. are responsive to the drug. Although the absolute change in DA efflux required to maintain self-administration may not be critical, it is clear that a rise in DA levels above a nominal reinforcement threshold is a necessary condition to support drug self-administration. Combined, the results from both experiments provide strong support for the positive reinforcement model of drug self-administration (Wise, 1987). Future hypotheses about ‘craving’ and drug abstinence will need to consider that a change in DA efflux is a critical component to the acquisition of both drug use and the maintenance 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 process of ‘craving’. The incentive-sensitization theory of drug addiction (Robinson and Berridge, 1993) maintains that activity in the mesolimbic DA system is critically involved in processes by which incentive salience is attributed to the drug and to conditioned stimuli paired with the unconditioned effects of drugs of abuse. Furthermore, the DA system becomes sensitized as a result of repeated exposure to drugs of abuse. By sensitization of the DA system, the salience of conditioned stimuli associated with drug taking is enhanced. Robinson and Berridge (1993) propose that the sensitization of the incentive salience of the conditioned stimulus, underlies the process of ‘craving’ in the absence of drug. Therefore, this theory, unlike the dopamine depletion hypothesis, accounts for both the long-term nature of ‘craving’ and ‘craving’ in the absence of  110  any systemic drugs or withdrawal symptoms. Consistent with the findings of experiment one and two reported here, ‘craving’ is linked to the increased efflux of DA, not to its depletion. Future work will likely focus on establishing an animal model of ‘craving’ and on testing the predictions of the incentive-sensitization theory. Specifically, the incentivesensitization theory has drawn its conclusions about the nature of sensitization from studies in which drugs of abuse were experimenter administered. A true test of the validity of this theory will be to establish that sensitization of the mesolimbic DA system can occur in animals permitted to self-administer drugs of abuse. The present set of studies suggest that the application of the intravenous and in vivo techniques presented here will prove valuable in determining the validity of this new theory.  111  REFERENCE S  Adams, R.N. and Marsden, CA. (1982). Electrochemical detection methods for monoamine measurements in vitro and in vivo. In: Handbook of Psychopharmacology, L.L Iversen, S.D. Iversen and S.H Snyder (Eds), Vol 15. Plenum, New York. pp 1-74. Beitner-Johnson, D., Guitart, X. and Nestler, E.J. (1991). Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins. Brain Research, 561: 147-150. Beninger, R.J. (1983). The role of dopamine in locomotor activity and learning. Brain Research Reviews, : 173-196. Blaha, C.D. (1992). Electrochemical evaluation of the microenvironment surrounding microdialysis probes in vivo. In: Monitoring Molecules in the Neurosciences. H. Rollema and B.H.C. Westerink (Eds.). Krips Repro, Meppel, the Netherlands. pp. 56-60. Blaha, C.D., Coury, A., Phillips, A.G. and Fibiger, H.C. (1990). Effects of neurotensin on dopamine release and metabolism in the rat striatum and nucleus accumbens: cross-validation using in vivo voltammetry and microdialysis. Neuroscience, j: 699-705. Blaha, C.D. and Jung, M.E. (1991). Electrochemical evaluation of stearate-modified graphite paste electrodes: selective detection of dopamine is maintained after exposure to brain tissue. Journal of Electroanalytical Chemistry, j: 3 17-334. Blaha, CD. and Lane, R.F. (1983). Chemically modified electrode for in vivo monitoring of brain catecholamines. Brain Research Bulletin, j.Q.: 861-864. Brady, J.V. (1991). Animal models for assessing drugs of abuse. :35-43. Neuro science and Biobehavioral Reviews, Brown, E.E. and Fibiger, H.C. (1992). Cocaine-induced conditioned locomotion: absence of associated increases in dopamine release. Neuroscience, 4j.:621-629. Caine, S.B. and Koob, G.F. (1993). Modulation of cocaine self administration in the rat through D-3 dopamine receptors. Science, 260:1814-1816.  