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Differential effects of the t-type calcium channel antagonist, Z944, on behaviours associated with amphetamine… Cunningham, Jonathan 2016

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    DIFFERENTIAL EFFECTS OF THE T-TYPE CALCIUM CHANNEL ANTAGONIST, Z944, ON BEHAVIOURS ASSOCIATED WITH AMPHETAMINE AND MORPHINE ADDICTION   by Jonathan Cunningham B.A. (Hons), St. Francis Xavier University, 2014  A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2016 © Jonathan Cunningham, 2016   ii Abstract  Mixed L-/T-type calcium channel antagonists attenuate morphine- and amphetamine induced conditioned place preference (CPP). Subtype specific antagonists for T-type calcium channels attenuate nicotine-reinforced behaviours in rats. This thesis investigated the effects of a novel T-type calcium channel antagonist, Z944, on the acquisition, expression, and reinstatement of amphetamine and morphine CPP. Furthermore, we examined Z944 for aversive or rewarding properties, and determined changes in locomotion with Z944 alone and in conjunction with amphetamine or morphine. CPP was induced with either morphine (5.0 mg/kg, IP) or amphetamine (1.5 mg/kg, IP) and Z944 (vehicle, 5.0, 7.5 mg/kg, IP) was administered 15 min prior to conditioning sessions for CPP acquisition experiments. For CPP expression experiments, Z944 was administered prior to the test session. In a related experiment Z944 was administered 15 min prior to the reinstatement injection of morphine after a period of extinction. Aversive and rewarding properties of Z944 were evaluated using a CPP/CPA procedure. Z944 dose-dependently attenuated the acquisition of morphine CPP, the expression of amphetamine CPP, and drug-induced reinstatement of both amphetamine and morphine CPP. Further, Z944 alone had no inherent rewarding or aversive effects, despite causing a decrease in spontaneous locomotor activity. It was also revealed that Z944 attenuated amphetamine-induced hyperlocomotion and potentiated morphine-induced hypolocomotion. These results suggest that T-type calcium channel antagonists differentially attenuate behaviours induced by several classes of drugs of abuse. iii Preface  Data were collected by Jonathan Cunningham for all CPP and locomotor experiments except where otherwise indicated. Maya Nesbit collected data on the effect of Z944 on the expression of amphetamine CPP (Experiment 2). David Montes assisted with the locomotor activity experiments with Z944 alone and Z944 in conjunction with morphine (Experiments 8, 10). Dr. Carine Dias oversaw data collection for the effect of Z944 on the acquisition of amphetamine CPP (Experiment 1). Drs. Dias and Phillips contributed to the experimental design of all experiments in this thesis. All statistical analyses were conducted by Jonathan Cunningham under the guidance of Dr. Dias. The manuscript was prepared by Jonathan Cunningham, and was reviewed and edited by Drs. Dias and Phillips. All experiments were conducted in accordance with the guidelines outlined by the Canadian Council of Animal Care and the University of British Columbia Animal Care Committee. Experiments were approved under the protocol A13-0323 iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Figures .............................................................................................................................. vii Acknowledgments ...................................................................................................................... viii 1. Introduction ......................................................................................................................... 1 1.1 The conditioned place preference model of addiction .................................................... 1 1.2  Psychopharmacology of amphetamine and morphine addiction .................................... 2 1.3  T-type channel antagonists as a treatment for drug addiction ........................................ 3 2. Materials and Methods ....................................................................................................... 6 2.1 Subjects ........................................................................................................................... 6 2.2 Drug preparation ............................................................................................................. 6 2.3  Conditioned place preference apparatus ......................................................................... 6 2.4  Amphetamine- and morphine-induced conditioned place preference ............................ 7 2.4.1 Effect of Z944 on the acquisition of amphetamine-induced conditioned place preference .............................................................................................................................. 8 2.4.2 Effect of Z944 on the expression of amphetamine-induced conditioned place preference .............................................................................................................................. 8 2.4.3 Effect of Z944 on the reinstatement of amphetamine-induced conditioned place preference .............................................................................................................................. 9  v 2.4.4 Effect of Z944 on the acquisition of morphine-induced conditioned place preference 9 2.4.5 Effect of Z944 on the expression of morphine-induced conditioned place preference 9 2.4.6 Effect of Z944 on the reinstatement of morphine-induced conditioned place preference ............................................................................................................................ 10 2.5  Measure of the intrinsic motivational effect of Z944 .................................................. 10 2.6  Locomotor Activity ...................................................................................................... 10 2.6.1 Effect of Z944 on amphetamine-induced locomotor activity .................................... 11 2.6.2 Effect of Z944 on morphine-induced locomotor activity ........................................... 11 2.6.3 Effect of Z944 on locomotor activity ......................................................................... 11 2.7 Statistical Analysis ........................................................................................................ 11 3. Results ................................................................................................................................ 13 3.1  Effect of Z944 on amphetamine-induced CPP ............................................................ 13 3.1.1 Experiment 1 – Effect of Z944 on acquisition of amphetamine CPP ........................ 13 3.1.2 Experiment 2 – Effect of Z944 on the expression of amphetamine CPP ................... 14 3.1.3 Experiment 3 – Effect of Z944 on amphetamine-induced reinstatement of amphetamine-induced CPP ................................................................................................. 15 3.2  Effect of Z944 on morphine-induced CPP.................................................................... 16 3.2.1 Experiment 4 – Effect of Z944 on the acquisition of Morphine CPP ........................ 16 3.2.2 Experiment 5 – Effect of Z944 on expression of morphine CPP ............................... 17 3.2.3 Experiment 6 - Effect of Z944 on morphine-induced reinstatement of morphine-induced CPP ........................................................................................................................ 18 3.3 Intrinsic motivational properties of Z944 ..................................................................... 19 3.4 Effect of Z944 on locomotor activity............................................................................ 20  vi 3.4.1 Experiment 8 – Effect of Z944 on amphetamine-induced locomotor activity ........... 20 3.3.2 Experiment 9 – Effect of Z944 on morphine-induced locomotor activity ................. 21 3.4.3 Experiment 10 – Effect of Z944 on locomotor activity ............................................. 23 4. Discussion ........................................................................................................................... 25 4.1 Neurochemical implications of Z944 effects on amphetamine and morphine conditioned place preference.................................................................................................... 26 4.2 Locomotor effects ......................................................................................................... 