STUDY OF THE ROLE OF THE BED NUCLEUS OF THE STRIA TERMINALIS D1- AND D2-LIKE DOPAMINE RECEPTORS IN COCAINE SELF- ADMINISTERING RATS

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1 STUDY OF THE ROLE OF THE BED NUCLEUS OF THE STRIA TERMINALIS D1- AND D2-LIKE DOPAMINE RECEPTORS IN COCAINE SELF- ADMINISTERING RATS by Chia-Jung Cindy Chiang A thesis submitted to the Centre for Neuroscience Studies In conformity with the requirements for the degree of Master of Science Queen s University Kingston, Ontario, Canada (August, 2010) Copyright Chia-Jung Cindy Chiang, 2010

2 Abstract Previous studies have suggested the importance of the bed nucleus of the stria terminalis (BST) in modulating drug seeking behaviors. With heavy reciprocal projections from the BST to various dopamine rich areas (ventral tegmental area (VTA), nucleus accumbens shell (NAc shell), retrorubral nucleus (RR), periaqueductal gray region (PAG)), BST dopamine receptors are indeed an important part of the highly integrated reward system and thus possess sensitivity to pharmacological rewards. In the present study, the effects of bilateral intracranial injections of the D1- dopamine receptor antagonist SCH (1.6, 3.2, and 6.4 µg total bilateral dose), the D2- dopamine receptor antagonist Sulpiride (3.2 µg total bilateral dose), and D2- dopamine receptor agonist Quinpirole (3.0µg total bilateral dose), administered into the BST immediately prior to self-administration sessions, were examined in both sucrose (180-minute sessions) and cocaine (270-minute sessions) self-administering rats. Injections of SCH (3.2 & 6.4 µg) to the cocaine group significantly reduced the final ratio of operant lever responding (0.75 mg/kg/i.v. injection; progressive ratio schedule of reinforcement (PR); timeout 20 sec); there was no effect of SCH in the sucrose group. Injections of Sulpiride (3.2µg) to the cocaine group produced a significant attenuation in the final ratio of operant lever pressing, with no effect in the sucrose group. Injections of Quinpirole, however, resulted in no alternations in both the cocaine and sucrose selfadministration group. These results suggest that both D1 and D2 dopamine receptor activation is occurring in the reinforcement of cocaine, with the possibility that D2 receptors are not as highly activated as the D1 receptors. This conclusion further ii

3 implicates the complexity of the neurobiological factors involved in drug addiction and the essential role of the BST in dopamine-facilitated reward reinforcement. iii

4 Acknowledgements I would like to thank my supervisor, Éric C. Dumont, for providing me with an opportunity to acquire my MSc degree in such a fun, loving lab. He has helped to keep me in check at times to ensure that I stay motivated and enlightened to finish my thesis on time. Most importantly, I would like to thank the people from the Dumont lab. Because of them, the atmosphere in the lab has been a lot of fun. I particularly would like to acknowledge Dasha Ianovskaia and Xenos Mason for providing me with useful suggestions for experiments and my thesis. I also would like to thank Michal Krawczyk for keeping things going smoothly in the lab. Last but not least, I would like to thank my friends and family for providing me with great support. Mom and Dad have always been there to encourage me. My friends, Winni, Asin, Bing, and Eunice: thank you all. iv

5 Table of Contents Abstract... ii Acknowledgments... iv Table of Contents... v List of Abbreviations... vii List of Figures... ix List of Tables... xi Chapter 1: Introduction What is drug addiction Definition Animal model of drug addiction Neural circuits of reward and motivation Dopamine in motivated behaviours Mechanisms of psychostimulants effects The BST: an important component of the reward and motivation pathway and a target for drugs of abuse Definition and anatomy BST dopamine and motivated behaviour Research aims and hypothesis Research aims Hypothesis Chapter 2: Materials and Methods Animals Drugs Apparatus Cannulation surgery Intra-BST drug micro-injection Behavioural procedure: operant conditioning-reinforcement paradigm Data analysis v

6 2.8 - Histology / brain cannulation placements Chapter 3: Results Effect of SCH on self-administration Effect of Sulpiride on self-administration Effect of Quinpirole on self-administration Histology / Brain cannulation placements Chapter 4: Discussion Summary of current results Selective D1 antagonist (SCH-23390) has no effect on sucrose self-administration, but decreases cocaine self-administration D2-like antagonist (Sulpiride) has no effect on sucrose self-administration, but decreases cocaine self-administration D2-like agonist (Quinpirole) has no effect on sucrose- and cocaine selfadministration Implications of current finding Conclusion References Appendix I: Behavioural traces showing drug pretreatment effects Appendix II: Summary table for breakpoint, final ratio, and drug effects vi

7 List of Abbreviations BP: Breaking Point BST: Bed Nucleus of the StriaTerminalis camp: Cyclic Adenosine Monophosphate CeA: Central Nucleus of the Amygdala CPP: Conditioned Place Preference DR: Dorsal Raphe Nucleus FR-1: Fixed Ratio-1 Schedule of Reinforcement GA: Gauge HCl: Hydrochloride IP: Intra-Peritoneal NAc: Nucleus Accumbens PAG: Periaqueductal Gray Substance Region PFA: Paraformaldehyde PFC: Prefrontal Cortex PR: Progressive Ratio Schedule of Reinforcement Quinpirole: (D2-like DA receptor agonist) a.k.a. (-)-Quinpirole hydrochloride; Full name: (-)-Quinpirole monohydrochloride, LY-171, 555, trans-(-)-(4ar)-4, 4a, 5, 6, 7, 8, 8a, 9-Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride; Linear formula: C 13 H 21 N 3 HCl; Molecular weight: RR: Retrorubral Nucleus SCH-23390: (selective D1-like DA receptor antagonist) a.k.a. R (+)-SCH hydrochloride; Full name: R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- vii

8 tetrahydro-1h-3-benzazepine hydrochloride; Linear formula: C 17 H 18 CINO HCl; Molecular weight: SNc: Substantia Nigra Pars Compacta Sulpiride: (D2-like DA receptor antagonist) a.k.a. (S)-(-)-Sulpiride; Full name: Levosulpiride, (S)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2- methoxybenzamide; Linear formula: C 15 H 23 N 3 O 4 S; Molecular weight: VTA: Ventral Tegmental Area viii

9 List of Figures Figure 1 Cocaine dose response curve... 8 Figure 2 Dose dependent effect of cocaine s reinforcing efficacy... 9 Figure 3 Extended amygdala and the drugs of abuse Figure 4 Schematic of dopamine projection from the ventral tegmental area (VTA) in reward pathways Figure 5 Primary dopaminergic inputs into the extended amygdala Figure 6 Reward circuitry involving the dopamine in the bed nucleus of the stria terminalis (BST) Figure 7 Fixed ratio-1 (FR-1) operant response for sucrose self-administering rats. 36 Figure 8 Fixed ratio-1 (FR-1) operant response for cocaine self-administering rats.. 36 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Operant response for sucrose self-administration under progressive ratio (PR) schedule of reinforcement Operant response for cocaine self-administration under progressive ratio (PR) schedule of reinforcement Bar graph showing the effects of vehicle and D1 selective dopamine receptor antagonist, SCH-23390, on final ratio in sucrose and cocaine selfadministering rats Effects of vehicle and selective D1 dopamine receptor antagonist on sucrose self-administration Effects of vehicle and selective D1 dopamine receptor antagonist on cocaine self-administration Bar graph showing the effects of vehicle and D2-like dopamine receptor antagonist, Sulpiride, on final ratio in sucrose and cocaine self-administering rats Effects of vehicle and D2-like dopamine receptor antagonist on sucrose selfadministration Effects of vehicle and D2-like dopamine receptor antagonist on cocaine selfadministration Bar graph showing the effects of vehicle and D2-like dopamine receptor agonist, Quinpirole, on final ratio in sucrose and cocaine self-administering rats Effects of vehicle and D2-like dopamine receptor agonist on sucrose selfadministration Effects of vehicle and D2-like dopamine receptor agonist on cocaine selfadministration ix

10 Figure 20 Figure 21 Figure 22 Figure 23 Brain maps showing intra-cranial cannulation placements in sucrose selfadministering rats Brain maps showing intra-cranial cannulation placements in cocaine selfadministering rats Sucrose brain placement depicted through fluorescent beads at BST injection site in actual rat brain under fluorescent microscopy Cocaine brain placements depicted through fluorescent beads at BST injection site in actual rat brain under transmitted microscopy and fluorescent microscopy x

11 List of Tables Table 1 Progressive ratio schedule of reinforcement table... 6 Table 2 Distribution of D1 and D2 receptors in the rat central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BST) Table 3 Pseudorandom design of intra-bst drug injections Table 4 Distribution of total intra-bst injections in sucrose- and cocaine- selfadministering rats Table 5 Outline of intra-bst injections received by each rat xi

12 Chapter 1: Introduction The bed nucleus of the stria terminalis (BST) is now considered an important component of the reward and motivation circuit in the brain and a target for drugs of abuse (Eiler et al., 2003; Walker et al., 2000; Epping-Jordan et al., 1998; Hasue and Shammah-Lagnado, 2002). The BST contains several key components of the dopaminergic system, including tyrosine hydroxylase positive terminals, dopamine and cyclic adenosine monophosphate (camp) responsive Phosphoprotein of 32kDa (DARPP- 32), and dopamine receptors (Gustafson, 1990; Hasue and Shammah-Lagnado, 2002). Given the key role of dopamine in motivated behaviours, we sought to determine whether pharmacological manipulations of BST dopamine receptors would influence the expression of naturally- or pharmacologically-driven operant behaviours in rats. Specifically, the objective of the present study was to determine the role of BST D1- and D2-like receptors in the expression of operant responding driven by either a natural (sucrose) or a pharmacological (cocaine) reward combining the rat model of selfadministration with intra-cranial pharmacological manipulations. The general objective of the study was to contribute to a better understanding of the neurobiological basis of drug addiction, a necessary step towards better therapies for this neurological disease. 1.1 What is Drug Addiction? Definition Addiction is a widespread pathological condition characterized by drug-seeking and taking, which is manifested as complex neural adaptations of learning the association between the rewarding stimuli and other motivational/emotional cues. The Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association 2000) 1

13 reports that the increase in energy and time drug users spend seeking access to drugs is a salient feature of addictive behaviour Animal Model of Drug Addiction The use of drug self-administration in animal models has helped understanding of certain elements of the process to drug addiction. Animal models of drug selfadministration have construct and predictive validity when it comes to its concordance with the human form of addiction. Construct validity refers to the ability of the animal model to provide explanatory power and to assess how various variables influence the development of addiction. Predictive validity refers to the model s ability to produce valid predictions about the human form of addiction based on results from the animal model (Koob and Le Moal, 2006). Another reason for using the animal model of drug self-administration is that both animals and humans self-administer the same classes of drugs. These drugs usually have very potent positive reinforcing properties such that animals expend a tremendous amount of energy to perform many different tasks and procedures to obtain them (i.e. pressing a lever multiple times to receive an intravenous injection of drug, associating environmental cues with the reinforcing property of drugs in conditioned place preference approaches) (Koob and Le Moal, 2006; Koob et al., 1998). In summary, the animal model of self-administration is attractive because it provides answers for a number of research questions: the abuse liability of reinforcing compounds and their dose-response functions, patterns of drug intake, the effect of response contingencies on drug intake, and the neural substrates of reinforcement (Arnold and Roberts, 1997). 2

14 Within the intravenous drug self-administration approach, several different schedules of reinforcement exist. The self-administration approach is based on an operant paradigm in which the reward is given and obtained in a response-contingent manner. Thus, the animal earns the drug reward by meeting predetermined response requirements (Skinner, 1938). Examples of schedules include the second-order schedule, progressive-ratio (PR), and fixed ratio (FR) schedules of reinforcement. Second-order schedule is where the completion of two successive schedules is required before a reward is delivered. Therefore, the animal s completion of the first schedule is followed by the onset of the cue light in the chamber (i.e. FR10, where every 10 lever responses is reinforced with the onset of light), which is contingent on the completion of the second schedule (i.e. FR20, where every 20 light onsets is followed by the delivery of a single reward). Thus, the animal gets the reward only after making 200 lever responses (Katz and Goldberg, 1987). The PR schedule of reinforcement requires the animal to achieve increasing number of lever responses for each subsequent reinforcer (Richardson and Roberts, 1996). The FR schedule is where a reward is delivered after every nth response (i.e. FR-1=every lever response is reinforced with a reward) (Richardson and Roberts, 1996). Using different schedules of reinforcement can provide important control procedures for nonspecific motor and motivational actions such as increases in exploratory activity and locomotion (Rowlett et al., 1996). Increasing the lever press value requirement for obtaining a reinforcer, such as in a PR schedule, ensures that the increase in behaviour is actually due to the animal maintaining the previous rate of drug infusion to maintain the high from the drug (Richardson and Roberts, 1996; Rowlett et al., 1996). Another approach is to introduce a second, inactive lever into the 3