112  Carr, G.D. and White, N.M. (1986). Anatomical dissociation of amphetamin&s rewarding and aversive effects: an intracranial microinjection study. PsychoDharmacology, 34O-346. Chait, L.D. (1993). Factors influencing the reinforcing and subjective effects of d-amphetamine in humans. Behavioural Pharmacology, :191199. Church, W.H., Justice, J.B. Jr., Neil!, D.B. (1987). Detecting behaviorally relevant changes in extracellular dopamine with microdialysis. Brain Research, 4i:397-399. Coury, A., Blaha, C.D., Atkinson, L.J. and Phillips, A.G. (1992). Cocaineinduced changes in extracellular levels of striatal dopamine measured concurrently by microdialysis with HPLC-EC and chronoamperometry. Annals of the New York Academy of Sciences, .4424-427. Dackis, C.A. and Gold, M.S. (1985). New concepts in cocaine addiction the dopamine depletion hypothesis. Neuroscience and Biobehavioral Reviews, :469-477. Damsma, G., Pfaus, J.G., Wenkstern, D., Phillips, A.G. and Fibiger, H.C. (1992). Sexual behavior increases dopamine transmission in the nucleus accumbens and the striatum of male rats: comparison with novelty and locomotion. Behavioral Neuroscience, i: 181-191. Di Chiara, G., Acquas, E. and Carboni, E. (1992). Drug motivation and abuse: A neurobiological perspective. Annals of the New York Academy of Sciences, :2O7-219. Fiorino, D., Coury, A., Fibiger, H.C. and Phillips, A.G. (1993). Electrical stimulation of reward sites in the ventral tegmental area increases dopamine transmission in the nucleus accumbens of the rat. Behavioural Brain Research, 55:131-141. Fischer, J.F. and Cho, A.K. (1979). Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model. Th& 203-209. Journal of Pharmacology and Experimental Therapeutics, Gawin, F.H. (1991). Cocaine addiction: psychology and neurophysiology. :1580-1586. Science,  113  Gawin, F.H. and Kieber, H.D. (1986). Abstinence symptomatology and psychiatric diagnosis in cocaine abusers. Archives of General Psychiatry, 41:107-113. Gilbert, S.G. and Rice, D.C. (1979). NOVA SKED II: A behavioral notation language utilizing the Data General Corporation real-time disk operating system. Behavior Research Methods and Instrumentation, 11:71-73. Graeff, F.G., Leme, J.G. and Silva, M.R.E. (1965). Role played by catechols and indolamines in the central actions of reserpine after monoamineoxidase inhibition. International Journal of Neuropharmacology, : 17-26. Grove, R.N. and Schuster, C.R. (1974). Suppression of cocaine selfadministration by extinction and punishment. Pharmacolo gy. Biochemistry and Behavior, :199-208. Hurd, Y.L., Weiss, F., Koob, G.F. and Ungerstedt, U. (1989). Cocaine: in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum. Synapse, :48-54. Iglauer, C., Llewellyn, M.E. and Woods, I.E. (1976). Concurrent schedules of cocaine injection in rhesus monkeys: dose-variation under independent and non-independent variable-interval procedures. Pharmacological Reviews, i: 367-383. Iwamoto, E. and Martin, W. (1988). A critique of drug self-administration as a method for predicting abuse potential of drugs. NIDA, U:457-465. Jackson, D.M., Anden, N-E. and Dahistrom, A. (1975). A functional effect of dopamine in the nucleus accumbens and in some other dopamine-rich parts of the rat brain. Psychopharmacology, 41: 139-149. Johanson, C.E. and Fischman, M.W. (1989). The pharmacology of cocaine related to its abuse. Pharmacological Reviews, 41:3-52. Joyce, E.M. and Koob, G.F. (1981). Amphetamine-, scopolamine- and caffeine-induced activity following 6-hydroxydopamine lesions of the mesolimbic dopamine system. Psychopharmacology, 71:311-313. Kalivas, P.W. and Duffy, P. (1990). Effect of acute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse, : 48-58.  114  Kelly, P.H., Seviour, P.W. and Iversen, S.D. (1975). Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Research, 9..4507522. Keppel, G. (1982). Design and Analysis: A Researcher’s Handbook. Prentice Hall, New Jersey. pp.468-473. Kokkinidis, L. and McCarter, B.D. (1990). Postcocaine depression and sensitization of brain-stimulation reward: analysis of reinforcement and performance effects. Pharmacology. Biochemistry and Behavior, 463471. Koob, G.F. (1992). Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends in Pharmacological Sciences, .11:177-184. Koob, G.F. (1992b). Neural mechanisms of drug reinforcement. Annals of the New York Academy of Sciences, .:171-191. Kosten, T.E. and Kosten, T.R. (1991). Pharmacological blocking agents for treating substance abuse. The Journal of Nervous and Mental Disease, j7.: 583-592. Kuczenski, R. (1986). Dose response for amphetamine-induced changes in dopamine levels in push-pull perfusates of rat striatum. Journal of Neurochemistry, 4: 1605-1611. Kuczenski, R. and Segal, D.S. (1989). Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis. fj Journal of Neuroscience, :205 1-2065. Kuczenski, R., Segal, D.S. and Aizenstein, M.L. (1991). Amphetamine, cocaine, and fencamfamine: relationship between locomotor and stereotypy response profiles and caudate and accumbens dopamine dynamics. The Journal of Neuroscience, 11:2703 -2712. Lane, R.F., Blaha, C.D. and Han, S.P. (1987). Electrochemistry in vivo: monitoring dopamine release in the brain of the conscious, freely moving rat. Brain Research Bulletin, li:19-27. Louilot, A., LeMoal, M. and Simon, H. (1986). Differential reactivity of dopaminergic neurons in the nucleus accumbens in response to different behavioral situations. An in vivo voltammetric study in free moving rats. Brain Research, i:395-400.  115  Lyness, W.H., Friedle, N.M. and Moore, K.E. (1979). Destruction of dopaminergic nerve terminals in nucleus accumbens: effect on d amphetamine self-administration. Pharmacology. Biochemistry and Behavior, 11:553-556. Martin-Iversen, M.T., Szostak, C. and Fibiger, H.C. (1986). 6hydroxydopamine lesions of the medial prefrontal cortex fail to influence intravenous self-administration of cocaine. Psychopharmacology, :3 10-314. Mogenson, G.J., Jones, D.L. and Yim, C.Y. (1980). From motivation to action: functional interface between the limbic system and motor system. Progress in Neurobiology, jA69-97. Mogenson, G.J. and Phillips, A.G. (1976). Motivation: a psychological construct in search of a physiological substrate. In: J.M. Sprague and A.N. Epstein (Eds.). Progress in Psychobiology and Physiological Psychology. Academic Press, New York. pp. 189-243. Mogenson, G.J. and Yang, C.R. (1991). The contribution of the basal forebrain to limbic-motor integration and the mediation of motivation to action. In: The Basal Forebrain: Anatomy to Function. T.C. Napier and P.W. Kalivas (Eds.). Plenum Press, New York. pp. 267-290. Moghaddam, B. and Bunney, B.S. (1989). Ionic composition of microdialysis perfusing solution alters pharmacological responsiveness and basal outflow of striatal dopamine. Journal of Neurochemistry, : 652-654. Moore, K., Chieuh, CC. and Zeldes, G. et al. (1977). Release of neurotransmitters from the brain in vivo by amphetamine, methyiphenidate and cocaine. In: Cocaine and Other Stimulants. (E.H. Ellinwood and M.M. Kilbey (Eds.). Plenum Press, New York. pp. 143160. Nicolaysen, L.C., Pan, H-T., Justice, J.B. (1988). Extracellular cocaine and dopamine concentrations are linearly related in rat striatum. Brain Research.456: 3 17-323. North, A. (1992). Cellular actions of opiates and cocaine. Annals of the New York Academy of Sciences, :1-6.  116  Paulson, P.E., Camp, D.M. and Robinson, T.E. (1991). Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentration during amphetamine withdrawal in rats. Psychopharmacology, j.Q:480-492. Paxinos, G. and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates. Academic Press, Australia. Pettit, H.O. and Justice, J.B. (1989). Dopamine in the nucleus accumbens during cocaine self-administration as studied by in vivo microdialysis. Pharmacology. Biochemistry and Behavior, :899-9O4. Pettit, H.O and Justice, J.B. (1991). Effect of dose on cocaine selfadministration behavior and dopamine levels in the nucleus accumbens. Brain research, i9:94-1O2. Pickens, R.W. and Johanson, C.E. (1992). Craving: consensus of status and agenda for future research. Drug and Alcohol Dependence, jQ.127131. Pickens, R. and Thomson, T. (1968). Cocaine-reinforced behavior in rats: effects of reinforcement magnitude and fixed-ratio size. The Journal of Pharmacology and Experimental Therapeutics, 161: 122-129. Rescorla, R.A. (1988). Behavioral studies of Pavlovian Conditioning. Annual Review of Neuro science, fl:3 29-353. Risner, M.E. and Jones, B.E. (1976). Role of noradrenergic and dopaminergic processes in amphetamine self-administration. Pharmacology. Biochemistry and Behavior, :477-482. Ritz, M.C., Lamb, R.J., Goldberg, S.R. and Kuhar, M.J. (1987). Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science, i:1219-1223. Roberts, D.C.S., Corcoran, M.E. and Fibiger, H.C. (1977). On the role of ascending catecholaminergic systems in intravenous self-administration of cocaine. Pharmacology. Biochemistry and Behavior, : 615-620. Roberts, D.C.S. and Koob, G.F. (1982). Disruption of cocaine self administration following 6-hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacology. Biochemistry and Behavior, 11:901-904.  117  Roberts, D.C.S., Koob, G.F., Kionoff, P. and Fibiger, H.C. (1980). Extinction and recovery of cocaine self-administration following 6hydroxydopamine lesions of the nucleus accumbens. Pharmacology. Biochemistry and Behavior, U:781-787. Roberts, D.C.S., Loh, E.A. and Vickers, G. (1989). Self-administration of cocaine on a progressive ratio schedule in rats: dose-response relationship and effect of haloperidol pretreatment. Psychopharmacology, 91:535-538. Roberts, D.C.S. and Vickers, G. (1984). Atypical neuroleptics increase self-administration of cocaine: an evaluation of a behavioural screen for antipsychotic activity. Psychopharmacology, : 135-139. Robinson, T.E. and Berridge, K.C. (1993). The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Research Reviews, 18:247-291. Segal, D.S. and Kuczenski, R. (1992). Repeated cocaine administration induces behavioral sensitization and corresponding decreased extracellular dopamine responses in caudate and accumbens. Brain Research, ii:351-355. Segal, D.S. and Kuczenski, R. (1992b). In vivo microdialysis reveals a diminished amphetamine-induced dopamine response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment. BrainResearch ij.: 330-337. Stamford, J.A. (1986). In vivo voltammetry: some methodological considerations. Journal of Neuro science Methods, j7: 1-29. van Ree, J.M., Slangen, J.L. and deWied, D. (1978). Intravenous selfadministration of drugs in rats. The Journal of Pharmacology and Experimental Therapeutics, 4: 547-557. Wallace, M. and Singer, G. (1976). Schedule induced behavior: A review of its generality, determinants and pharmacological data. Pharmacology. Biochemistry and Behavior. :483-490. Westerink, B.H.C. (1979). Sequence and significance of dopamine metabolism in the rat brain. Neurochemistry International, 2:221-227.  118  Weiss, F., Hurd, Y.L., Ungerstedt, U., Markou, A., Plotsky, P.M. and Koob, G.F. (1992). Neurochemical correlates of cocaine and ethanol self-administration. Annals of the New York Academy of Sciences,:22O-241. White, N.M. and Mimer, P.M. (1992). The psychobiology of reinforcers. Annual Review of Psychology, 4.1:443-471. Willner, P., Ahlenius, S., Muscat, R. and Scheel-Kruger, J. (1991). The mesolimbic dopamine system. In: The Mesolimbic Dopamine System: From Motivation to Action, P. Willner and J. Scheel-Kruger (Eds.). pgs. 3-15. Wilson, M.C., Hitomi, M. and Schuster, C.R. (1971). Psychomotor stimulant self-administration as a function of dosage per injection in the 1-281. rhesus monkey. Psychopharmacologia, Wise, R.A. (1978). Catecholamine theories of reward: a critical review. Brain Research, 152:215-247. Wise, R.A. (1987). The role of reward pathways in the development of drug dependence. Pharmacological Therapeutics, :227-263. Wise, R.A. and Hoffman, D.C. (1992). Localization of drug reward mechanisms by intracranial injections. Synapse, jQ.:247-263. Wise, R.A., Spindler, J., deWit, H. and Gerber, G.J. (1978). Neuroleptic induced ‘anhedonia’ in rats: pimozide blocks reward quality of food. Science, .Q.j:262-264. Yokel, R.A. and Pickens, R. (1973). Self-administration of optical isomers of amphetamine and methamphetamine by rats. Journal of Pharmacology and Experimental Therapeutics, ji7.:27-33. Yokel, R.A. and Pickens, R. (1974). Drug level of d- and 1-amphetamine during intravenous self-administration. Psychopharmacologia, 14.:255264. Yokel, R.A. and Wise, R.A. (1975). Increased lever pressing for amphetamine after pimozide in rats: implications for a dopamine theory of reward. Science, jji:547-549. Yokel, R.A. and Wise, R.A. (1976). Attenuation of intravenous amphetamine reinforcement by central dopamine blockade in rats. Psychopharmacology, 41:311-318.  

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