33 4.3 Speculative mechanism of Z944’s observed effects on addictive behaviours .............. 34 5. Conclusion .......................................................................................................................... 36 References .................................................................................................................................... 38   vii  List of Figures Figure 1. Effect of Z944 on the acquisition of amphetamine-induced conditioned place preference .......................................................................................................................................13 Figure 2. Effect of Z944 on the expression of amphetamine-induced conditioned place preference .......................................................................................................................................14 Figure 3. Effect of Z944 on amphetamine-induced reinstatement of amphetamine conditioned place preference .............................................................................................................................16 Figure 4. Effect of Z944 on the acquisition of morphine conditioned place preference ..............17 Figure 5. Effect of Z944 on the expression of morphine conditioned place preference ...............18 Figure 6. Effect of Z944 on the morphine-induced reinstatement of morphine conditioned place preference .......................................................................................................................................19 Figure 7. Effect of Z944-induced conditioned place preference ..................................................20 Figure 8. Effect of Z944 on amphetamine-induced locomotor activity ........................................21 Figure 9. Effect of Z944 on morphine-induced locomotor activity ..............................................22 Figure 10. Effect of Z944 on spontaneous locomotor activity .....................................................23   viii Acknowledgments  Professor Anthony Phillips has been an invaluable supervisor during my tenure at UBC, providing didactic feedback and unwavering support. Dr. Carine Dias deserves an unprecedented ovation for her guidance in designing experiments and her expertise in behavioural neuroscience. Her commitment to science and to her students is greatly appreciated and I wish her the best of luck in her future career endeavors.  My colleagues in the Centre of Disease Modeling provided a productive and fun work-environment. In this regard I would like to thank Dr. Kris Martens, Dr. Cole Vonder Haar, and Jacqueline-Marie Ferland for their expertise in behavioural neuroscience; and the staff at CDM, in particular Brian Ryomoto, Carlos Barron, and Kelly Ryan. Additionally, I would like to thank Maya Nesbit who is one of the most kind, caring, and thoughtful individuals I have had the pleasure of working beside. Her intelligence and keen mind was an invaluable asset in interpreting our results in the lab. I extend a warm thank you and well wishes to Maya in her future career.  Lastly, I would like to thank all those who have indirectly supported me throughout my academic career. My family has always provided a strong sense of encouragement and support when at times graduate school became overwhelming. Most of all, I would like to thank the love of my life and wife, Keely. You have made me a better person, have strengthened my commitment to science, and made every day a ray of sunshine with your smile. We took a commitment to learning and that has meant two very long years apart, but the best of our lives is just now before us. 1 1. Introduction 1.1 The conditioned place preference model of addiction  Human drug addiction is a chronic disorder characterized by a cycle of reoccurring behaviours such as drug use, abstinence, and relapse to drug use (Koob & Volkow, 2010). Currently, there are no medications approved for human use that effectively treat drug addiction to psychostimulants, and medications for opioid addiction are intended as maintenance therapies (Kreek et al., 2012). Hence, there is a distinct need for effective pharmacotherapies for individuals addicted to psychostimulants or opioids.  Drug-mediated contextual learning is an important factor in mediating addiction as the drug-associated context contributes to the acquisition and eventual development of the habitual behaviours of addiction (Bardo & Bevins, 2000). Indeed, environmental stimuli often act as triggers for the chronic compulsion in drug seeking, and are a salient obstacle in the treatment of drug addiction (O’Brien et al., 1998). In humans that abuse drugs such as heroin, specific environmental cues induce a desire, craving, or compulsion to use drugs, and individuals addicted to drugs of abuse often report feelings of euphoria, withdrawal, or craving when exposed to these cues (O’Brien et al., 1998).  Context-dependent addictive behaviours can be modeled in rodents with the conditioned place preference (CPP) paradigm. In the CPP paradigm, repetitive pairing of a reward with a compartment having distinctive visual and olfactory stimuli that are different from those in a second environment paired with vehicle injection will induce preference for the environment associated with the initial reward (Rossi & Reid, 1976). Place preference is inferred when an animal spends more time in the environment previously associated with the reward versus the neutral context (Phillips & LePaine, 1980). Drugs of abuse such as amphetamine and morphine  2 have incentive value for the rodent (Bindra, 1974) and can be used to induce a CPP, which is used frequently to study the acquisition and expression of a preference, as well as the reinstatement of the conditioned preference following extinction (Bardo & Bevins, 2000). CPP can also be used to study the neurobiological mechanism that mediates drug reward, and to test novel pharmacotherapies for anti-addictive properties (Tzschentke, 2007). 1.2  Psychopharmacology of amphetamine and morphine addiction  The defining neurochemical characteristic of drugs of abuse is their propensity to increase efflux of the neurotransmitter dopamine (DA) in the mesocorticolimbic system (Wise, 1996). Neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and prefrontal cortex (PFC) release DA in the terminal regions and are the primary circuits that underlie the rewarding effects of drugs of abuse (Wise, 1996; Roberts et al., 1977, 1980; Lyness et al., 1979). Amphetamine increases DA efflux by preventing DA reuptake from the synapse, whereas morphine indirectly activates DA neurons by removing tonic inhibition from gamma-aminobutyric acid (GABA) interneurons in the VTA (Kelley & Berridge, 2002; Gysling & Wang, 1983; Johnson & North 1992). Additionally, both drugs of abuse increase the burst firing of DA neurons (Covey et al., 2014; Melis et al., 2000).  Distinct circuits mediate the separate phases of conditioned place preference (CPP): acquisition, expression, and reinstatement of CPP. The acquisition of conditioned place preference measures the primary rewarding properties of a drug. Research indicates that the rewarding properties of both amphetamine and morphine require the activation of DA neurons in the VTA, and subsequent efflux of DA in the NAc (Nutt et al., 2015). However, there is a body of literature that indicates glutamate neurotransmission is also necessary for morphine reward (Nutt et al., 2015).  3  In line with the observation that addiction is neither a homogenous behaviour, or neurochemically homogenous across drugs, a number of studies have attempted to attenuate addiction by modulating synaptic transmission with calcium channel antagonists. There are five distinct classes of calcium channels (L-, P-, N-, R-, and T-type) that selectively regulate Ca2+ entry into the cell and are characterized by unique biophysical properties (Cain & Snutch, 2011). Compounds that antagonize multiple classes of calcium channels can attenuate a variety of addictive behaviours including CPP induced by ethanol, amphetamine, morphine, and nicotine, along with self-administration of ethanol and morphine, and reinstatement of ethanol self-administration (Newton et al., 2008; Pucilowski et al., 1995; Kuzmin et al., 1992, 1996). Additionally, numerous DA antagonists previously investigated for anti-addictive properties also block the T-type calcium channel (Heady et al., 2001; Santi et al., 2002; Traboulsi et al., 2006; Kraus et al., 2007). In an effort to clarify the exact mechanism by which mixed and non-selective calcium antagonists attenuate addictive behaviours, more specific antagonists are being employed in preclinical addiction psychopharmacology. In keeping with this approach, this thesis will present data from experiments using a specific T-type channel antagonist.  