15 experimental chamber so the selective increase in responding on the active lever can be measured (Koob and Le Moal, 2006). In the current study, animals were trained and tested under the FR-1 and PR schedules of reinforcements. FR-1 schedule of reinforcement is where each lever response is reinforced with a drug reward. Under this schedule, the rate of drug selfadministration is often the dependent measure for the drug s reinforcing efficacy (Richardson and Roberts, 1996; Arnold and Roberts, 1997). However, the rate of drug self-administration in the FR-1 schedule is insensitive to changes in reinforcement efficacy, especially with pharmacological manipulations of the reinforcing efficacy of cocaine (Arnold and Roberts, 1997). Therefore, for the purpose of our experiment, the FR-1 schedule was useful for training animals to associate lever pressing with drug delivery. Once the rats attained stable pressing behaviour under the FR-1 schedule, they graduate onto PR schedule (criteria outlined in materials and methods section of thesis). The PR schedule is useful for evaluating the reinforcing efficacy of a self-administered drug (Richardson and Roberts, 1996; McGregor and Roberts, 1993; Roberts et al., 1989). In the PR schedule, the response requirements for each successive reinforcer increases systematically until the animal stops responding within a certain time window. This point, defined as the breaking point (BP), is an indication of the maximum effort that an animal is willing to expend to receive a certain number of drug infusion at a specific concentration. Although the BP is defined as the point when the animal fails to attain a drug infusion within a 60-minute period in most cases, this arbitrary rule is not applicable in every case (Richardson and Roberts, 1996). Due to individual differences in pressing patterns for each rat and differing levels of potency in neuropharmacological 4

16 manipulations, the criteria for defining BP vary substantially across studies. In some cases, the BP criteria is set as the fixed amount of time for each subject to complete each successive ratio, with variation from 12 minutes to 48 hours (Yanagita, 1973). (i.e. if the fixed amount of time was set at 12 minutes, and the subject takes longer than 12 minutes to achieve the response requirement for the second reward, then the second reward is defined as the BP). In our study, however, we defined our BP as the point when the full expression of operant behaviour has occurred during the experimental session (i.e. in Sucrose self-administration group, the sessional length is set as 180 minutes; in Cocaine self-administration group, the sessional length is set as 270 minutes). Thus, the BP for sucrose- and cocaine- self-administering rats is the last reward received during their sessional length time. Therefore, the BP would be defined as the final ratio completed for the whole session length. The response ratio is the number of total lever presses required for each reward, whereas the final ratio is the number of additional lever presses between each successive reward (i.e. if the response ratio for the first reward is 1, and the response ratio for the second reward is 3, then the final ratio between the first and the second reward is 2. See table 1 for a table of final ratio and response ratio (total pressing)). The PR schedule used in our study employed the PR series derived from the equation: Response ratio = 5[ee (iiiiiiiiiiiiiiiiii nnnnnnnnnnnn 0.2) ] 5. Since the final ratio is exponential in nature, a ln transformation was done on the final ratio so this value conforms to the assumptions of parametric statistics (i.e. resulting in homogeneous variance). Lastly, the most attractive reason for using PR schedule is that stable lever pressing behaviour requires only 3 to 10 days to acquire. Once attained, stable behaviour can be maintained for long periods of time, usually over 50 sessions (Richardson and 5

17 Roberts, 1996). The use of PR schedule of drug self-administration in animals provide usable data because: the use of PR schedule is a valid and flexible method for measuring drug reinforcement in humans, and stimulant drugs with abuse potential maintain responding in humans under this schedule (Stoops, 2008). Table 1. The progressive ratio schedule of reinforcement. Infusion is the breakpoint (BP) determined from raw behavioural traces with lever presses. BP is the last infusion before session ends. Each infusion value corresponds to a final ratio value, which indicates the additional number of lever presses between each successive infusion. Total pressing is the cumulative number of lever presses. To use this table, match the number of infusions (BP) to its final ratio value and calculate the effect of the drug (expressed as change in final ratio in percentage) by dividing the final ratio of the injection day by the average final ratio of the 3 pre-injection days. Infusion Final Ratio Total Pressing

18 For the current study, we chose to use the self-administration approach. This is because the self-administration paradigm, combined with the use of the PR schedule of reinforcement, more closely resembles the progressive and deteriorating human condition of the drug addiction disorder. Human addicts often self-administer drugs intravenously, and the degree of addiction slowly progresses with escalating dosage and time, up to a certain point. More specifically, they self-administer cocaine to the extent of funds available and within the limits of tolerable side effects (i.e. nervousness, dysphoria, and depression) (Koob and Kreek, 2007; Liu et al., 2005). In addition, under the PR schedule, it has been demonstrated that dose (Roberts et al., 1989, 2007), injection speed (Liu et al., 2005), drug deprivation (Morgan et al., 2002), and circadian rhythm can all interact to influence BP. Therefore, the animal model of self-administration can be used to model the human condition very closely with some modifications in these factors. The relationship between the stimulant effects of cocaine to dose can be best depicted on an inverted U-shaped dose-response function (Figure 1) (Richardson and Roberts, 1996). On the curve, the 1.5 mg/kg/injection cocaine dose at the tip of the U-shaped curve suggests that this is the dose with the highest acute reinforcing efficacy. On the other hand, the 3.0 mg/kg/injection dose is over the top, where it is starting to be on the descending limb of the curve. Therefore, rats self-administering cocaine at this dose may not find cocaine to be reinforcing. This is evident in an experiment showing that the increases in cocaine s reinforcing effects are dose-dependent. Breakpoints (BP) maintained by 0.75 and 1.5 mg/kg/injection increase progressively over days, whereas 0.38 or 3.0 mg/kg/injection shows not much change (Figure 2) (Liu et al., 2005; Roberts et al., 2007). Consequently, the rats in the current study self-administered a cocaine dose 7

19 of 0.75 mg/kg/injection. This concentration is ideal because it is still on the ascending limb of the curve, located approximately at about 25% away from the top dose (1.5 mg/kg/injection) (see Figure 1). With this dose, rats will still find cocaine relatively reinforcing and their pressing behaviour can still increase or decrease with pharmacological manipulations. Figure 1. Cocaine dose-response curve showing the relationship between dose of cocaine given (mg/kg/injection) and the final ratio completed on a progressive ratio (PR) schedule. Graph depicts an inverted U-shape, showing the maximal tolerable dose of cocaine that can be given to a rat before the appearance of aversive effects at 1.5mg/kg/injection. To facilitate the observation of the effects of intra-bed nucleus of stria terminalis (BST) drugs given on cocaine self-administration behaviour, the dosage used in the current experiment was set as 0.75mg/kg/injection. (Adapted from Richardson and Roberts, 1996) 8

20 Figure 2: Depiction of the dose-dependent effect of cocaine s reinforcing efficacy. Breakpoints maintained by 0.75 and 1.5mg/kg/injection increased across days, showing that cocaine is reinforcing at these dosages (still on the ascending limb of the cocaine dose-response curve). Cocaine dosages that are either too low (0.38mg/kg/injection) or too high (3.0mg/kg/injection) are not able to induce reinforcing feelings of cocaine self-administration (so it shows stable response over days). (Adapted from Roberts et al., 2007) Neural Circuits of Reward and Motivation The reward circuitry involved in obtaining reinforcing stimuli (i.e. sex, cocaine, food, water) requires the dopamine mesocorticolimbic system (Caine and Koob, 1994; Di Chiara and Imperato, 1988; Thomas et al., 2008). This system comprises primarily of the ventral tegmental area (VTA), the nucleus accumbens (NAc), the prefrontal cortex (PFC), and associated limbic structures (i.e. hippocampus, amygdala) (Kauer and Malenka, 2007). The major site of dopamine neurons in the midbrain region is in the VTA. The VTA is involved in the processing of rewarding and aversive properties of environmental 9

21 stimuli. It provides information for the reward value of an action or stimulus to modify future behaviour and addictive behaviours (Fields et al., 2007; Thomas et al., 2008; Schultz, 1997; Wise, 1996). The NAc is the principal target of VTA dopamine neurons and it mediates the rewarding properties of survival related natural stimuli (i.e. food and sex) and drugs of abuse (Kelley and Berridge, 2002; Di Chiara and Imperato, 1988; Wise and Rompre, 1989). Furthermore, the NAc contains two subregions with distinctive functions, namely the core and the shell of the NAc. The NAc core is important for instrumental learning and cue-induced reinstatement of drug-seeking behaviour (relapse), and the NAc shell is involved with mediating the primary reinforcing effects of addictive drugs (Beurrier, et al., 2001; Pennartz et al., 1994). The rewarding effect of drugs of abuse is heavily dependent on the dopamine signaling in the NAc. Cocaine can be selfadministered into the rat NAc (McKinzie et al., 1999; Rodd-Henricks et al., 2002), and reinstatement can be induced with injection of amphetamine into the NAc (Stewart and Vezina, 1988). The PFC correlates the overall motivational significance of the stimuli to determine the intensity of behavioral responding (Goldstein and Volkow, 2002). Thus, the cognitive processes required for goal-directed behaviours depend on the PFC function (Goldstein and Volkow, 2002; Hyman, 2005). The amygdala, on the other hand, is associated with fear conditioning and the processing of negative emotions. However, since this structure is also involved in the processing of positive emotions and the learning about the positive value of stimuli, the amygdala may act within the mesocorticolimbic dopamine system to integrate the positive and negative value of an environmental stimulus (i.e. drug of abuse, natural food reward, sex, stress). 10

22 The dopaminergic connection between the mesolimbic dopamine system and the extended amygdala is hypothesized to be responsible for the reinforcing effects of abused drugs (Ettenberg et al., 1982; Koob 1999, 2003). The extended amygdala is made up of several basal forebrain regions that share similar morphology, immunoreactivity, and connectivity (Sun et al., 1991; Freedman and Cassel, 1994). The regions include the BST, the central nucleus of the amygdala (CeA), and the shell of the NAc (Figure 3) (Koob, 1999). The extended amygdala receives several afferents from limbic structures (i.e. basolateral amygdala and hippocampus) and sends efferents to the VTA, and lateral hypothalamus. Thus, the unique positioning and connectivity that the extended amygdala has with other brain regions enables it to serve as an interface for emotional and motor system (Koob, 2003). Consequentially, the extended amygdala works in close association with the mesolimbic dopamine system for the generation of goal directed motivational behaviour for rewards (see Figure 4). Figure3: The involvement of each component of the extended amygdala in drugs of abuse. Specifically, the bed nucleus of the stria terminalis (BST), the shell of nucleus accumbens (NAc), and the central extended amygdala (CeA) are all heavily associated with the reinforcing properties of cocaine. They mediate these changes by regulating the level of dopamine neurotransmission associated with cocaine addiction. (Adapted from Koob, 1999) 11

23 Figure 4: Schematic showing the dopaminergic projections from the ventral tegmental area (VTA) to various areas of the brain implicated in coordinating the reward and motivation neural circuit (i.e. basolateral amygdala, prefrontal cortex (PFC), nucleus accumbens (NAc), and the extended amygdala). The BST, a key part of the extended amygdala, projects to several areas of the brain (i.e. somatomotor, central autonomic control, neuroendocrine) and thus is strategically placed in the brain to process afferent inputs and efferent outputs of emotions, motivation and drugs of abuse (Koob, 1999; Dong et al., 2000, 2001; Dong and Swanson, 2003, 2004). The BST is highly interconnected with elements of the reward circuitry, the mesolimbic dopamine system. The dorsal BST projects to the NAc shell and VTA (Dong et al., 2001), therefore the BST also regulates the firing of dopaminergic cells within the VTA (Georges and Aston-Jones, 2002). In addition, the BST also receives strong 12

24 dopaminergic inputs from the VTA, substantia nigra pars compacta, retrorubral area (RR), and periaqueductal gray (PAG) through the medial forebrain bundle (Deutch et al., 1988; Phelix et al., 1992; Freedman and Cassell, 1994; Hasue and Shammah-Lagnado, 2002; Meloni et al., 2006) (See Figure 5). In accordance to the fact that the BST is heavily innervated by dopamine neurons, drugs of abuse such as opiates, ethanol, amphetamine, nicotine, and cocaine have been shown to increase dialysate dopamine levels in the BST, at an even higher level than in the NAc shell (Di Chiara and Imperato, 1988; Di Chiara et al., 1999; Carboni et al., 2000). The dopamine receptors in the BST exist in a 3:2 ratio of D1: D2 receptors (Scibilia et al., 1992; Mengod et al., 1992) (See Table 2), showing that there is a higher concentration of D1 receptors in the BST (See Figure 6). Thus, the projections from and to the BST implicate the importance of this structure in modulating the physiological and pathological reward-related behaviours such as cocaine and food self-administration. (Aston-Jones and Harris, 2004; Dumont et al., 2005). 13