1.3  T-type channel antagonists as a treatment for drug addiction  This particular line of inquiry was encouraged by the aforementioned studies indicating that DA antagonists with T-type antagonist activity and compounds that antagonize multiple or all calcium channels are able to attenuate addictive behaviours. Recently, the subtype specific T-type antagonist TTA-A2 was shown to attenuate the motivation to self-administer nicotine, and prevent drug- and cue-induced reinstatement of nicotine self-administration (Uslaner et al., 2010). TTA-A2 also blocks amphetamine-induced hyperlocomotion, and decreased amphetamine-induced c-fos expression in the NAc (Uslaner et al., 2012). The mechanism by  4 which T-type channel antagonists may attenuate addictive behaviours could be explained by the unique biophysical properties of the T-type channel.  The T-type channel is distinct from other calcium channels as it causes a rapid influx of Ca2+ into the cell at a relatively negative membrane potential between -80 and -60 mV (McRory et al., 2001). T-type channels also show fast inactivation and slow deactivation (Zhange et al., 2013). In this regard, T-type channels open as a result of small depolarizations near the resting membrane potential, driving the membrane towards action potential threshold (Lechleiter & Clapham, 1992). These properties indicate that T-type channels uniquely regulate the neuronal excitability and burst firing of neurons. Anatomical localization of T-type channels indicates that while they are expressed ubiquitously throughout the CNS, they are highly expressed in the VTA (Talley et al., 1999).  This thesis will present experiments that examine the effects of a novel T-type calcium antagonist, Z944, on amphetamine and morphine-induced CPP. Initial investigation with this compound indicates that it has high affinity for the T-type channel in the nano-molar range, is 50-600 times greater affinity for the T-type over other calcium channels, and attenuates T-type action by holding the channel in the inactive state (Tringham et al., 2010). Preclinical investigations with Z944 demonstrate that it is effective at attenuating the progression of seizures, improves cortical synchrony in neuropathic pain, and rescues memory deficits in genetic models of absence epilepsy (Tringham et al., 2010; Casillas-Espinosa et al., 2015; Le Blanc et al., 2016; Marks et al., 2016). Initial Phase I clinical trials also indicate that Z944 is well tolerated by humans (Lee, 2014). Based on the biophysical properties and neuronal location of T-type channels, and the ability of T-type antagonists to attenuate the phasic firing of neurons and attenuate behaviours associated with nicotine reinforcement, we hypothesize that the T-type  5 channel antagonist Z944 will attenuate both amphetamine- and morphine-induced CPP. Given that impaired locomotion can bias the CPP protocol and can be an indicator of serious side effects such as sedation we also examined whether Z944 has any locomotor effects when given alone or in combination with amphetamine or morphine.    6 2. Materials and Methods 2.1 Subjects  Male Sprague-Dawley rats (Charles River, Quebec) weighed 250g-300g at the start of experiments and were kept on a 12-hour reverse light cycle (lights on 8pm-8am), housed according to standards of the Canadian Council on Animal Care, and given an ad libitum diet of standard rat chow and fresh tap water. Upon arrival, pairs of rats were housed in a single cage and acclimatized to the colony environment for 7 days during which daily handling occurred.  All experiments followed the standards of laboratory animal care in accordance with the standards of the Canadian Council on Animal Care and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003). Experimental procedures were approved by the Committee on Animal Care, University of British Columbia. 2.2 Drug preparation  d-amphetamine (US Pharmacopeia, Rockville, MD) and morphine sulfate (Unipharm Wholesale Drugs Ltd., Richmond, BC) were dissolved in 0.9% saline. Z944 (N-[[1-[2-(tert-butylamino)-2-oxoethyl]piperidin-4-yl]methyl]-3-chloro-5-fluorobenzamide) was dissolved in saline, adjusted to a pH of 4.2 with 0.1M H2SO4, then diluted to the final volume with 0.9% saline. The final solution had a pH of 5.5. Control groups received a 0.9% saline injection (ml/kg) as a vehicle control. All drugs were administered via intraperitoneal (IP) injection. 2.3  Conditioned place preference apparatus  Conditioned place preference boxes (total 6) were designed with two large contextually distinct compartments (47.2 x 24.6 x 31.5 cm) connected by a smaller  neutral compartment (21 x 16 x 31.5 cm). One of the large compartments had grey walls with a floor made of Plexiglas  7 bars; the other had black-and-white striped walls with a metal grid floor. Both large compartments contained Aspen Chip bedding underneath the floors. The middle compartment had white walls, smooth Plexiglas floor, and no bedding. A red lamp (40 Watts) was positioned 41 cm above the middle compartment and illuminated all three compartments with no shadows. Behaviour in all three compartments was recorded with overhead digital cameras and tracked with EthoVision 3.1 (Noldus, Wageningen, The Netherlands). This method provided an accurate measure of the amount of time spent in each individual compartment. 2.4  Amphetamine- and morphine-induced conditioned place preference Rats were first habituated to the testing room in 15 min sessions for two days prior to the start of CPP protocol. During habituation, rats remained in their home cage and were placed in the procedure room with the lights and white noise activated. On the third day, a pre-conditioning session consisted of allowing the rat to explore all three compartments for 15 min in order to determine any biased preference for a compartment. These data were used to establish randomized, unbiased, counterbalanced groups prior to conditioning. Conditioned place preference was initially established by pairing amphetamine (1.5 mg/kg, IP) or morphine (5.0 mg/kg, IP) with one compartment and saline with the other compartment on alternating days (8 days total, 4 pairings each). During the conditioning session, guillotine doors confined the rats to one compartment (30 min for amphetamine CPP and 45 min in the morphine CPP).  Twenty-four hrs after the last day of conditioning, CPP expression tests were conducted and rats were allowed to explore all compartments for 15 min.  Time spent in each compartment was used to assess preference for the compartment associated with drug-reward.   8 After the CPP acquisition test, a sub-set of experiments continued into an extinction phase where a rat’s time spent in each compartment was measured each day for 15 min until the rats no longer displayed a preference for the drug-paired compartment. The extinction criterion was reached when a rat no longer spent more time in the drug-associated compartment for 2 consecutive days. After CPP was extinguished, a drug-induced reinstatement procedure was performed in which a single injection of the drug used during conditioning was administered. Preference for drug- or saline-associated compartments was assessed during a 15 min test session. Rats that did not show CPP during the initial expression test prior to extinction (Experinemt 3, n=2; Experiment 6, n=7) were eliminated from the study. Six separate experiments examined the effects of Z944 on the acquisition, expression, and drug-induced reinstatement of either amphetamine or morphine CPP. 2.4.1 Effect of Z944 on the acquisition of amphetamine-induced conditioned place preference To assess the effects of Z944 on the acquisition of amphetamine CPP, rats (n=31) were injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior to the injection of amphetamine during conditioning. All rats were administered vehicle 15 min prior to saline on the saline conditioning days. Twenty-four hrs after the end of conditioning, rats were tested for CPP. 2.4.2 Effect of Z944 on the expression of amphetamine-induced conditioned place preference In Experiment 2, the rats (n= 28) were initially conditioned as described in 2.4 and the effect of Z944 on the expression of amphetamine CPP was assessed. Specifically, rats were injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior to the expression session. No  9 amphetamine was given on this day. The time spent in each compartment was recorded for 15 min.  2.4.3 Effect of Z944 on the reinstatement of amphetamine-induced conditioned place preference Experiment 3 determined the effects of Z944 on amphetamine-induced reinstatement of amphetamine CPP. After conditioning, rats (n=38) went through an extinction phase, which lasted no more than 21 days. On the reinstatement day, Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) was administered 15 min prior to an amphetamine injection (1.5 mg/kg, IP) and rats were placed into the CPP apparatus for 15 min and time spent in each compartment was recorded. 2.4.4 Effect of Z944 on the acquisition of morphine-induced conditioned place preference In Experiment 4, rats (n=40) received Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior to the injection of morphine (5.0 mg/kg, IP) during the conditioning phase. Twenty-four hrs after the last conditioning session all rats were tested for CPP during a 15 min test session. No morphine was administered on this day. 2.4.5 Effect of Z944 on the expression of morphine-induced conditioned place preference In Experiment 5, animals (n=41) were conditioned with morphine during the conditioning phase as described in 2.4. Twenty-four hrs following completion of the conditioning phase, animals received Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior being placed in the CPP apparatus. The CPP session lasted 15 min.   10 2.4.6 Effect of Z944 on the reinstatement of morphine-induced conditioned place preference In experiment 6, after completing the conditioning phase with morphine, rats (n=33) underwent daily extinction sessions for no more than 21 days. On the reinstatement day, rats received Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior to an injection of morphine (5.0 mg/kg, IP). The rats explored the apparatus for 15 min and the time spent in each compartment was recorded. 2.5  Measure of the intrinsic motivational effect of Z944  A separate experiment (Experiment 7) was conducted to assess whether Z944 is inherently rewarding or aversive. As described in 2.4, rats (n=46) were given a preconditioning test to determine any biased preference for either compartment and rats were assigned to groups based on these data. During the conditioning phase, rats were injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) immediately before being placed and isolated in one of the compartments for 30 min; all rats were injected with vehicle on the alternating conditioning days (8 days total, 4 pairings each). Similar to other CPP experiments, 24 hrs after the last conditioning day rats were allowed to explore all compartments freely for 15 min and time spent in each compartment was recorded. 2.6  Locomotor Activity Locomotor activity can be measured by assessing horizontal movement. Separate experiments examined the locomotor activity induced by Z944 itself, as well as the effect of Z944 when administered in conjunction with morphine (5.0 mg/kg, IP), or amphetamine (1.5 mg/kg, IP). Locomotor activity boxes were constructed of black Plexiglas (40 x 40 x 40 cm) and illuminated by red ribbon lighting on the ceiling. Each box (total 16) was filled with 5 cm of  11 CareFresh bedding. Overhead digital cameras recorded behaviour and locomotion was tracked with Noldus EthoVision 3.1.  2.6.1 Effect of Z944 on amphetamine-induced locomotor activity Experiment 8 assessed the effect of Z944 on amphetamine-induced hyperlocomotion. Rats (n=32) were first given a 30 min habituation session, then injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 min prior to an injection of amphetamine (1.5 mg/kg, IP) or saline. Horizontal activity was recorded for 3 hrs and analyzed in 10 min bins.  2.6.2 Effect of Z944 on morphine-induced locomotor activity Experiment 9 examined the effects of Z944 on morphine-induced locomotor activity. Rats (n=31) were injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 prior to an injection of morphine (5.0 mg/kg) or saline, and then immediately placed in the testing box. Horizontal activity was recorded for 3 hrs and analyzed in 10 min bins. 2.6.3 Effect of Z944 on locomotor activity Finally in Experiment 10, rats (n=24) were injected with Z944 (vehicle, 5.0, or 7.5 mg/kg, IP) 15 prior to being placed in a locomotor activity box for 2 hrs. Horizontal activity was recorded and analyzed in 10 min bins. 2.7 Statistical Analysis Behavioural data from the CPP experiments (Experiments 1-7) were analyzed using a two-way analysis of variance (ANOVA) with treatment as the between-subjects factor and compartment as the within-subjects factor. The dependent variable examined in these experiments was time spent in the drug-associated compartment versus the vehicle-associated compartment. Where appropriate, post hoc analysis (Fishers LSD) was used to determine if there is a conditioned preference as indicated by significantly greater time spent in drug-associated  12 compartment. Two-way ANOVA with treatment as the between-subjects factor and bin as the within-subjects factor was used to analyze the data from locomotor activity experiments (Experiments 8-10) across the entire testing session in 10 min bins, and where appropriate post hoc analysis followed (Fishers LSD). Data from Experiments 8-10 were also combined into 30 or 60 min bins and subsequently analyzed with one-way ANOVA followed by post hoc analysis (Fishers LSD). Differences were considered significant if p<0.05.     13 3. Results 3.1  Effect of Z944 on amphetamine-induced CPP 3.1.1 Experiment 1 – Effect of Z944 on acquisition of amphetamine CPP In Experiment 1, rats were administered Z944 15 min prior to the injection of amphetamine during the conditioning period. The time spent in each compartment 24 hrs after the 8th conditioning session is presented in Fig. 1. A two-way ANOVA revealed a significant compartment effect (F(1,56)=59.20, p <0.0001) indicating that each group preferred the amphetamine-associated chamber over the saline-associated chamber. Furthermore, no significant group X compartment interaction (F(2, 56)=0.82, p<0.45) was observed. Thus, Z944 had no effect on the acquisition of amphetamine CPP.   Figure 1. Effect of Z944 on the acquistion of amphetamine-induced conditioned place preference. Animals received saline (n=8), 5.0 mg/kg (n=15), or 7.5 mg/kg (n=8) of Z944 15 min prior to amphetamine (1.5 mg/kg) during conditioning. Data represents group means (+SEM) of time spent in the amphetamine and saline-associated compartments on the test day 24 hrs after conditioning. Significant difference between amphetamine and saline compartment: ***p<0.001.    14  3.1.2 Experiment 2 – Effect of Z944 on the expression of amphetamine CPP Experiment 2 assessed the effects of an acute injection of Z944 given 15 min prior to the expression of CPP on the post-conditioning test day. Group means for time spent in each compartment is presented in Fig 2. Two-way ANOVA revealed a significant group X compartment interaction (F(2,50)=3.40, p<0.042) and post-hoc analysis revealed that there was a significant difference between the amphetamine and saline-associated compartments in both the vehicle and 5.0 mg/kg Z944 groups, but not the 7.5 mg/kg Z944 group. Therefore, an acute injection of Z944 at 7.5 mg/kg blocked the expression of amphetamine-induced CPP.  Figure 2. Effect of Z944 on the expression of amphetamine-induced conditioned place preference. Animals were injected with vehicle (n=9), 5.0 mg/kg (n=10), or 7.5 mg/kg (n=9) of Z944 15 min prior to the testing session, which followed 24 hrs after the last conditioning day. Data represents group means (+SEM) of time spent in the amphetamine- and saline-associated compartment on the test day 24 hrs after conditioning. Significant difference between amphetamine and saline compartment: *p<0.05.   