25 RR VTA SNc PAG DR Figure 5: Bar graph showing the relative dopaminergic input into the extended amygdala. (A8 = retrorubral nucleus (RR); A9 = substantia nigra pars compacta (SNc); A10 = ventral tegmental area (VTA); periaqueductal gray substance region (PAG); dorsal raphe nucleus (DR)). (Adapted from Hasue and Shammah-Lagnado, 2002) 14

26 Table 2. The distribution of D1 and D2 dopamine receptors in subregions of the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BST). The BST contains relatively higher densities of dopamine receptors than the CeA. There are more D1 receptors than the D2 receptors in the BST. (Adapted from Scibilia et al., 1992) Figure 6: Schematic showing the connection between the extended amygdala circuit and the mesolimbic dopamine system in generating goaldirected motivational behaviours for attaining rewards. Specifically, the bed nucleus of the stria terminalis (BNST) is innervated by dense dopaminergic projections from several areas of the brain (i.e. ventral tegmental area (VTA), retrorubral nucleus (RR), substantia nigra pars compacta (SN), nucleus accumbens (NAcc), and the basolateral amygdala). (Adapted from Koob and Le Moal, 2006) 15

27 1.1.4 Dopamine in Motivated Behaviours Dopamine, as a catecholamine neuromodulator in the mammalian brain, controls a variety of functions including positive reinforcement of drugs, food intake, locomotor activity and endocrine regulation (Pierce and Kumaresan, 2006). Dopamine receptors are classically divided into five G protein-coupled receptor subtypes, namely the D1-like and the D2-like receptor subtypes. The D1-like receptors consist of the D1 and the D5 dopamine receptors, and they generally couple to the G s protein to activate adenylyl cyclase. The D2-like receptors consist of the D2, D3, and D4 dopamine receptors, and they couple to the G i protein to inhibit adenylyl cyclase (Missale et al., 1998). With chronic administration of opiates, cocaine and alcohol, there is an up-regulation of the camp pathway in the NAc (Nestler and Aghajanian, 1997; Terwilliger et al., 1991; Ortiz et al., 1995). Increased dopamine transmission in the NAc is critical for the maintenance of the psychostimulant self-administration behaviour (Pierce and Kumaresan, 2006; Di Chiara and Imperato, 1988). On the other hand, dopamine-depleting lesions in the NAc decrease self-administration of cocaine (Caine and Koob, 1994; Gerrits and Van Ree, 1996; Di Chiara and Imperato, 1988). Drugs not abused by humans (i.e. atropine, imipramine) do not modify synaptic dopamine concentrations (Di Chiara and Imperato, 1988). In addition, during cocaine self-administration sessions, NAc dopamine levels decline between cocaine infusions. This decrease may trigger responding for cocaine self-administration in order to maintain extracellular dopamine above a certain threshold level (Ranaldi, et al., 1999; Wise et al., 1995). Furthermore, self-administration studies consistently show that systemically administered D1-like antagonists decrease the 16

28 reinforcing efficacy of cocaine (Bergman et al., 1990; Hubner and Moreton, 1991; Winger, 1994) and D2-like antagonists reduce cocaine reinforcement (Bergman et al., 1990; Caine et al., 2002). More specifically, bilateral intracerebral injections of the D1 receptor antagonist, SCH-23390, into the NAc attenuated the reinforcing effects of the self-administered cocaine under both FR and PR schedule of reinforcement (Caine et al., 1995; McGregor and Roberts, 1993). Thus, both the D1-like and the D2-like dopamine receptors are essential for the reinforcing efficacy of psychostimulants. Dopamine transmission in the brain is also responsible for the ability of food to establish and maintain response habits and conditioned preferences. The auditory, visual, tactile, olfactory and gustatory stimuli of foods activate the dopaminergic system in the brain to energize feeding and reinforce food-seeking behaviour (Wise, 2006). Dopamine neurons do not discriminate amongst various appetitive stimuli (i.e. between different foods, liquids, sensory modalities) (Schultz, 1997; Romo and Schultz, 1990; Schultz and Romo, 1990; Mirenowicz and Schultz, 1996). Pretreatment with dopamine antagonists results in a dose-dependent decrease in how quickly the animals learn to lever-press for food (Wise and Schwartz, 1981). Thus, sucrose operant-responding acts as a control for the effect of dopamine on natural reinforcers in this study. The dopamine inputs to the extended amygdala originate from three cell groups labeled as A8 (retrorubral nucleus (RR)), A9 (substantia nigra pars compacta), and A10 (VTA). The BST and the CeA, the two major parts of the extended amygdala, are also heavily innervated by the dopamine neurons. Thus, the extended amygdala is suggested to be involved in the motor expression of drives and motivation through its projections to 17

29 the brainstem areas and the lateral hypothalamus (Di Chiara, 1995; Bjorklund and Lindvall, 1984; Hasue and Shammah-Lagnado, 2002) Mechanisms of Psychostimulants Effects Psychostimulant drugs, such as cocaine, are drugs of high-abuse potential. They can be ingested via several routes of administration: intranasally, intravenously, and smoked. When administered, cocaine produces stimulant effects such as sustained performance, heightened arousal, alertness, and motor activity ( stereotyped behaviour, characterized by increasing rate of repetitive behaviour), accompanied by feelings of intense euphoria (Koob and Le Moal, 2006). They bind to monoamine transporters located on monoaminergic nerve terminals. Cocaine inhibits all three monoamine transporters: dopamine, serotonin, and norepinephrine; thereby, potentiating the monoaminergic transmission. The relative monoamine transporter affinity of cocaine is serotonin > dopamine > norepinephrine, where the interaction of cocaine with dopamine best predicts the reinforcing potency of cocaine (Pierce and Kumaresan, 2006; Ritz and Kuhar, 1989). The prevention of the reuptake of dopamine by the monoamine transporter inhibitors results in the accumulation of dopamine levels within the synaptic cleft of monoamine synapses. The use of cocaine as a positive reinforcer in our experiment is beneficial because the pharmacological manipulation of cocaine reinforcement can be observed more easily due to its fast kinetics. Cocaine has a short half-life of about 40 minutes and thus it can be excreted much faster (Javid et al., 1983). In addition, cocaine has a more robust reinforcing effect than other drugs. Animals learn to self-administer cocaine more readily than any other drug and they work harder for it. This is shown through an 18

30 experiment done by Yanagita (1975), where the reinforcing potency of cocaine was compared with other drugs, under the PR schedule of reinforcement. In the BST, cocaine dose dependently increases the extracellular dopamine (Carboni et al., 2000), suggesting that the BST is indeed sensitive to the dopamine stimulant actions of cocaine. 1.2 The BST: An Important Component of the Reward and Motivation Pathway and A Target for Drugs of Abuse Definition & Anatomy The BST, as an important part of the reward motivational pathway, lies next to the anterior commissure in the basal forebrain to make up the rostral part of the extended amygdala (Fudge and Haber, 2001; McDonald et al., 1999). It is a complex forebrain structure that is composed of heterogeneous cell groups (Ju and Swanson, 1989). These different subnuclei are contained within the anterior and the posterior divisions of the BST (Ju and Swanson, 1989). The anterior division consists of the anterolateral (consisting of oval, juxtacapsular, rhomboid, and fusiform nuclei) (Larriva-Sahd, 2004), anterodorsal, and anteroventral (consisting of dorsomedial, dorsolateral, magnocellular, and ventral nuclei) cell groups (Ju and Swanson, 1989). The posterior division, however, lies caudal to the primary bundles of the dorsal and commissural bundles of the stria terminalis and contains six recognizable components: the principal nucleus, interfascicular nucleus, transverse nucleus, premedullary nucleus, dorsal nucleus, and cell-sparse zone (Ju and Swanson, 1989). Each of the BST subnuclei, with its heterogeneous cytoarchitectural nature and projections to various parts of the brain, is responsible for coordinating different combinations of vital functions. 19

31 Specifically, with the dopaminergic inputs that the BST receives from the VTA, substantial nigra pars compacta, RR and periaqueductal gray (Hasue and Shammah- Lagnado, 2002), the BST may thus be critical for the reward and motivation behaviours BST Dopamine and Motivated Behaviour Several studies have demonstrated the importance of both the D1- and the D2-like dopamine receptors in modulating the reinforcing efficacy of both the natural food and cocaine, with most of the research done via systemic, intra-nac injections, intra-vta injections, intra-amygdala injections, intra-hippocampus injections, intra-pfc injections, and intra-striatum injections (Weissenborn et al., 1996; Khroyan et al., 2003; Bergman et al., 1990; Kuo, 2002; Bari and Pierce, 2005; Terry and Katz, 1992; Caine et al., 1995; Johnson and Kenny, 2010; McGregor and Roberts, 1993, 1995; Ranaldi and Wise, 2001; Berglind et al., 2006; Andrzejewski et al., 2006). In most of these studies, both the D1 and D2 antagonists decreased the reinforcing value of cocaine (Koob et al., 1987; Egilmez et al., 1995; Caine and Koob, 1994; Hubner and Moreton, 1991). Studies with knock-out mutant mice suggest that D1 receptors are essential (Caine et al., 2007) and D2 receptors might be less involved in cocaine self-administration than the D1 receptors (Caine et al., 2002). However, studies using dopamine agonists gave less conclusive results. Depending on the dose of the cocaine self-administered and the dosage of the agonists given, the agonists can either produce a decrease or an increase in the rewarding effects of the drugs (Weissenborn, et al., 1996; Katz et al., 2006; Caine et al., 1999). Up to now, only a few studies demonstrate the potential significance of BST dopamine in the regulation of rewarding behaviours. As mentioned above, the BST, the CeA, and the NAc shell are homologous in nature and share a role in the acquisition and 20

32 expression of emotions and of appetitive behaviour (de Olmos and Heimer, 1999; Dumont et al., 2005). Due to the dense dopamine innervations in the BST, it is suggested that cocaine dose dependently increases the extracellular dopamine in the BST. This implicates the fact that the BST, similar to the shell of the NAc, is particularly sensitive to dopamine stimulant actions of drugs of abuse (Carboni et al., 2000). In addition, it is suggested that the development of addiction may coincide with alternations of dopaminergic activity following chronic exposure to substances of abuse. D1 and D2 receptor densities within the extended amygdala (i.e. in the NAc core, shell, and amygdala) increase with both continuous alcohol intake and deprivation of alcohol (Sari et al., 2006). Furthermore, the D1 receptors in the BST are particularly essential for modulating alcohol-motivated behaviours. Specifically, SCH dose-dependently reduces both alcohol- and sucrose-motivated responding; however, the D2 antagonist only elicits small decreases in sucrose responding (Eiler II et al., 2003). D1 dopamine receptor in the BST may be important in cocaine self-administration. Specifically, intra- BST injections of SCH significantly increase the rate of cocaine selfadministration under the FR-5 schedule of reinforcement, implicating partial attenuation of the reinforcing effects of cocaine (Epping-Jordan et al., 1998). However, the use of the rate of lever pressing under the FR-5 schedule as the only dependent measure is equivocal. This is because the rate of lever responding is not sensitive to changes in reinforcement efficacy, especially with pharmacological manipulations of the reinforcing efficacy of cocaine (Arnold and Roberts, 1997). In addition, in the Epping-Jordon et al. (1998) study, the use of only cocaine infusions as the sole reinforcer and SCH as the sole pharmacological manipulation agent may be intrinsically flawed. Since cocaine 21

33 is a highly abused drug, the decrease of the reinforcing efficacy of cocaine seen in rats treated with SCH is predictable. Thus, testing the effect of SCH in a different kind of reinforcer (i.e. food, sucrose) will provide a more complete picture of the effect of SCH on BST dopamine receptors. With this in mind, we will use the PR schedule of reinforcement to train and test our rats to self-administer either sucrose pellets or cocaine infusions. With the increase in response requirement for each successive reinforcer, it can be assured that our measure of BP and final ratio is truly a reflection of the reinforcing efficacy of either sucrose or cocaine rewards, not due to the animals random lever pressing behaviour (Richardson and Roberts, 1996; Koob and Le Moal, 2006). In addition, the stable lever pressing response under the PR schedule and the sensitivity of the PR schedule to various parameters (i.e. cocaine dose, dopamine antagonists, injection speed) will enable us to quantify the rats behaviour as a reflection of the effect of our pharmacological manipulations (Roberts et al., 1989, 2007; Liu et al., 2005). Furthermore, as there are also D2 receptors in the BST, we will also test the contribution of the BST D2 receptors to the reinforcing efficacy of both cocaine and sucrose rewards. Therefore, in this study, we will be focusing on elucidating the potential dopamine transmission mechanisms in the BST through the D1 and D2 receptors. This will be demonstrated via rats that self-administer cocaine infusions and sucrose pellets. 1.3 Research Aims and Hypothesis Research Aims Since previous research showed that intra-bst injection of the selective D1- receptor antagonist SCH decreased the reinforcing efficacy of cocaine in self- 22

34 administering rats under the FR schedule of reinforcement (Epping-Jordan et al., 1998; Eiler II et al., 2003), and electrophysiological data (Krawczyk et al., 2010) demonstrated an upregulation of BST D1 receptors and a downregulation of D2 receptors in rats with chronic cocaine treatment, this behavioral study was designed to determine the relative contributions of the BST D1- and D2 receptors to the expression of operant responding driven by either a natural (sucrose) or pharmacological (cocaine) reward (Krawczyk et al., 2010). Given that the BST contains both the D1-like and D2-like dopamine receptors, rats will receive intra-bst microinjections of the selective D1 antagonist SCH , the D2-like antagonist sulpiride, and D2-like agonist quinpirole. All of the selfadministration training and experiments will be performed under the PR schedule of reinforcement Hypothesis Based on the fact that rats with chronic cocaine treatment exhibited an upregulation of BST D1 receptors and a downregulation of BST D2 receptors (Krawczyk et al., 2010), and because the BST is critical in natural and pharmacological reward selfadministration in rats due to its heavy dopaminergic inputs from neighboring structures and its expression of dopamine receptors, we hypothesize that pharmacological manipulation of the BST D1 and D2 receptors influences the reinforcing efficacy of pharmacological (cocaine) rewards under the PR schedule of reinforcement. 23

35 Chapter 2: Materials and Methods Animals Twenty-seven male Long-Evans rats (Charles River Laboratories, St. Constant, Quebec, Canada) weighing between g upon arrival were housed in pairs and were kept on a 12-hour reversed light-dark cycle (09:00 hour lights off 21:00 hour lights on) in a temperature-controlled animal holding room (temperature: ~22 C; humidity: ~34%). They were handled every day for 3 days before cannulation surgeries. After the surgery, rats were moved into the experimental room, and they had 3 days of postoperational recovery period before the start of the experiment. All behavioral testing was conducted during the dark active phase, where water and food were provided ad libitum. Cages and bedding were changed once a week. Treatment and housing of the rats were in accordance to the guidelines of the Canadian Council on Animal Care (CCAC) and all conducted experiments were approved by the Queen s University Animal Care Committee (UACC). Some rats (n = 5) were not included in the data analysis because of complications after cannulation surgeries. The total number of rats used in the final data analysis was Drugs The three drugs used in the present study were: SCH [R(+)-7-Chloro-8- hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1h-3-benzazepine hydrochloride (R (+)- SCH hydrochloride; Sigma-Aldrich, Oakville, Ontario, Canada) in a total bilateral dose of either 1.6, 3.2, or 6.4 µg/µl]; Sulpiride [ Levosulpiride, (S)-5-Aminosulfonyl-N- [(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide ((S)-(-)-Sulpiride; Sigma-Aldrich, Oakville, Ontario, Canada) in a total bilateral dose of 3.2 µg/µl], and Quinpirole [(-)- 24

36 Quinpirole monohydrochloride, LY-171, 555, trans-(-)-(4ar)-4, 4a, 5, 6, 7, 8, 8a, 9- Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride ((-)-Quinpirole hydrochloride; Sigma-Aldrich, Oakville, Ontario, Canada) in a total bilateral dose of 3.0 µg/µl. All of the drugs prepared for intra-bst microinjections were dissolved and diluted in sterile saline. Intra-BST saline microinjections were also given to serve as a vehicle control. All the drugs and saline solutions were stored in 20µL aliquots at -20 C until experiment day. Cocaine-hydrochloride (HCl) (MediscaPharmaceutique, Saint-Laurent, QC) was dissolved in sterile saline (0.9% NaCl isotonic saline) in a concentration of half-strength (0.75 mg/kg/injection) and adjusted to the rats physiological ph of Apparatus Behavioral data were collected using six operant conditioning chambers (MedAssociates, St-Albans, VT) with access to food and water ad libitum. Each chamber was equipped with a Plexiglass front and back panel (W(30cm) x H(30cm) x L(30cm)), a cue light, and an operant retractable lever mounted 5 cm above the floor. With each depression on the operant lever, it would administer either one 45mg sucrose pellet (Test Diet, Richmond, IN) for the sucrose self-administration group, or a 0.12 ml infusion of 2.5mg/mL (equivalent to 0.75 mg/kg/injection) cocaine-hcl for the cocaine selfadministration group. Following each reward, the lever was retracted for 20 seconds (time-out period of 20 sec), followed by the simultaneous activation of the cue light. After the time-out period of 20 seconds, the operant chamber went dark again, with the re-appearance of the operant lever. Intravenous infusions of cocaine were delivered by a Razel infusion pump (MedAssociates) via polyethylene-50 tubing (PE-50) (HRS Plastics 25

37 One) enclosed within a metal spring tether that was reaching towards a swivel to allow free movement of the animal, and the intravenous catheter port linking onto the dorsal region of the rat. Raw behavioral data traces for each experimental session were recorded using the MedPCIV software (MedAssociates) for later analysis Cannulation Surgery Upon arrival, rats were allowed a minimum of 3 days to acclimatize to the new surroundings before the cannulation surgery. Bilateral intracranial guide cannula implantation surgery was performed to allow direct infusion of the drug solution into the oval region of the dorsal BST. In addition, intravenous cannulation surgery was done to allow intravenous infusion of cocaine for the cocaine self-administration group of rats. When rats were ready for surgery, they were weighed and anesthetized with ketamine-xylazine (75mg/kg: 10mg/kg, intra-peritoneal (IP) injection). Stock ketamine hydrochloride injection (100mg/mL) (Bioniche Animal Health, Belleville, ON, Canada). Stock xylazine (20mg/mL) sterile injectable (Bayer HealthCare Animal Health Division, Toronto, ON, Canada) via intraperitoneal injection in the animal holding room and then transported in their home cages into the surgery room. The use of injectable induction agents facilitated stereotaxic placement (TSE Systems) into the ear bars, and decreased peripheral bleeding to allow for the maintenance of surgical site clearance. Sucrose self-administration rats received intracranial surgery. After the rats have been anesthetically induced, 2% xylocane jelly (Mississauga, ON, Canada) was applied to their ears, then their heads were shaved and fixated into the stereotaxic instrument and secured with nose-tooth bar and non-rupture ear bars where they received 1.75% isoflurane gas (from 99% isoflurane inhalation anaesthetic, distributed by Benson, 26

38 Markham, ON, Canada) mixed with oxygen in a vaporizer system (Forane vaporizer system, Ohio Medical Products, Madison, WIS). The oxygen (Praxair, Mississauga, ON, Canada) was delivered inside the chamber at 5.0 L/min for the entire duration of the surgery to maintain anaesthesia. Throughout the whole duration of the surgery, the rat s respiratory rate was closely monitored so the level of isoflurane gas may be adjusted to ensure the rat stayed under constant stable anaesthesia. Tear gel (Novartis Pharmaceuticals, Mississauga, ON, Canada) was applied to the eyes and reapplied during surgery to prevent rat s eyes from drying out. Then, an injection of Anafen (5mg/mL, subcutaneous; Merial Canada, Quebec, Canada) was given, followed by an injection of 10mL of saline (0.9% NaCl, subcutaneous; Baxter Corporation, Mississauga, ON, Canada). Before making the incision for the intracranial surgery, 4% Germi-stat (Germiphene Corporation, Brantford, ON, Canada), 70% isopropyl alcohol (HealthCare Plus Jedmon Products, LTD, Toronto, ON, Canada), and iodine (1% free iodine, Rougier Pharma, Canada) was applied twice in this sequence to sanitize the surgical site and prevent potential infections during surgery. A vertical incision was made from between the eyes to slightly behind the ears. Then, the incision site was cleaned up so Bregma could be identified. Two holes were drilled through which two single guide cannulae (6.5mm in length, Plastics One Inc, HRS Scientific, Roanoke, VA) were bilaterally implanted into the oval region of the dorsal BST (-0.6 A.P. ± 1.9 M.L D.V., measured relative to Bregma). [**Sucrose self-administration rats with Rat IDs that are before L143 were implanted with a shorter set of bilateral guide cannuale: 5.8mm in length, Plastics One Inc, HRS Scientific, Roanoke, VA] Four additional holes were drilled around the first two where anchoring screws (0.080 x 0.125, Plastics One Inc, 27

39 HRS Scientific, Roanoke, VA) were inserted, and dental acrylic cement was filled around the screws and guide cannulae to hold the head attachment securely in place. To protect from pathogens and ensure patency, an autoclaved dummy cannula (30GA stylette, 0.014in-0.35mm, with 0 protrusion) was inserted into the guide cannulae and covered with a sterile dust cap. Once the dental acrylic had hardened, the head incision was sutured up, the anaesthetic gas was turned off and oxygen (1.0 L/min) was given for 5 minutes. An injection of 10mL of saline (0.9% NaCl, subcutaneous; Baxter Corporation, Mississauga, ON, Canada) was given to supplement for fluid loss during surgery. For the whole duration of the surgery, the rats were kept warm while they were under anaesthesia on the stereotaxic apparatus with a heating pad beneath them (medium heat). For analgesia following surgery, Anafen (5mg/mL, subcutaneous; Merial Canada, Quebec, Canada) was given for three days after the surgery, during which they were kept in the experimental room for recovery. After three days of recovery, the rats started their experiments. Cocaine self-administration rats received intravenous cannulation surgery right after the completion of the intracranial cannulation surgery. The jugular cannula implantation surgery started with the transport of the rat from the stereotaxic apparatus to the intravenous surgery station. Rats were kept under anaesthesia by continuing to receive 1.75% isoflurane gas (from 99% isoflurane inhalation anaesthetic, distributed by Benson, Markham, ON, Canada) mixed with oxygen in a vaporizer system (Forane vaporizer system, Ohio Medical Products, Madison, WIS). The oxygen was delivered inside the chamber at 5.0 L/min for the entire duration of the surgery. The right jugular vein was isolated so a silastic cannula (cannula made up of: Polypropylene mesh

40 micron (28mm in diameter), Silicone tubing (Outer diameter: 0.63mm x Inner diameter: 0.30mm x Wall: 0.17mm), silicon bead that is 90mm away from main catheter, stainless steel tubing: (Outer diameter: 0.71mm x Inner diameter: 0.406mm), CamCaths, Ely, UK) could be inserted 32mm into the vein, towards the right atrium. The cannula was connected to a port secured onto the dorsal side of the rat via a hernia mesh sutured to the muscle wall using 3-0 silk thread (Ethicon, Somerville, NJ, USA). After the mesh was sutured in place, the rat s back was sutured so that his skin is tightly lying against the dorsal port. In addition, the neck incision from the insertion of the silastic cannula was sutured up using both internal suture and a normal suture to ensure the threads stayed in place. After the surgery, anaesthetic gas was turned off and oxygen (1.0 L/min, Praxair, Mississauga, ON, Canada) was given for 5 minutes or longer to allow rats to recover from surgery faster. Then, they received an injection of 10mL of saline injection (0.9% NaCl, subcutaneous; Baxter Corporation, Mississauga, ON, Canada). Afterwards, they were returned to the experimental room, where they received three days of postoperational Anafen injections (5mg/mL, subcutaneous; Merial Canada, Quebec, Canada) for analgesia. After surgery, intravenous cannulae were flushed with heparin (70U in 0.2mL sterile saline) twice a day throughout the rats whole experimental career to prevent clotting and conserve patency Intra-BST Drug Micro-Injection Rats received micro-injections based on pre-established criteria. Firstly, rats were trained on the progressive ratio (PR) schedule of reinforcement on the operant paradigm for equal or more than 10 days before they could receive their first micro-injection. Secondly, the candidate must reach a stable BP over a period of one to three consecutive 29