15 3.1.3 Experiment 3 – Effect of Z944 on amphetamine-induced reinstatement of amphetamine-induced CPP Data presented in Fig 3. represent time spent in the amphetamine- and saline-associated compartments during each phase of the experiment. Expression represents the time spent in each compartment on the post-conditioning test day for all rats in the experiment. Similarly, Extinction shows the average time spent in each compartment for all rats across the last two days of extinction. Data from the reinstatement session are presented as time spent in the saline- and drug-associated compartments based on the treatment received 15 min prior to the priming injection of amphetamine. The two-way ANOVA revealed a significant group X compartment interaction (F(2,79)=3.37, p<0.04). Post-hoc analyses revealed that rats significantly preferred the amphetamine-associated compartment on test day, and second, there was no significant difference between time spent in the amphetamine- or saline-associated compartment at the end of extinction. Additionally, rats that received either vehicle or 5.0 mg/kg of Z944 prior to the priming dose of d-amphetamine spent significantly more time in the amphetamine-associated compartment. However, no significant difference was observed between the time spent in the amphetamine- versus saline-associated compartment in the group given that 7.5 mg/kg of Z944. Therefore, high doses of Z944 administered 15 min prior to a priming injection blocked the reinstatement of drug-induced amphetamine CPP.  16  Figure 3. Effect of Z944 on amphetamine-induced reinstatement of amphetamine conditioned place preference. All animals showed strong preference for the amphetamine compartment on test day (Expression) after 8 days of conditioning, and this preference was eliminated after no more than 21 consecutive daily extinction sessions (Extinction). Twenty-four hrs after the last extinction session, rats were administered vehicle (n=16), 5.0 mg/kg (n=11), or 7.5 mg/kg (n=11) 15 min prior to an injection of amphetamine (1.5 mg/kg) and subsequently place in the apparatus. Data represent the group means (+SEM) of time spent in each compartment during the 15 min sessions. Significant difference between amphetamine and saline compartment: **p<0.01, ***p<0.001.  3.2  Effect of Z944 on morphine-induced CPP 3.2.1 Experiment 4 – Effect of Z944 on the acquisition of Morphine CPP Experiment 4 assessed the effects of Z944 (5.0, or 7.5 mg/kg) or vehicle administered 15 min prior to the injection of morphine (5.0 mg/kg, IP) during the CPP conditioning phase. Time spent in each compartment on the test day 24 hrs after conditioning are presented in Fig. 4. A two-way ANOVA revealed a significant group X compartment interaction (F(2,74)=4.00, p<0.03). Subsequent post-hoc analyses revealed that rats pretreated with vehicle or 5.0 mg/kg of Z944 during conditioning spent more time in the morphine-associated compartment compared to the saline-associated compartment on the test day However, rats pretreated with 7.5 mg/kg did  17 not show a significant difference between time spent in each compartment. Therefore, Z944 7.5 mg/kg administered during conditioning blocked the acquisition of morphine CPP.   Figure 4. Effect of Z944 on the acquisition of morphine conditioned place preference. Animals received vehicle (n=12), 5 mg/kg (n=15), or 7.5 mg/kg (n=13) of Z944 15 min prior to morphine (5 mg/kg) during conditioning. Data represents group means (+SEM) of time spent in the morphine- and saline-associated compartment on the test day 24 hrs after the conditioning phase. Significant difference between morphine and saline compartment: ***p<0.001.  3.2.2 Experiment 5 – Effect of Z944 on expression of morphine CPP Experiment 5, assessed the effect of acute administration of Z944 given prior to the test day on the expression of morphine CPP. Time spent in each compartment on test day is presented in Fig. 5. A two-way ANOVA revealed a significant group X compartment interaction (F(2,76)=3.94, p<0.024). Post-hoc analyses revealed that all treatment groups spent significantly more time in the morphine-associated compartment compared to the saline compartment. Therefore, Z944 did not block the expression of morphine CPP.   18  Figure 5. Effect of Z944 on the expression of morphine conditioned place preference. Animals were injected with vehicle (n=10), 5 mg/kg (n=16), or 7.5 mg/kg (n=15) of Z944 15 min prior to the testing session, which followed 24 hrs after the last conditioning day. Data represents group means (+SEM) for time spent in the morphine- and saline-associated compartment. Significant difference between morphine and saline compartment: **p<0.01, ***p<0.001.  3.2.3 Experiment 6 - Effect of Z944 on morphine-induced reinstatement of morphine-induced CPP Experiment 6 assessed the effect of an acute injection of Z944 on the morphine-primed reinstatement of morphine-induced CPP. Data in Fig. 6 represent group means for time spent in each compartment during the three phases of this experiment: 1) on the post-conditioning test day (Expression), 2) mean time during the last two days of extinction (Extinction), and 3) from the three treatment groups during the 15 min drug-prime induced reinstatement session (Vehicle, 5.0, 7.5 mg/kg of Z944). The two-way ANOVA significant main effect of compartment (F(1,60)=7.52, p<0.008). Post hoc analysis revealed that all rats showed a significant preference for the morphine-associated compartment on test day (Expression). Additionally, there was no significant difference between the compartments when averaged across the last two days of extinction (Extinction). Post hoc analysis also revealed that rats that received vehicle 15 min  19 prior to a priming injection of morphine spent significantly more time in the morphine-associated compartment, but there was no significant difference between compartments in rats that received 5.0 or 7.5 mg/kg of Z944. Therefore, an acute injection of 5.0 or 7.5 mg/kg Z944 blocked the morphine-primed reinstatement of morphine-induced CPP.  Figure 6. Effect of Z944 on the morphine-induced reinstatement of morphine conditioned place preference. All animals and were tested for time spent in each compartment 24 hrs after the last conditioning day (Expression). Subsequent daily extinction sessions occurred until the time spent in each compartment was comparable across two days (Extinction). During the reinstatement day, vehicle (n=11), 5.0 mg/kg (n=9), or 7.5 mg/kg (n=13) of Z944 was administered 15 min prior to a priming injection of morphine (5 mg/kg) and time spent in each compartment was recorded for 15 min. Bars represent the average of time spent in each compartment (+SEM) during the three phases of the experiment. Significant difference between morphine and saline compartment: *p<0.05, ***p<0.001.  3.3 Intrinsic motivational properties of Z944 Experiment 7 assessed whether Z944 possess any inherent rewarding or aversive properties. Data presented in Fig. 7 shows the time spent each compartment associated with either Z944 or saline during conditioning. A two-way ANOVA revealed no significant group X compartment interaction (F(2,86)=0.67, p>0.05) or main effect (F(1,86)=1.31, p>0.05), indicating that  Z944 did not induce a conditioned place preference or aversion.  20  Figure 7. Effect of Z944-induced conditioned place preference. Animals were assigned to three groups to received either vehicle (n=15), 5.0 mg/kg (n=15), or 7.5 mg/kg (n=16) in one compartment and saline in the other compartment for 8 days, 4 pairings each. Bars (+SEM) represent time spent in each compartment on the test day, 24 hrs after the last conditioning day. There are no significant differences between time spent in each compartment.  3.4 Effect of Z944 on locomotor activity 3.4.1 Experiment 8 – Effect of Z944 on amphetamine-induced locomotor activity Experiment 8 assessed the effects of Z944 on amphetamine-induced locomotor activity. Vehicle or Z944 (5.0, 7.5 mg/kg, IP) was administered 15 min prior to an injection of saline or amphetamine (1.5 mg/kg, IP) and rats were allowed to explore the open field for 180 min. Data in Fig. 8A represent average distance traveled (+SEM) during 10 min intervals across the 180 min test, or total distance moved for the first, second, or third 60 min period (Fig. 8B-D) The two-way ANOVA on locomotor activity over the full 180 min revealed a significant group X bin interaction (Fig. 8A; F(51,476)=3.96, p<0.001). Subsequent pot-hoc tests revealed that rats administered both Z944 (5.0, or 7.5 mg/kg, IP) and amphetamine moved significantly less than rats that received only amphetamine, but moved significantly more than rats that only received saline. This effect was most pronounced in the first 60 min, but is reflected in all three hrs of testing (Fig. 8B-D)   21  Figure 8. Effect of Z944 on amphetamine-induced locomotor activity. Rats were randomly assigned to receive vehicle or Z944 (5.0, 7.5 mg/kg) 15 min prior to an injection of saline or amphetamine, and then allowed to explore an open field box for 180 min (n=8 per group). Symbols on the line graph (A) represent average distance moved (+SEM) per 10 min bin based on treatment group after the second injection. Data in the bar graphs represent total distance moved (+SEM) during the first 60 min (B), the second 60 min (C), and the last 60 min (D). * and + in line graph represent significant differences compared to the Vehicle-Saline group and Vehicle-Amphetamine group respectively, p<0.05.  