41 days (total rewards received at BP ±3, based on a criterion of 8% deviation from the LN final ratio average) (see data analysis section of the thesis for explanation and formulae used to calculate the LN final ratio average deviation). In this experiment, a BP was defined as the last infusion received before the end of the session, at which the full expression of operant responding has occurred (i.e. in Sucrose self-administration group, the session length is set as 180 minutes; in Cocaine self-administration group, the session length is set as 270 minutes). Therefore, the BP is the final ratio completed for the whole session length. This is used as a measure of the maximum motivational effort that the rat is willing to exert to attain the reward. Final ratio is the number of presses in the last bout of pressing required to achieve BP. If all the criteria were met, the rat would receive intracranial injections into the BST. Micro-injections occurred immediately prior to self-administration experiments and were delivered over a 90-second interval. For the microinjection, a pair of 10µL microsyringes (Microliter #1701; Hamilton, Reno, Nevada, USA), cleaned and flushed with 70% ethanol (diluted from 100% reagent alcohol, Fisher Scientific, NJ, USA) and distilled water, were mounted on a Razel micro-infusion pump via a 1RPM motor. An injector set (28GA, 6.5mm with 1.2mm protrusion=7.7mm total length, HRS Scientific, Plastics One, Roanoke, VA), was 1.2mm longer than the bilateral guide cannula. [**Sucrose self-administration rats with Rat IDs that are before L143 used shorter injector sets: 21GA, 5.8mm with 0.3mm protrusion=6.1mm total length, HRS Scientific, Plastics One, Roanoke, VA] It was inserted into polyethylene tubing PE50 (thin walled,0.023x0.041 HRS Scientific, Plastics One, Roanoke, VA). The tubing and injector were flushed and filled with sterile saline (0.9% NaCl saline, Baxter Corporation, 30

42 Mississauga, ON, Canada) before they were attached to the microsyringes. A small volume (about 5µL) of sterile saline (0.9% NaCl saline, Baxter Corporation, Mississauga, ON, Canada) was drawn back into the injector and tubing, followed by 1µL of air to ensure the integrity of the saline and drug. Lastly, 1µL of drug was drawn up into the injectors, ready for intra-bst micro-injection. Before each injection, a test run of the Razel micro-infusion pump was performed to make sure that the drug solution was coming out of the injector set. Then, the injectors were inserted into the bilateral guide cannulae on the head of each rat, and 0.5 µl of the diluted drug (or saline vehicle) was injected into the BST on each side of the brain over 60 seconds. Injectors were left in the guide cannulae for an additional 30 seconds, and the injectors were retracted slowly to minimize the amount of residual drug due to suction force. The stylets were put back in the guide cannulae and the rat was immediately placed inside an operant chamber to start the self-administrative experiment. The procedure was repeated for all the rats that met the pre-established criteria for microinjections. A different set of injectors was used for different drugs and rats to prevent cross-contamination across rats. Experimental design for the order of intra-bst micro-injected drugs: At the initial stages of our experiment, we started out with a pseudorandom design for the order of the intra-bst drugs given to each animal. This is a double blind procedure, where both the animal and the experimenter were blind to the drugs given, so potential experimental bias and manipulation could be minimized. Under this design, each animal received a maximum of 5 intra-bst drug injections. Among these 5 injections, the animal received 2 injections of the target drug at the same concentration, 1 31

43 injection of the same drug at a different concentration, and 2 injections of saline. Each injection was coded by a letter and a number, and the order of the injections was mixed randomly (see Table 3 for an example of the pseudorandom design of drug injections). Under this design, each drug injection group, represented by a letter, only contains one kind of the drug at 2 different concentrations. The ordering of the injections was designed to counterbalance any potential carry-over effects from previous injections and to provide good experimental control. Table 3. Pseudorandom design of drug injections. Each drug injection group is designated by a letter and contains 5 injections. Of these 5 injections: 2 injections are vehicle saline, the other 2 injections are the drug at one concentration, and 1 injection is the same drug at a different concentration. The order of these injections is randomized to avoid experimenter bias. Group A (SCH-23390) A1. SCH 1.6 μg/μl A2. Saline (Vehicle) A3. SCH 3.2 μg/μl A4. Saline (Vehicle) A5. SCH 1.6 μg/μl From the middle towards the end stages of our experiments, as we realized that the order of the injections did not affect the behaviour of the animals, and experimenters were not biased by knowing what the drugs were, we maximized the use of our rats by giving them many injections until they were too old for more experiments. Therefore, each rat could receive more than one kind of drugs. The injections from various rats were pooled into individual drug treatment groups and the resulting dependent measures 32

44 were analyzed using one-way ANOVA (see Table 4 and Table 5 for the distribution of injection N numbers for each animal and drug treatment group). Table 4. Distribution of total intra-bst injections in sucrose- and cocaineself-administering rats. For both groups, the same saline treatment data was used to compare to other drug treatment groups (SCH-23390, Sulpiride, Quinpirole). Numerical data from all our drugs of interest were pooled from various rats. Drug # of injections in each group (N numbers) Sucrose self-admin Cocaine self-admin Saline (vehicle) SCH 1.6μg/μL 4 5 SCH 3.2μg/μL 6 4 SCH 6.4μg/μL 4 5 Sulpiride 3.2μg/μL 5 6 Quinpirole 3.0μg/μL

45 Table 5. An outline of the intra-bst injections received by each rat (in terms of the kinds of drugs received, the concentrations of the drugs, and the number of injections received). The number in each cell represents the number of injections given in each condition. Rat ID Saline SCH 1.6μg/μL SCH 3.2μg/μL SCH 6.4μg/μL Sulpiride 3.2μg/μL Quinpirole 3.0μg/μL Sucrose Rats (number in each cell represents the number of injections given in each condition) L L L L L L L L L194 1 L L198 2 L196 1 Total N Cocaine Rats (number in each cell represents the number of injections given in each condition) L L L

46 L176 1 L L L L L L215 1 Total N Behavioural Procedure: Operant Conditioning-Reinforcement Paradigm Operant Training All experiments and training were run between 9:00 hour to 21:00 hour in the dark phase (active) of the reversed light/dark cycle. For both the cocaine and sucrose self-administration groups, rats were trained on a fixed-ratio-1 (FR-1, one lever press gives one reward) schedule until they achieved a controlled, titrating pattern of pressing (40 lever presses for 40 rewards for sucrose (see Figure 7) and 25 lever presses for 25 infusions for cocaine (see Figure 8), where infusions were self-administered at equal time intervals for 3 consecutive days. The rats then graduated to the progressive atio training (PR) for both sucrose (see Figure 9) and cocaine (see Figure 10) self-administration groups. 35

47 Figure 7: Example behavioural trace showing the acquisition of operant lever pressing response under the fixed ratio-1 (FR-1) schedule of reinforcement in sucrose self-administering rats. Rats demonstrate consistent pressing behaviour (40 presses for 3 consecutive days) in a controlled manner. Actual lever presses (operant response) are demonstrated by vertical increments, and the receipt of each reward (45mg sucrose pellet) is represented by tick marks. On the FR-1 schedule, each operant response is paired with one reward. Figure 8: Example behavioural trace showing the acquisition of operant lever pressing response under the fixed ratio-1 (FR-1) schedule of reinforcement in cocaine self-administering rats. Rats demonstrate consistent pressing behaviour (25 presses for 3 consecutive days) in a controlled and titrating manner. Actual lever presses (operant response) are demonstrated by vertical increments, and the receipt of each reward (0.12mL of 0.75mg/kg/injection of cocaine) is represented by tick marks. 36

48 Figure 9: Example behavioural trace showing the expression of operant lever pressing behaviour under the progressive ratio (PR) schedule of reinforcement for the sucrose self-administration group. The behaviour of each rat is graphed as cumulative number of lever presses against time in minutes. The receipt of each sucrose reward (45mg/pellet), is represented by tick marks. Typically, rats on the PR schedule are trained for at least 10 days until stable operant responding is achieved so they can receive intracranial microinjections of our drugs of interest. Under the PR reinforcement schedule, each reward after the first requires a progressively greater number of operant responses. On each experimental day, the data collected for quantification of behaviour is breakpoint (BP), which is the last reward received before session ends (in the sucrose self-administering rats, the whole session lasts for 180 minutes). The ln (final ratio) scale on the right corresponds to the total number of lever presses on the left. From the total number of lever presses, the number of infusions (breakpoint) is derived and final ratio (the number of additional lever presses between each reinforcer) is determined from Table 1. The ln (final ratio) value is used to calculate the 8% deviation from the LN final ratio average for the 1 to 3 consecutive days before the actual injection day. This formula is outlined in detail in the method section. Drug effect is calculated as the change in final ratio in percentage. This is calculated by dividing the final ratio of the injection day by the average of the 3 pre-injection day final ratio. 37

49 Figure 10: Example behavioural trace showing the expression of operant lever pressing behaviour under the progressive ratio (PR) schedule of reinforcement for the cocaine self-administration group. The behaviour of each rat is graphed as cumulative number of lever presses against time in minutes. The receipt of each cocaine infusion (0.75mg/kg/infusion) is represented by tick marks. Typically, rats on the PR schedule are trained for at least 10 days until stable operant responding is achieved so they can receive intracranial microinjections of our drugs of interest. Under the PR reinforcement schedule, each reward after the first requires a progressively greater number of operant responses. On each experimental day, the data collected for quantification of behaviour is breakpoint (BP), which is the last reward received before session ends (in the cocaine self-administering rats, the whole session lasts for 270 minutes). The ln (final ratio) scale on the right corresponds to the total number of lever presses on the left. From the total number of lever presses, the number of infusions (breakpoint) is derived and final ratio (the number of additional lever presses between each reinforcer) is determined from Table 1. The ln (final ratio) value is used to calculate the 8% deviation from the LN final ratio average for the 1 to 3 consecutive days before the actual injection day. This formula is outlined in detail in the method section. Drug effect is calculated as the change in final ratio in percentage. This is calculated by dividing the final ratio of the injection day by the average of the 3 pre-injection day final ratio. 38

50 Experimental Testing (Intra-BST Micro-Injection of Drugs) For each experimental session, a maximum number of 2 rats were micro-injected (if there were that many rats ready at the time). After the rats received intra-bst microinjected drugs, they were put into the experimental chambers and the selfadministrative sessions were initiated. The rats were observed through the transparent plexiglass of the chambers and any irregularities in their behaviours due to potential effects of the drugs were recorded. For the sucrose self-administration group, both the FR-1 and PR sessions were 180 minutes in length. The cocaine self-administration group lasted 270 minutes in length for both the FR-1 and PR sessions. MedPCIV software (MedAssociates, St-Albans, VT) was used to record the sessional behaviour data for analysis (i.e. number of lever presses, BP) Data Analysis In total, 22 rats were used in the final data analysis. All of these rats were used for intra-bst drug micro-injections and histology was done to ensure proper cannulae placement. Behavioural changes (i.e. traces) recorded by MedPCIV were compared within the two test groups: sucrose self-administration and cocaine self-administration (example behavioural traces used to calculate the effects of dopamine drugs administered are shown in Figures 9 and 10). On each behavioural trace, each tick represents the receipt of one reward, either in the form of a single sucrose pellet or a single cocaine infusion. The number of lever presses on the left vertical axis of the behavioural trace corresponds to the total pressing column in Table 1, which is calculated using the response ratio 39

51 formula for the PR schedule series (Response ratio = 5[ee (iiiiiiiiiiiiiiiiii nnnnnnnnnnnn 0.2) ] 5). Each value of total pressing corresponds to an infusion number, which is taken as the BP in our experiment since we defined our BP as the last infusion reward received before experiment session ends. If the animal s total pressing value is less than the specified total pressing value outlined in Table 1, then the BP will be one less (i.e. if the animal achieved total pressing of 10, the BP for this animal would be 3, not 4, because 10 is less than the specified total pressing value of 13 on the table, which corresponds to a BP of 4). Each BP value then corresponds to a final ratio value (Table 1), which is then ln transformed to ensure it conforms to the assumptions of parametric statistics. This is because the final ratio is derived from a natural ln equation, so values at the higher end of the scale will have a larger variance than values at the low end. Consequentially, the ln(final ratio) scale on the right side of the behavioural trace (Figure 9 and 10) was used in the calculation for deciding if an animal could receive an intra-bst drug injection (our criteria was that the candidate must reach a stable BP over a period of one to three consecutive days, with total rewards received at BP ±3, based on a criterion of 8% deviation from the LN final ratio average). The formula used to determine if there was a 8% deviation from the LN final ratio average is outlined as follows: ((ln[final ratio Day1 ])-(ln[final ratio Day2 ])) + ((ln[final ratio Day2 ])-(ln[final ratio Day3 ])) (ln[final ratioday1]) (ln[final ratio Day2 ]) x100 2 Once the intra-bst micro-injection was given, we determined the effect of our dopaminergic drugs of interest. The change in final ratio relative to 3 pre-injection day 40