3.3.2 Experiment 9 – Effect of Z944 on morphine-induced locomotor activity Data in Fig. 9 represents average distance traveled (+SEM) during 10 min sections across the 180 min test (Fig. 9A), or total distance moved for the first, second, or third 60 min periods (Fig. 9B-D). A two-way ANOVA using data presented in Fig. 10A revealed a significant group X time bin interaction (F(51,476)=7.01, p<0.001). Post-hoc analysis revealed that co-administration of Z944 and morphine significantly potentiated hypolocomotion induced by morphine alone, compared to saline alone. Additionally, rats given morphine alone, displayed  22 significantly more locomotor activity than saline controls after first 60 min of the 180 min session. This hyperlocomotive phase was delayed for rats that received 5.0 and 7.5 mg/kg of Z944 and morphine. Similar results were observed when the data were analyzed by 60 min blocks (see Fig. 9B-D).  Figure 9. Effect of Z944 on morphine-induced locomotor activity. Rats were injected with either vehicle or Z944 15 min prior to an injection of either saline or morphine, then allowed to explore an open field box for 180 min (Vehicle-Saline, n=7; Vehicle-Morphine, n=9; 5.0 mg/kg Z944-Morphine, n=8; 7.5 mg/kg Z944-Morphine, n=8). Symbols on the line graph (A) represent average distance traveled (+SEM) for the corresponding 10 min segment for each treatment group. Bar graphs represent average distance traveled (+SEM) for the first (B), second (C), and third (D) 60 min segments. * and ^ in the line graph indicated significant differences compared to the Vehicle-Saline group and Vehicle-Morphine group respectively, p<0.05.    23  3.4.3 Experiment 10 – Effect of Z944 on locomotor activity Data presented in Fig. 10 represent the average distance traveled during either 10 min bins (Fig. 10A) or during the first and last 30 min of the experiment (Fig. 10B and C). A two-way ANOVA of total locomotor activity over the 2-hr test session (Fig. 10A) revealed only a significant main effect of time (F(11,242)=26.78, p<0.0001). A one-way ANOVA followed by post hoc test on the total distance travelled during the first 30 min revealed that rats treated with 5.0 or 7.5 mg/kg of Z944 had significantly lower locomotor scores than control animals (F(2,22)=4.20, p<0.029). A one-way ANOVA followed by post hoc test on the total distance travelled during the last 30 min bin revealed that rats given a 7.5 mg/kg dose of Z944 had significantly lower locomotor scores than rats given either saline, or 5.0 mg/kg of Z944 (F(2,22)=3.73, p<0.040). Therefore, Z944 decreased horizontal locomotion in the first and last 30 min bins of the 2 hr test session.  Figure 10. Effect of Z944 on spontaneous locomotor activity. Vehicle (n=8), 5.0 mg/kg (n=8), or 7.5 mg/kg (n=8) of Z944 was injected 15 min prior to being place in an open field arena for 120 min. Points on line graph represents average distance moved (+SEM) in 10 min bins across  24 120 min (A). Bar graphs show average distance moved (+SEM) in the first 30 min of testing (B), and in the last 30 min of testing (C). Significant difference between treatments: *p<0.05.   25 4. Discussion This thesis addressed the hypothesis that the T-type calcium channel antagonist Z944 would attenuate addictive properties of both the psychostimulant drug d-amphetamine and the opiate drug morphine as assessed by the preclinical CPP model of addiction. Although Z944 administered during conditioning had no effect on the acquisition of amphetamine CPP, it did block the acquisition of morphine CPP. These data suggest that Z944 may decrease the primary rewarding effects of morphine, but not amphetamine. Conversely, acute injections of Z944 prior to a post-conditioning test session attenuated the expression of an amphetamine-induced CPP, but not a morphine-induced CPP. Given the important role of the recall of context-associated memories in the expression of CPP, one possible explanation for the present results is that Z944 may block recall of associations between environmental context and the rewarding effect of amphetamine, but not morphine. Of even greater importance, was our finding that Z944 attenuated drug-induced reinstatement of both amphetamine and morphine CPP after a period of extinction. This is the first investigation to examine the effects of a sub-type specific T-type calcium channel antagonist on morphine and amphetamine-induced CPP. These results complement other investigations which demonstrate that T-type antagonists can attenuate the motivation to self-administer nicotine, and nicotine-induced reinstatement of nicotine-seeking behaviour (Uslaner et al., 2010). Experiments in this thesis also provide additional information about other behavioural effects of Z944, including possible rewarding or aversive effects as assessed by the CPP protocol. Z944 at doses of 5.0, or 7.5 mg/kg did not induce a conditioned preference or aversion. Therefore, Z944 does not appear to have intrinsic rewarding properties and therefore would not have abuse liability. Z944 decreased horizontal movement in the first 30 minutes of an open field  26 test, indicating that either Z944 has mild sedative effects or it attenuates the effects of novelty-induced exploratory behaviour. When co-administered with amphetamine, Z944 attenuated the hyperlocomotion induced by amphetamine. Furthermore, morphine-induced hypolocomotion was potentiated by Z944 and the hypolocomotive phase was prolonged in the 7.5 mg/kg group. This effect may be attributed to an interaction between the sedative effects of morphine and Z944. This combined sedative effect of Z944 in combination morphine raises the possibility that such sedation may confound the interpretation of our observation that Z944 blocked the reinstatement of morphine CPP (Experiment 7). 4.1 Neurochemical implications of Z944 effects on amphetamine and morphine conditioned place preference Psychostimulants and opioids both increase DA neurotransmission (Harris & Baldessarini, 1973; Phillips & LePiane, 1980). Psychostimulants such as amphetamine, cocaine, and methamphetamine block the reuptake of DA from the synapse (Harris & Baldessarini, 1973). In addition, amphetamine increases DA efflux by reversing the DA reuptake transporter and stimulating further release of DA from vesicles into the cytosolic compartment (Cao et al., 2016). Accordingly, amphetamine may elevate DA efflux without an increase in the firing of the DA neuron. However, new evidence suggests that amphetamine-induced efflux is dependent on action potentials, modulated by cannabinoids (Covey et al., 2014, 2016). The relative contribution of these two mechanisms of action on reward and reinforcement requires further investigation.  The phases of CPP are analogous to separate aspects of addictive behaviour that can be differentiated based on the neurotransmitters and neural circuits. It is well established that DA  27 efflux in the NAc is necessary to induce amphetamine CPP. Pharmacological studies demonstrate that DA agonists administered systemically or injected directly into the NAc induce CPP (Khroyan et al., 1999; Abrahams et al., 1998; Houchi et al., 2005; Tzschentke, 2007; Aujla & Beninger, 2003; Gerdjikov et al., 2004, 2006), DA antagonists block the acquisition of amphetamine CPP (Spyraki et al., 1983; Bardo et al., 1999; Wu & Zhu, 1999; Garcia Horsman & Paredes, 2004), and lesion of dopaminergic cells in the NAc prevents amphetamine CPP (Sellings & Clarke, 2003). In Experiment 1, Z944 did not block the preference for the amphetamine-paired compartment when administered prior to the amphetamine conditioning sessions, which suggests that Z944 did not alter the primary rewarding properties of amphetamine, and did not reduce DA efflux in the NAc. However, other studies have demonstrated that the T-type antagonist TTA-A2 attenuates the motivational properties of nicotine as measured by a progressive ratio schedule of reinforcement in a self-administration study (Uslaner et al., 2010). This difference is likely to be dependent upon both the mechanism of action of amphetamine versus nicotine, and the differences between progressive ratio drug-delivery and CPP. The results from Experiment 1 are not surprising considering that the classic mechanism of action of amphetamine is action-potential independent (Cao et al., 2016), and as such there would be little effect on DA release by altering the firing pattern of DA neurons. Similar to acquisition, DA is also necessary for the initial expression of amphetamine CPP, even though the animal is tested in a drug-free state. Evidence shows that exposure to environments previously associated with drug delivery increase DA efflux in the absence of any drug (Di Ciano et al., 1998; Duvauchelle et al., 2000; Lin et al., 2007). Furthermore, DA antagonists block the expression of amphetamine (Liao et al., 1998; Mechanic et al., 2003; Aujlia  28 & Beninger, 2005), cocaine, and methamphetamine CPP (Adams et al., 2001; Mizoguch et al., 2004; Duarte et al., 2003; Vorel et al., 2002; Macdonald et al., 2003); and chemical lesion of DA neurons in the NAc reduces the expression of amphetamine CPP (Sellings & Clarke, 2003). Pharmacological manipulation of the expression of amphetamine CPP suggest that blocking this behaviour with Z944 would indicate a decrease in DA efflux when the animal is re-exposed to the context associated with drug delivery.  In Experiment 2, rats treated with the vehicle prior to test day consistently demonstrated a preference for the compartment previously associated with amphetamine, and a single injection of Z944 (7.5 mg/kg) blocked the expression of this behaviour. One interpretation of this result is that Z944 attenuates the initial approach behaviours that occur upon exposure to the contextual cues associated with amphetamine injection. Increases in DA during expression are necessarily dependent on increased firing of DA neurons as there are no drugs on-board to account for an increase. As such, there is a strong likelihood that concomitant with the behavioural changes, Z944 attenuated the release of DA in the NAc via altering the firing pattern of DA neurons.  Very few studies have used amphetamine to study drug-induced reinstatement of CPP. Consequently, much of the neurobiology and pharmacology of reinstatement of psychostimulant CPP is based on cocaine. Cocaine only blocks the DA reuptake transporter, causing a build-up of DA in the synaptic cleft (Ritz et al., 1987). Similar to amphetamine, it has been postulated that the phasic DA release induced by cocaine is mediated by cannabinoids (Wang et al., 2015). Drug-induced reinstatement of cocaine CPP is reliably induced by cocaine itself and other compounds that increase dopaminergic synaptic activity (Romieu et al., 2004; Graham et al., 2007). In contrast to acquisition and expression, however, DA antagonists block reinstatement when administered directly into the mPFC (Sanchez et al., 2003). This is supported by self- 29 administration studies, which suggest that the mPFC and amygdala, but not the NAc mediate cue and drug-induced reinstatement (Shaham et al., 2003; Chao & Nestler, 2004; See, 2002). Several studies also indicate that glutamate antagonists attenuate reinstatement of cocaine seeking (Maldonado et al., 2007; Kelley & Berridge, 2002; Mcgeehan & Olive, 2006). Therefore, reinstatement of psychostimulant CPP is dependent on both DA and glutamate neurotransmission, and that the mPFC and amygdala mediated this behaviour. In the reinstatement of amphetamine CPP (Experiment 3), the vehicle control group showed robust place preference induced by a priming injection of amphetamine after many days of extinction. Importantly, Z944 blocked amphetamine-induced reinstatement only when administered at 7.5 mg/kg. As such, it is interpreted that a single acute injection of Z944 may decrease the likelihood of amphetamine-seeking after a period of extinguished behaviour. These results are consistent with investigation showing TTA-A2 prevents nicotine- and cue-induced reinstatement of nicotine self-administration (Uslaner et al., 2012). Furthermore, based on the aforementioned studies on the reinstatement of psychostimulant CPP, Z944 would likely affect diverse neurotransmitter systems beyond DA in the VTA-NAc/-mPFC projections. Compared to psychostimulants, opiates have a very different behavioural and neurochemical profile. The opioid class of abused drugs includes morphine and heroin, which increase DA efflux by indirectly activating dopaminergic neurons in the VTA (Gysling & Wang, 1983; Johnson & North 1992; Phillips & LePiane, 1980). Specifically, morphine binds to µ-opioid receptors on inhibitory interneurons in the VTA, which hyperpolarizes the cell and decreases the release of the inhibitory neurotransmitter GABA. As a result of decreased GABA, DA neurons in the VTA are disinhibited, increasing firing rates and DA efflux (Melis et al., 2000; Bechara et al., 1992; Nader & van der Kooy, 1997).  30 CPP induced by opiates also differs with regard to the circuits necessary to acquire, express, or reinstate CPP behaviour. However, one similarity between amphetamine and morphine is the characterization of the VTA as the focal point of reward (see Fields & Margolis, 2015 for review). For example, morphine CPP is induced by systemic morphine, or by microinjection of morphine into the VTA or NAc shell (David & Cazala, 2000; Phillips & LePiane, 1980; Bozarth & Wise 1984; Bals-Kubik et al., 1993; Nader & van der Kooy, 1997; Castro & Berridge, 2014), and morphine is also reliably self-administered into the VTA and NAc shell (Zangen et al., 2002; Bozarth & Wise, 1981; David & Cazala, 1994). Furthermore, morphine CPP is blocked by systemic injection of DA antagonists (Manzanedo et al., 2001; Liu et al., 2003; Ashby et al., 2003; Olmstead & Franklin, 1997), by microinjection of DA antagonists into the NAc shell (Fenu et al., 2006; Zarrindast et al., 2006; Laviolette et al., 2002; Liu & Zhang, 1999) and amygdala (Rezayof et al., 2002), and genetic knockdown of the µ-opioid receptor in the VTA (Zhang et al., 2009). However, it has also been found that lesion of dopaminergic cells in the NAc shell has no effect on morphine CPP (Sellings & Clarke, 2003).  In line with observations that CPP is not affected by NAc shell lesion, morphine CPP can also occur in the absence of DA function. Studies have shown that DA depleted mice still acquire CPP (Hanasko et al., 2005), and DA antagonists (systemic or microinjected into the ventral striatum) will only block CPP in opioid-dependent, but not opioid-naïve rats (Laviolette et al., 2002). Additionally, studies have indicated that glutamate neurotransmission is a necessary component for morphine-induced increases in VTA DA neuron firing (Jalabert et al., 2011), and compounds that reduce glutamate neurotransmission block the acquisition of morphine CPP (Suzuki et al., 2000, 1999; Riberio Do Couto et al., 2004; Popik et al. 2003a, 2003b; Popik &  31 Wrobel, 2002; Tzschentke &Schmidt 1998; Nakagawa et al., 2005; Ma et al., 2006). Based on these studies, it is clear that both DA and glutamate mediate morphine reward in the NAc. Our results from Experiment 4 indicate that Z944 blocked the acquisition of morphine CPP. Therefore, it is possible that Z944 was able to block the rewarding properties of morphine. However, it is unclear whether Z944 decreased DA in the NAc, had an effect on glutamate neurotransmission, or possibly affected both neurotransmitter systems simultaneously. It is known that morphine disinhibits neuronal activity (Bechara et al., 1992), and based on the ability to T-type channels to regulate neuronal firing, blocking these channels with Z944 would likely inhibit neuronal activity. Therefore, at the superficial level, treatment with Z944 opposes the neuronal firing effects induced by morphine. However, we have little evidence to identify if this process occurs in the VTA to mitigate disinhibition, or occurs in upstream nuclei to decrease excitatory input onto the VTA.  In parallel to the acquisition of morphine CPP, both DA and glutamate neurotransmission seem to contribute to the expression of morphine CPP. Systemic injection of both DA and glutamate antagonists block the expression of morphine CPP (Ashby et al., 2003; Popik et al., 2003a; Yonghui et al., 2006; Wei et al., 2005; Popik & Kolasiewicz, 1999; Kotlinsak & Biala, 1999; Zhu et al., 2006; Popik & Worbel, 2002). Interestingly, microinjection of DA antagonists into the NAc shell have no effect, but block expression when injected into the central nucleus of the amygdala or hippocampus (Fenu et al., 2006; Rezayof et al., 2002, 2003; Zarrdinast et al., 2003). In contrast, glutamate antagonists block expression when injected into either the VTA or NAc (Popik & Kolasiewicz, 1999; Harris et al., 2004). Based on the pharmacological manipulation of the expression of morphine CPP it is clear that while DA is important, it may play a minor role compared to glutamatergic neurotransmission.  