52 average baseline was measured for each drug treatment group: Vehicle (sterile saline), SCH (1.6, 3.2, or 6.4 µg/µl), Sulpiride (3.2 µg/µl), and Quinpirole (3.0 µg/µl). For each injection, the final ratio of the intra-bst injection day was compared with the average final ratio of the 3 pre-injection days and expressed as a change in percentage. The formula used in the calculation of the drug effect is outlined as follows: final ratio injection day Average (final ratio preinjection day1to3 ) - 1 x 100 The reason for not doing a ln(final ratio) transformation for the calculation of the drug effect using the above formula is because the numerical values generated by this formula is more reflective of the actual drug effect on the behaviour of the animals. After the drug effects were calculated, the numerical results from each animal were pooled into separate drug treatment groups. So, within one drug treatment group, the injections could come from a various drugs and some animals were used for more than one kind of drug injection (see Table 4 and Table 5 for clarification). All the data were checked for normality and equality of variances and then all the values were compared by using the one-way ANOVA test and post hoc comparisons using Dunnett s method for comparison with control to test for statistical significance between the saline treatment group and the individual drug treatment groups. Because of the way that the intra-bst drug injection data were collected, the drug effect values obtained were not independent measures. Therefore, we performed one-way ANOVA for our statistical analysis. 41

53 2.8 - Histology / Brain Cannulation Placements Once an experiment was completed, the brains of the rats were examined in order to determine whether the bilateral guide cannulae were placed within the oval BST. To prepare fluorescent microscope slices, rats were given intra-bst microinjections of 1.0µL (0.5µL in each side of brain) of micro-fluorescent beads (FluoSpheres carboxylate modified microspheres; 0.04µm, red-orange fluorescent; actual size: 0.036µm; invitrogen Molecular Probes, Oregon, USA) using the same procedure as previous intra-bst microinjections. To prepare for brain extraction, rats were anaesthetized with sodium pentobarbital (60mg/ml; from Euthanyl stock solution of 240mg/ml; Bimeda-MTC Animal Health Inc, Cambridge, ON) until they reached surgical plane for perfusion (each animal started at 0.6mL, and were gradually given additional incremental amounts of 0.2mL, depending on their weight). A horizontal incision was made across the rat s belly, with the underlying muscle tissue cut open to expose the interior organs. The sternum was clamped down to expose the diaphragm. Then, the diaphragm was cut open to expose the heart. To attain a better surgical view of the heart, the rib cage was cut open even further up from both the left and right sides. The right atrium was clipped to release the potential pressure that might build up during perfusion. Then, the tip of a 16G needle (Becton Dickinson, Franklin Lakes, NJ, USA) was inserted into the apex of the heart (at the left ventricle) for saline and 4% paraformaldehyde (PFA) solution to be pumped through. After this, the rats were then perfused transcardially with 250 ml of saline or until the liquid dripping down from the rat s body was transparent in appearance. This was then followed by pumping 500 ml of 4% PFA solution 42

54 transcardially into the rat. Perfusion was only considered successful if the rat s body was solid hard by the end of the 4% PFA solution. Then, the brains were extracted and placed in the 4% PFA solution for postfixation overnight (in 4 C refrigerator). At this stage, a successfully extracted brain would appear white in color, with no blood vessels showing on the cortex. To ensure cryoprotection, the brains were then transferred to a 30% sucrose-pfa solution for at least 2-3 days (in 4 C refrigerator) to ensure complete infusion. Then, using a cryotome (Leica SM2000R, Germany), the brain was partitioned into 50µm slices taken from the dorsolateral BST. The slices were then mounted and viewed under a fluorescing microscope (Leica Mikroskopie and Systeme GmbH Wetzlar, Germany) via the projection of green fluorescent light (ebq100, Germany) to confirm the placement of the cannulae guides. 43

55 Chapter 3: Results Due to individual variations in behavioral responses and for general statistical analysis considerations, first saline injections for cocaine rats L191, L159, L215, and L213 were excluded. In addition, the first SCH (1.6µg/µL) injection for cocaine rat L188, and the first Sulpiride (3.2 µg/µl) injection for cocaine rat L162 were excluded because the resulting drug effects were more than 2 standard deviations away from the mean for those injections and treatment groups. For the calculation of the change in baseline final ratio or BP (to show the effect of the drug), the average of the final ratio from 1 to 3 days before the injection date was used. For more detailed behavioural traces and data of all the rats with each individual drug treatment, see appendix I, and II Effect of SCH on Self-Administration In the sucrose self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = ±7.34%, n = 12), and the D1 selective antagonist SCH (1.6µg/µL = ±12.13%, n = 4; 3.2µg/µL = ±8.53%, n = 6; 6.4µg/µL = 21.61±14.45%, n = 4). Oneway ANOVA test revealed no statistical significance between the drug treatment groups, F (3, 21) = 2.57, p = (see Figure 11). Example behavioral traces for the effect of saline and SCH in sucrose self-administering rats were shown in Figure 12. In the cocaine self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and the D1 selective antagonist SCH (1.6µg/µL = ±18.99%, n = 5; 3.2µg/µL = ±5.81%, n = 4; 6.4µg/µL = ±5.48%, n = 5) (Figure 11). Oneway ANOVA test (using Dunnett s method for comparison with control) revealed that there was 44

56 statistical significance in cocaine rats treated with SCH at concentrations of 3.2µg/µL and 6.4µg/µL when compared to the saline treatment group (SCH µg/µL: p = 0.024; SCH µg/µL: p = 0.028) (see Figure 11). Example behavioral traces for the effect of saline and SCH in cocaine self-administering rats were shown in Figure 13. Delta final ratio pressing (%) Effect of SCH (D1 antagonist) on Reinforcement N=11 N=5 N=4 N= N=12 N=4 N=6 * N= saline 1.6 μg/μl 3.2 μg/μl 6.4 μg/μl Cocaine self-admin Sucrose self-admin Cocaine self-admin Sucrose self-admin Figure 11: Bar graph showing the effects of vehicle (saline) and SCH (at total bilateral dosages of 1.6, 3.2, and 6.4 µg/µl) on the breakpoint (percentage change in final ratio) of rats self-administering sucrose and cocaine under the progressive ratio (PR) schedule of reinforcement. Mean (±SEM) changes in final ratio pressing in percentage (%) were averaged across all rats in each drug treatment group, with N numbers outlined on the graph for each treatment group. The effect of the drug is calculated by comparing the final ratio on the day of the injection with the average of the 3 day pre-injection final ratio. Asterisk (*) indicates that responding for SCH at concentrations of 3.2 µg/µl, and 6.4 µg/µl in the cocaine self-administering rats was significantly different from responding under the saline vehicle treatment ( p < 0.05) by post hoc comparisons using Dunnett s method for comparison with control. 45

57 Figure 12: The effects of intra-bst micro-injections on sucrose self-administration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate the change (black dashed line) from the 3-day pre-injection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the selective D1- dopamine receptor antagonist, SCH-23390, at a total bilateral dosage of 3.2µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (180 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 46

58 Figure 13: The effects of intra-bst micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the selective D1-dopamine receptor antagonist, SCH-23390, at a total bilateral dosage of 6.4µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (270 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 47

59 3.2 - Effect of Sulpiride on Self-Administration In the sucrose self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = ±7.34%, n = 12) and the D2-like antagonist Sulpiride (3.2µg/µL = 2.74±16.57%, n = 5). Oneway ANOVA test revealed no statistical significance between the drug treatment groups, F (2, 18) = 2.52, p = 0.11 (see Figure 14). Example behavioral traces for the effect of saline and Sulpiride in sucrose self-administering rats were shown in Figure 15. Delta final ratio pressing (%) Effect of Sulpiride (D2 antagonist) on Reinforcement N=11 N= N=12 N= * Saline 3.2 μg/μl Cocaine self-admin Sucrose self-admin Cocaine self-admin Sucrose self-admin Figure 14: Bar graph showing the effects of vehicle (saline) and Sulpiride (at total bilateral dosage of 3.2µg/µL) on the breakpoint (percentage change in final ratio) of rats self-administering sucrose and cocaine under the progressive ratio (PR) schedule of reinforcement. Mean (±SEM) changes in final ratio pressing in percentage (%) were averaged across all rats in each treatment group, with N numbers outlined on the graph for each treatment group. The effect of the drug is calculated by comparing the final ratio on the day of the injection with the average of the 3 day pre-injection final ratio. Asterisk (*) indicates that responding for Sulpiride at the concentration of 3.2 µg/µl in the cocaine self-administering rats was significantly different from responding under the saline vehicle treatment ( p < 0.05) by post hoc comparisons using Dunnett s method for comparison with control. 48

60 Figure 15: The effects of intra-bst micro-injections on sucrose selfadministration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate the change (black dashed line) from 3-day pre-injection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the D2-like dopamine receptor antagonist, Sulpiride, at a total bilateral dosage of 3.2µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (180 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 49

61 Figure 16: The effects of intra-bst micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the D2-like dopamine receptor antagonist, Sulpiride, at a total bilateral dosage of 3.2µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (270 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 50

62 In the cocaine self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and the D2-like antagonist Sulpiride (3.2µg/µL = ±6.20%, n = 6) (Figure 14). Oneway ANOVA test (using Dunnett s method for comparison with control) revealed that there was statistical significance in cocaine rats treated with Sulpiride 3.2µg/µL when compared to the saline treatment group (Sulpiride 3.2µg/µL: p = ) (see Figure 14). Example behavioral traces for the effect of saline and Sulpiride in cocaine selfadministering rats were shown in Figure Effect of Quinpirole on Self-Administration In the sucrose self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = ±7.34%, n = 12) and the D2-like agonist Quinpirole (3.0µg/µL = -5.69±12.08%, n = 4) (Figure 17). Oneway ANOVA test revealed no statistical significance between the drug treatment groups F(1, 14) = 0.20, p = 0.67 (see Figure 17). Example behavioral traces for the effect of saline and Quinpirole in sucrose self-administering rats were shown in Figure 18. In the cocaine self-administration group, the change in final ratio pressing was calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and the D2-like agonist Quinpirole (3.0µg/µL = ±11.61%, n = 7) (Figure 17). Oneway ANOVA test revealed no statistical significance between the drug treatment groups F(1, 16) = 2.50, p = 0.13 (see Figure 17). Example behavioral traces for the effect of saline and Quinpirole in cocaine self-administering rats were shown in Figure

63 Delta final ratio pressing (%) Effect of Quinpirole (D2 agonist) on Reinforcement N=11 N= N=12 N= Saline 3.0 μg/μl Cocaine self-admin Sucrose self-admin Cocaine self-admin Sucrose self-admin Figure 17: Bar graph showing the effects of vehicle (saline) and Quinpirole (at total bilateral dosage of 3.0 µg/µl) on the breakpoint (percentage change in final ratio) of rats self-administering sucrose and cocaine under the progressive ratio (PR) schedule of reinforcement. Mean (±SEM) changes in final ratio pressing in percentage (%) were averaged across all rats in each treatment group, with N numbers outlined on the graph for each treatment group. The effect of the drug is calculated by comparing the final ratio on the day of the injection with the average of the 3 day pre-injection final ratio. There were no significant differences between the saline and the Quinpirole treatment groups. 52

64 Figure 18: The effects of intra-bst micro-injections on sucrose selfadministration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate the change (black dashed line) from 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the D2-like dopamine receptor agonist, Quinpirole, at a total bilateral dosage of 3.0µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (180 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 53

65 Figure 19: The effects of intra-bst micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement. These graphs illustrates the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top graph) or the D2-like dopamine receptor agonist, Quinpirole, at a total bilateral dosage of 3.0µg/µL (bottom graph). Each reward received is represented by ticks on the graph and breakpoint (BP) is defined as the last reward received before the session ends (270 minutes). BP is used to determine the final ratio for calculation of drug effects expressed as percentage change in final ratio pressing (see methods section for explanation and formulae). 54

66 3.4 - Histology / Brain Cannulation Placements The 2 groups (sucrose and cocaine self-administration) included a total of 22 rats. Of these, 12 rats were sucrose rats and 10 rats were cocaine rats. In the sucrose selfadministration group, 4 rats had traces of fluorescent beads in the ventricle, suggesting inaccurate cannulation placements. However, this only occurred with a few rats in the very initial stage of our experiment. Our cannulation placements were much more accurate later on. The majority of our intra-cranial cannulations were directly in the BST, with different rats showing its optimal placement in the BST at somewhat different brain levels. In the cocaine self-administration group, 1 rat had traces of fluorescent beads in the ventricle. However, the rest of the rats in this group all had injection tracts and sites directly in the BST. The cannulae placements for each rat in each group are shown in Figure 20 (a)~(g) (sucrose rats) and Figure 21 (a)~(d) (cocaine rats), and photographs of coronal sections showing typical BST cannulae placement are presented in Figure 22 (sucrose rats) and Figure 23 (cocaine rats). 55

67 Figure 20. These brain maps show the intra-cranial cannulation placements in sucrose self-administering rats at different brain levels. Parts (a) ~ (g) depict various rat brain levels, marked with anterior-posterior coordinates from Bregma. Cannulae placements in the bed nucleus of the stria terminalis (BST) are marked by thicker lines along the ventricle and ovals in the BST region. Each rat has a representative brain level for which its optimal injection site is located, which is marked on the brain maps with the rat s ID. The localization of the intra-bst injection is outlined by the existence of the fluorescent beads. The IDs of the rats are labelled on the arrows pointing to the lines along the ventricle and in the ovals in the BST region. Although only one side of the brain is marked, all of these are bilateral brain placements. The right side of the brain map facilitates identification of the anatomy of the brain, especially the BST. (Adapted from L. W. Swanson Brain Maps, 3 rd Edition) a) Level 15 (AP = from Bregma) b) Level 16(AP = from Bregma) 56

68 c) Level 18(AP = from Bregma) d) Level 19(AP = from Bregma) e) Level 20(AP = from Bregma) 57

69 f) Level 21(AP = from Bregma) g) Level 23(AP = from Bregma) 58

70 Figure 21. These brain maps show the intra-cranial cannulation placements in cocaine self-administering rats at different brain levels. Parts (a) ~ (d) depict various rat brain levels, marked with anterior-posterior coordinates from Bregma. Cannulae placements in the bed nucleus of the stria terminalis (BST) are marked by thicker lines along the ventricle and ovals in the BST region. Each rat has a representative brain level for which its optimal injection site is located, which is marked on the brain maps with the rat s ID beside it. The localization of the intra- BST injection is outlined by the existence of the fluorescent beads. The IDs of the rats are labelled on the arrows pointing to the lines along the ventricle and in the ovals in the BST region. Although only one side of the brain is marked, all of these are bilateral brain placements. The right side of the brain map facilitates identification of the anatomy of the brain, especially the BST. (Adapted from L. W. Swanson Brain Maps, 3 rd Edition) a) Level 15 (AP = from Bregma) b) Level 19(AP = from Bregma) 59

71 c) Level 20(AP = from Bregma) d) Level 21(AP = from Bregma) 60

72 Level (between AP = ~ from Bregma) Figure 22. Picture showing actual rat brain under fluorescent microscopy for sucrose self-administering rats. The shiny beads outline the injection site and tract along the BST region. Specifically, the injection site was located at brain level (between AP = ~ from Bregma). 61

73 Ventricle BST aco a) Level 20(AP = from Bregma). Fluorescent beads and injection tract at BST injection site on both sides of the brain. Ventricle b) Level 20(AP = from Bregma). No fluorescent beads lining the ventricle on either side of the brain. Figure 23. Pictures showing actual rat brain under both transmitted and fluorescent microscopy for cocaine self-administering rats. In picture (a), the photo on the left shows the rat brain with beads under transmitted microscope; the photo on the right depicts the same picture under fluorescent microscope. The shiny beads outline the injection site and tract along the bed nucleus of the stria terminalis (BST) region. Specifically, the injection site was located at around rat brain level 20 (AP = from Bregma). In picture (b), the photo shows that there are no beads lining the ventricle. Abbreviations for this figure are as follows: aco, anterior commissure; BST, bed nucleus of the stria terminalis; ventricle, lateral ventricle. 62

74 Chapter 4: Discussion Summary of Current Results In this study, our results suggested the significance of dopaminergic response to cocaine rewards in the BST. Dopaminergic drugs administered directly into this structure manipulated the expression of addictive behaviours related to pharmacological (i.e. cocaine), but not natural (i.e. sucrose) rewards. Delivery of dopamine antagonists into the BST decreased motivated behaviours: both the D1 selective antagonist SCH (3.2 & 6.4 µg/µl) and the D2-like antagonist Sulpiride (3.2 µg/µl) significantly reduced operant behaviour in animals self-administering cocaine. However, none of these dopamine agents had any significant effect on sucrose self-administration. The D2-like agonist, Quinpirole (3.0 µg/µl) also failed to induce any changes in operant responding. This general finding is in partial concordance with previous studies demonstrating changes in the reinforcing properties of both food and drugs resulting from blockade of dopamine receptors in other brain structures: the VTA (Shibata et al., 2009), the amygdala (Berglind et al., 2006; McGregor et al., 1994; McGregor and Roberts, 1993), and the NAc (McGregor and Roberts, 1993; Hodge, et al., 1997; Anderson et al., 2003; Anderson et al., 2006; Schmidt and Pierce, 2006). Since relatively few studies have examined the contribution of the BST dopamine receptors in the reinforcing properties of sucrose and pharmacological rewards (Epping-Jordan et al., 1998; Walker et al., 2000; Eiler II et al., 2003), our results will provide further confirmation of the sensitivity of the BST dopamine receptors on reinforcement, especially in cocaine. Furthermore, the results will confirm that the BST is well positioned for the role of addiction. The BST s connection to the mesolimbic dopamine system, its heavy innervations to and from 63

75 midbrain dopamine releasing areas (i.e. VTA, PAG, RR, SNc), and its expression of D1- and D2- dopamine receptors (Freedman and Cassell, 1994; Hasue and Shammah- Lagnado, 2002; McDonald et al., 1999; Scibilia et al., 1992) all serve to facilitate rewardelicited dopamine related modulation at the BST (Carboni et al., 2000) Selective D1 Antagonist (SCH-23390) Had No Effect on Sucrose Self- Administration, But Decreased Cocaine Self-Administration In our experiment, rats pre-treated with SCH before their selfadministration sessions produced very different results. In the sucrose self-administration group, treatment of SCH at various concentrations (1.6, 3.2, & 6.4 µg/µl) produced no significant effects compared to the vehicle (saline) treatment group. Although saline and SCH treatment at 1.6 µg/µl and 3.2 µg/µl generated slight decreases in responding compared to baseline, which was somewhat consistent with previous research (Eiler II et al., 2003), the slight increase in responding at 6.4 µg/µl of SCH made the role of the BST in modulating natural reinforcement more obscure. However, the fact that there were still decreases in sucrose maintained operant responding indicated that BST dopamine receptors maybe still be partially blocked or manipulated by the D1 dopamine receptor antagonist following chronic sucrose intake. Since food reward foraging is imperative for survival and reproduction, brain systems have evolved pathways to direct and motivate this behaviour. Studies have shown that dopaminergic projection from the VTA to the NAc is not only essential for modulating goal-directed behaviour, but the changes in the firing rate of NAc neurons also encode information related to the operant response for food reward (Wise et al., 1978; Carelli, 2002; Roitman et al., 2004). In our case, the BST projects moderately to other brain 64

76 areas involved in locomotor or exploratory behaviour control, reward prediction and ingestive behaviour control (Dong and Swanson, 2003). Therefore, it is not surprising that dopamine released in the BST can have a modifying effect on food seeking. The fact that there was no influence on operant responding maintained by food suggests that the decrease of cocaine seeking by intra-bst SCH injection was not due to general motor impairment (Stairs et al., 2010; Anderson et al., 2003). Other studies showing an effect of sucrose responding under SCH (Eiler II et al., 2003; Barrett et al., 2004) may be inconsistent with our results due to different schedules of reinforcement employed (i.e. they used FR schedules with differing time-out periods) and different methods of food / sucrose delivery (i.e. sucrose solution was self-administered instead of sucrose pellets) under the self-administration paradigm. The use of a range of sucrose solution concentrations may be maintained by different rates of responding which will subject the results to more variability in interpretation. In the cocaine self-administration group, treatment of saline produced no change in responding, however, treatment of SCH at concentrations of 3.2 µg/µl and 6.4 µg/µl yielded statistically significant attenuations in responding. This finding is in full concordance with the study conducted by Epping-Jordan et al (1998), which showed decreases in the reinforcing efficacy of cocaine (0.75mg/kg/intravenous injection) following intra-bst injection of SCH at concentrations of 3.2 µg/µl and 6.4 µg/µl. However, all experimental trials in this study were carried out under the FR-5 schedule of reinforcement with timeout periods of 20 seconds, while the PR schedule of reinforcement was used in our study. With the FR schedule, the dependent variable is the rate of self-administration. Since the rate of drug intake is inversely proportional to unit 65

77 injection dose, Yokel and Wise (1975) have proposed that the increase in rate of drug self-administration serves as a compensatory response to reflect the decrease in drug reinforcing efficacy; conversely, the decrease in rate reflect an increase in reinforcing efficacy (Richardson and Roberts, 1996; Yokel and Wise, 1975). On the other hand, with the PR schedule, the dependent variable is the breaking point, which is defined as the final ratio completed by an animal where responding ceases as the animal reaches the maximum number of infusions for the effort it is willing to put in for the drug reward. Therefore, a decrease in breaking point, as observed in our study, is indicative of attenuation in the ability of the drug to maintain patterns of self-administration (Richardson and Roberts, 1996). The effect of SCH was calculated as percentage change in baseline final ratio by comparing the final ratio of the drug treatment day with the average final ratio of three-day before drug treatment. The fact that we included the whole 270-minute session data in our analysis for final ratio and breaking point ensured the full potency of our drug of interest (i.e. both the psychostimulant effect of cocaine and the dopamine receptor antagonist and agonists) was scrutinized for their effect on the motivational aspect of drug seeking. In the Epping-Jordan et al. study, results were expressed as both percentage change in rate of cocaine self-administration for the first 20-minute of the experiment and the whole 3-hour experimental session. With both of these times, there were significant increases in the rate of cocaine self-administration under the FR schedule. In our study, SCH decreased both the cumulative number of lever presses and the breaking point under the PR schedule. The orderly interreinforcer time shown on the trace (Figure 13) suggests that the animals were able to maintain a controlled, titrated self-administering behaviour without inducing a 66

78 cumulative dosing effect. Since SCH treatment yielded similar inter-reinforcer time compared to the 3 day pre-injection baseline, it is suggestive that SCH does not generate a general impairment of the motor functioning in the animals. In addition, the fact that the breaking point from the SCH treatment day occurred so much earlier than the non-drug treatment days, where the breaking point spans the whole duration of the experimental session, implicates that this is due to blockade of the D1 receptor in the BST. Other studies also showed that SCH injected into the BST dose-dependently decreased alcohol motivated responding under FR4 operant schedule, both in responding rates and cumulative responding (Eiler II et al., 2003). Although the suppression of responding signifies an increase in reinforcing efficacy of cocaine with SCH treatment in the BST, this phenomenon may not be completely pertinent to our results due to the researchers use of FR operant schedule, the training of rats to selfadminister increasing dosages of ethanol, and the difference in the dosage of SCH infused into the BST via a different injection rate. Overall, our results are supported by studies conducted under the PR schedule of cocaine reinforcement with injection of SCH into the NAc (McGregor and Roberts, 1993), VTA (Ranaldi and Wise, 2001), and subcutaneously (Ward et al., 1996) D2-like Antagonist (Sulpiride) Has No Effect on Sucrose Self-Administration, But Decreases Cocaine Self-Administration Pretreatment of rats with Sulpiride did not affect operant responding in the sucrose self-administration group when compared to the pattern of responding in the saline treated group. This is in agreement with previous studies (Caine et al., 2002; Eiler II et al., 2003; Bari and Pierce, 2005), where it was shown that D2 receptors were not as 67

79 important in modulating natural reinforcer motivated behaviours. Again, this no effect of natural reinforcer with the D2 receptor antagonist treatment serves as a very good experimental control for the D2 receptors to be not involved in motor function in our case. For the cocaine self-administration group, only Sulpiride at 3.2 µg/µl produced significant attenuations in operant responding. This is not surprising according to studies of D2 receptor knock-out mice showing that they were still able to self-administer cocaine at higher rates than heterozygous and wild type mice when high doses of cocaine on the descending limb of the dose response curve is available. The effect of increased response rates with pretreatment of D2 antagonist (eticlopride) and the effect of D2 agonist (quinelorane) as a positive reinforcer for cocaine was only present in wild type mice. Taken together, these results suggest that the D2 receptor is not necessary for cocaine self-administration (Caine et al., 2002). In our study, however, the use of Sulpiride as our D2-receptor antagonist provided us with some significance of D2 receptor in the cocaine reinforcement because Sulpiride is about 100 times more selective for D2 receptors as for D3 receptors and about 400 times more selective for D2 receptors than for D4 receptors (Vallone et al., 2000). The specificity of Sulpiride generated reliable decreases in both the number of cumulative decreases in lever press and breaking point under PR schedule. In addition, the specificity of Sulpiride as a hydrophilic compound for D2 receptors is especially apparent when administered at lower dosages, whereas higher dosages of Sulpiride may result in non-specific binding for both D2 and D3 receptors (Arnt, 1985). Although the BST projects heavily to areas of the brain involved in generating locomotor (foraging) behaviour for reward acquisition (Dong and 68

80 Swanson, 2004; Dong et al., 2000; Dong and Swanson, 2003; Dong et al., 2001), and studies have suggested that the D2-like receptor antagonist are prone to produce a greater effect on motor impairment function than the D1-like antagonist (Fowler and Liou, 1994; Neisewander et al., 1995), our results showed no such complication. The fact that the actual behavioral traces (Figure 16) showed similar pressing pattern and decreases in time to reach final breaking point on the Sulpiride injection day compared to the 3 day baseline suggests that the abrupt cessation of responding on the Sulpiride treatment day was purely due to attenuations in motivation. Furthermore, by looking at our baseline traces with no Sulpiride treatment (Figure 16) the responding also plateaued in the latter half of the session. Since there was no antagonist given, the cessation of responding here must be a reflection of the maximal effort the animal was willing to put in. The trace on the Sulpiride injection day did not show a uniform decrease or cessation of responding, instead, there was still gradual increase in responding in the initial stage of the session followed by a gradual tapering off of responding, all achieved at lower cumulative lever presses. Overall, our results suggest that the significance of the D2 dopamine receptor in mediating reward (i.e. sucrose or cocaine) rely greatly on its localization in the brain. Since the D2 dopamine receptor has been shown to modulate the reinstatement of cocaine seeking in the NAc shell by Sulpiride (Anderson et al., 2006; Schmidt and Pierce, 2006), and in the basolateral amygdala (Berglind et al., 2006), our results in the BST may well serve to be the first experiment to show that the D2-like dopamine receptor in the BST plays a role in mediating cocaine seeking behaviour in rats. 69

81 4.4 - D2-like Agonist (Quinpirole) Has No Effect on Sucrose- And Cocaine Self- Administration Pre-treatment of the sucrose self-administration group with the D2-like agonist, Quinpirole, had no effect on both the breaking point and the cumulative number of lever presses. As shown on the behavioral trace (Figure 18), responding on the Quinpirole injection day persisted for the whole duration of the drug taking session and the general pattern of self-administration was identical to the 3-day baseline traces. A comparison between the saline treated group with the Quinpirole treated group revealed no statistical difference and significance. As for the cocaine self-administration group, Quinpirole resulted in decreases in lever pressing and breaking point. However, no statistical significance was found between the saline and Quinpirole treated groups. A possible explanation for this effect could be the dosage of Quinpirole used was not enough to modulate the psychostimulant effect elicited by the amount of cocaine ingested during the experiment. As it was shown previously that Quinpirole (0.3 to 3.0 µg) administered into the NAc increased locomotor activity (Dreher and Jackson, 1989), reinstated cocaine self-administration behaviour (Khroyan et al., 2000), and reduced electrically evoked dopamine release in slice preparation studies (Yamada et al., 1994), we would have expected to see Quinpirole to alter the cocaine-maintained responding by changing the cocaine-evoked dopamine release in the BST. However, our results could have occurred due to Quinpirole s D3-preferring nature as a D2/D3 receptor agonist (Vallone et al., 2000). Therefore, it is probable that Quinpirole could produce a biphasic effect of cocaine-reinforced responding through its dose-related differential action at the various dopamine receptor subtypes. At the low dose (up to 3.0 µg- ) of Quinpirole, D3 receptors 70

82 might have been more involved, whereas at the higher doses (3.0 to 10 µg), the D2 receptors might have been activated as well. At the concentration used in our study (3.0 µg/µl), however, both the D2 and D3 receptors could have been involved to produce the response seen in both of our sucrose and cocaine rats Implications of Current Finding In this study, the use of different pharmacological agents and schedules of reinforcement served to facilitate our understanding of the relative contributions of dopamine receptor subtypes to different reinforcing behaviours within the BST. It should be noted that our approach to combine FR-1 and PR schedules of reinforcement was well chosen. Firstly, FR-1 schedule enabled the animal to rapidly acquire robust associations of the contextual cues in the operant chamber (i.e. cue light, time-out period, lever retraction / protrusion) with the acquisition of a reward. Following this, under the PR schedule, the effect on the reinforcing properties of the rewards after the administration of various dopaminergic agents was quantified. Since the BP under PR schedule was sensitive to various pharmacological (i.e. dose, injection speed, pretreatment of dopaminergic agents intracranially) and non-pharmacological manipulations (i.e. environmental cues, food restriction) (Stoops, 2008; Arnold and Roberts, 1997), this schedule is ideal for studying and evaluating the reinforcing effects of rewarding compounds. The FR and PR schedule serve to measure different aspects of dopamine receptor functioning within the central nervous system (McGregor and Roberts, 1993). Specifically, the changes in the rate of drug self-administration under the FR schedule are not as sensitive or predictive to alternations in drug reinforcement efficacy as assessed by the PR schedule (Arnold and Roberts, 1997; McGregor et al., 1994). In addition, 71

83 depending on the method and the area of manipulation, the inverse relationship between rate and BP does not always hold true. In addition, under our experimental condition, cocaine served as a far more potent reinforcer than sucrose. Consequently, the reinforcing efficacy of cocaine was assessed under PR schedule. This is because it was shown previously that highly preferred stimuli (i.e. cocaine) were usually the more effective reinforcers under high schedule requirements, whereas both the highly and the less preferred stimuli (i.e. sucrose) were equally effective under low schedule requirements (Penrod et al., 2008). Another parameter that served to eliminate the possibility that the effects obtained from the current study were partially due to impairment in motor functioning was inter-reinforcer time (i.e. inter-reinforcer time refers to the time in between each successive reward). Despite studies showing the contribution of dopamine modulators on motor functioning (Neisewander et al., 1995), by looking at the time between each reward (represented by ticks on our behavioral traces), it is apparent that in some cases, this time interval was shortened by the dopamine agent administered. If motor deficits occurred, we would have seen a lengthening of this time interval. Lastly, by looking at the 3-day post injection traces, we can conclude that there was no cumulative carrying over effects of the dopamine agents that could potentially influence self-administration responding after the treatment. Since the post-treatment traces showed rebounds of the responding back to baseline on the first day after injection day (i.e. pressing pattern before treatment), the effects of the dopamine agents lasted for only one day. Conditioned place preference (CPP), another approach that is commonly used to evaluate the reinforcing properties of drugs, scrutinizes very different aspects of drug 72

84 addiction. It appears that the CPP specifically examines the association between environmental cues and the additive properties of the drug. With sufficient training, it was shown that dopamine agonists were able to modulate the length of time spent in environments paired with either aversive or preferred stimuli (Hoffman and Beninger, 1988; Graham et al., 2007; Hoffman et al., 1988). However, for the purpose of this study, it is more logical and beneficial to use the self-administration paradigm. This is because in order for us to elucidate the contribution of the BST dopamine receptor in the reinforcing efficacy of the sucrose and cocaine reinforcer, we need to keep track of the alternations of the extracellular dopamine concentration before and after dopamine receptor modulation. In the self-administration approach, studies have shown that dopamine concentrations in the NAc change at different points of the drug taking behaviour. For both sucrose and cocaine self-administration, there is an increase in dopamine concentration before the lever presses, which coincided with the initiation of drug seeking behaviours. After the lever presses when sucrose was delivered and consumed, there is no further increase in dopamine (Roitman et al., 2004). For cocaine, there are still increases in dopamine after the lever presses with the concurrent presentation of cocaine related cues (Phillips et al., 2003). On the other hand, however, under the CPP approach, there is no discrete timing of dopaminergic signals during contextual conditioning and expression of conditioned behaviour (Graham et al., 2007). Therefore, the animal model of self-administration under PR schedule of reinforcement enabled us to elucidate the potential dopaminergic contribution and mechanisms within the BST through quantified behavioral responses. 73

85 Since the BST is surrounded by dopamine rich areas both laterally (globus pallidus/caudate putamen) and cranially (NAc shell), we produced brain cannulation brain slices to support the fact that the effects seen in our study was not due to dopamine diffusion from adjacent areas, but was due to changes in the BST dopamine. Figure 20 and figure 22 show the BST cannulation placements for sucrose self-administering rats, and figure 21 and figure 23 show the BST brain placements for the cocaine selfadministering rats. Although there were variations in the accuracy of the placement of the cannula injectors into the BST region, the overall results showed that the approximate localization of the injection was in the BST and there was minimal diffusion into the lateral ventricle Conclusion In summary, the major findings in this study are: 1) cocaine operant responding decreased with blockade of both D1- and D2- dopamine receptors, 2) sucrose operant responding did not show significant change with either blockade or activation of dopamine receptors. Our results have partially confirmed our hypothesis that pharmacological manipulation of dopamine receptors influences the reinforcing efficacy of natural (sucrose) or pharmacological (cocaine) rewards. This implies that manipulation of the dopamine receptors in the BST contributes to the associative pairing of pharmacological rewards to operant behaviour. This observation is also supported by other work from the Dumont lab. Behaviorally, bilateral administration of the BP897 (a selective D3 partial agonist) into the BST produced a significant reduction in the final ratio of operant response in cocaine rats and no effect in sucrose rats. In addition, electrophysiological data in our lab have shown that rats with chronic cocaine treatment 74

86 exhibited an upregulation of D1 receptor, accompanied by a change from D2-mediated pre-synaptic decrease (in normal rats) to post-synaptic D1 mediated increase in GABAergic neurotransmission. Taken together, it was implicated that chronic cocaine treatment was probably ensued by the BST undergoing changes in synaptic strength (Dumont et al., 2005; Weitlauf et al., 2004). This is due to dopamine s critical contribution in increasing the strength of neuronal connections at the synaptic level within the reward pathways of the brain in performing reward related behaviours (Hyman et al., 2006; Dong et al., 2004; Winder et al., 2002; Dumont et al., 2005). Collectively, these results highlighted the importance of BST dopamine receptor mediation in the maintenance of cocaine addiction. Specifically, the effect of both D1 and D2 receptor antagonism on cocaine reinforcement seemed to have affected self-administration behaviour throughout the whole session. With the incomplete inhibition of behaviours shown in this study, the BST may very well be just a part of the complex reward system with various selective and non-selective antagonists and agonists working with several reinforcing agents. However, with the BST s strategic placement in the brain, this research serves to generate knowledge that will serve as key for developing treatments for drug addiction. 75

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96 Appendix: I (Behavioural traces showing the effects of dopamine antagonists and agonist pretreatment in all the rats) **Dashed black trace = behavioural effect for drug treatment day **Red regular trace = lever pressing for days before actual drug treatment day** Sucrose Rats (Operant control): L111: 85

97 March 10 (SCH1.6): only has one day preinjection because there was decreased trend of breakpoint to injection day (March 7-March 8). This is to minimize drug effect due to instability of behavior in this case. 86

98 L112: Exclude Feb 10 baseline trace to remove effect of upward trend of breakpoint and make drug effect more realistic. 87

99 Exclude March 12 baseline trace to remove downward trend of breakpoint and make drug effect more realistic. L113: 88

100 L126: Exclude May 2 baseline trace to make time to reach breakpoint more uniform with other days. L135: 89

101 March 29 baseline line trace missing, but data for that day was recorded and calculated accordingly. L138: 90

102 91

103 L169: Exclude June 13 baseline trace to eliminate downward trend of breakpoint to injection date and make drug effect more realistic. 92

104 August 30 baseline line trace missing, but numerical data for that day was still recorded and calculated accordingly. L170: 93

105 94

106 L194: L196: L197: Aug 30 baseline line trace missing, but numerical data for that day was still recorded and calculated accordingly. 95

107 L198: Aug 30 baseline line trace missing, but numerical data for that day was still recorded and calculated accordingly. 96

108 Cocaine Rats: L159: Exclude June 24 baseline trace to minimize effect of downward trend of breakpoint to make drug effect more realistic. 97

109 L160: 98

110 L162: 99

111 L176: Aug 28 baseline line trace missing, but numerical data for that day was still recorded and calculated accordingly. 100

112 L188: 101

113 L189: 102

114 103

115 L191: 104

116 105

117 106

118 L207: 107

119 L213: 108

120 L215: 109