32 In Experiment 5, Z944 did not block the expression of morphine CPP, and we observed a trend to potentiated preference for the morphine compartment. In considering why this might be the case, it is important to highlight that expression of morphine CPP requires DA release in the amygdala and hippocampus, and glutamate into the NAc. Complementary evidence demonstrates that DA binding to post synaptic receptors in the central nucleus of the amygdala in anxiolytic, and decreasing DA in these areas can increase anxiety-like behaviours (see Forster et al., 2012 for review), and other studies have suggested that Z944 may increase anxiety-like behaviours (Marks et al., 2016). Based on our hypothesis, we would anticipate that Z944 would attenuate the efflux of DA into this area, thereby increasing anxiety-like behaviours. In CPP, anxiety and stress augment preference for the drug-associated compartment (Prast et al., 2012; McLaughlin et al., 2006; Haile et al., 2001; Capriles & Cancela, 1999, 2002). Therefore, it is possible that Z944 decreased DA efflux into the central amygdala, thereby increasing anxiety-like behaviours, which augmented the behavioural expression of morphine CPP. This explanation of Z944-induced anxiety in an opioid-dependent state likely does not affect other phases of morphine CPP because Z944 is not given on the test day for the acquisition experiment, and would be partially blunted by the anxiolytic effects of acute morphine in the reinstatement experiment. The neurocircuity of the reinstatement of morphine CPP relies primarily on glutamate neurotransmission (Ribeiro Do Couto et al., 2005). For example, systemic injection of DA antagonists SCH23390, raclopride, and haloperidol fail to block morphine-induced reinstatement of morphine CPP (Ribeiro Do Couto et al., 2005), but glutamate receptor antagonists MK-801, memantine, and dizocilpine do block the behaviour (Ribeiro Do Couto et al., 2005). Lesions of the VTA and NAc also block reinstatement, while mPFC lesions do not (Wang et al., 2002; Hao et al., 2008). Taken together, reinstatement of morphine CPP appears to require glutamate  33 neurotransmission in the VTA and NAc. This conclusion is supported by the finding that projections from the VTA to the mesocorticolimbic system can also release glutamate and GABA (Carr et al., 2000; Yamaguchi et al., 2011). Results from Experiment 6 indicate that Z944 is able to block morphine-induced reinstatement of morphine CPP. This would initially imply that Z944 reduces drug-induced reinstatement of morphine CPP. Indeed these results are consistent with Experiment 3 that shows that Z944 blocks amphetamine-induced reinstatement of amphetamine CPP. However, the data may be confounded by a subsequent finding that combined doses of Z944 and morphine induce significant sedative effects greater than either drug alone (Experiment 9). As such, Z944 may not have specific effects on the morphine-induced reinstatement but may be masking the approach behaviour by impairing locomotion (see below). 4.2 Locomotor effects  Our experiments also show that a single injection of amphetamine (1.5 mg/kg, IP) substantially increased locomotion, and that 5.0 and 7.5 mg/kg of Z944 attenuated the hyperlocomotion induced by this dose of amphetamine (Experiment 8). These data extend previous investigations showing that mibefradil and 2-octanol (non-specific T-type channel antagonists) attenuate cocaine-induced hyperlocomotion (Bisagno et al., 2010) and TTA-A2 attenuates amphetamine-induced hyperlocomotion (Uslaner et al., 2012).  The importance of testing the combined effects of Z944 and morphine on locomotor activity are highlighted in investigations with the GABAb agonist Baclofen. While baclofen showed initial promise in preclinical investigation of drug addiction (Brebner et al., 2005), when taken into clinical trials for cocaine addiction the drug failed due to significant sedative effects (Cryan et al., 2004; Shoptaw et al., 2003). Our results show that Z944 potentiated the  34 hypolocomotion induced by morphine, and in the high dose Z944 group, prolonged the hypolocomotion (Experiment 9). Therefore, it is possible that based on the side-effect profile of Z944 and morphine, the combination of these compounds would be ill suited for treating morphine addicts.  Finally, results from Experiment 7 demonstrate that Z944 was neither aversive nor rewarding. Furthermore, in Experiment 10 it was observed that Z944 decreased locomotion compared to saline treated rats, which is consistent with other studies characterizing Z944 on rotorod performance (Tringham et al., 2012; Casillas-Espinosa et al., 2015; LeBlanc et al., 2016). Taken together, the characterization of Z944 indicates that the doses used in this experiment are well tolerated by rats, and the observed behavioural effects are not due an aversive response to the compound. 4.3 Speculative mechanism of Z944’s observed effects on addictive behaviours Although experiments in this thesis did not address the underlying neural mechanism related to the effects of Z944 on CPP and locomotor behaviours, we offer the following conjecture. Our overall hypothesis postulated that Z944 would attenuate addictive behaviours by attenuating the phasic firing of neurons, specifically in the mesocorticolimbic system, in response to drugs of abuse.  Most of our experiments are consistent with this hypothesis. Starting with the expression experiments (Experiments 2 & 5), Z944 is postulated to reduce the firing of DA and glutamate neurons that project from the VTA and in turn decrease the amount of neurotransmitter released. It is reasonable to make this assertion because T-type calcium channels are found in the VTA (Talley et al., 1999), T-type channels are primarily responsible for a burst firing pattern of neurons (Perez-Reyes, 2003), and drugs of abuse cause burst firing of mesocorticolimbic neurons  35 (Covey et al., 2014; Melis et al., 2000). The reinstatement experiments also support the possibility that Z944 attenuates the firing of neurons, although the projections responsible for these behaviours are more wide-spread compared to expression behaviour.  While the effect of Z944 on the acquisition of morphine CPP is consistent with the general hypothesis, in that attenuating neuronal firing reduces neurotransmitter efflux and in turn the addictive behaviour, the acquisition of amphetamine CPP was not attenuated by Z944. Nevertheless, this effect may still be consistent with our hypothesis given the classic view that action-potential mediated synaptic events are not necessary for amphetamine’s effect (Harris & Baldessarini, 1973). Considering that Z944 is likely modulating the pattern of action potentials, it is likely to have little effect on amphetamine and other psychostimulants that work pre-synaptically to prevent DA reuptake. Note however, a recent proposal that amphetamine’s mechanism of action may indeed be action potential-dependent (Covey et al., 2016). As an alternate theory, Z944 might affect upstream nuclei that input onto the VTA. For example, T-type channels are expressed in the subthalamic nucleus and pedunculopontine tegmental nucleus (Talley et al., 1999), areas that project to the VTA and are involved in processing reward (Keiflin & Janak, 2015). Furthermore, lesion of the subthalamic nucleus or lesion of NMDA receptors in the pedunculopontine tegmental nucleus block cocaine CPP, and morphine and amphetamine CPP respectively (Baunes et al., 2005, Leri & Franklin, 2000). Therefore, it is possible that Z944 would alter the firing pattern of upstream projections, thus altering mesocorticolimbic function in CPP behaviours.    36 5. Conclusion  The experiments presented in this thesis support a number of broad conclusions, and may contribute to understanding a possible mechanism of action for Z944. However, future study would greatly benefit from direct measurement of neurotransmitter efflux in response to Z944 alone, and in combination with either amphetamine or morphine. Additionally, we have postulated that would alter the firing pattern of mesocorticolimbic neurons, which would also benefit from direct electrophysiological measurement in vivo. Finally, there remains the possibility that T-type channels in the VTA are not located on the projection neurons (possibly on interneurons), a hypothesis that requires cellular localization of the T-type channel using either molecular markers or an in vitro patch-clamp assay. Our results indicate that Z944 is an effective compound for attenuating the craving and relapse of amphetamine-induced contextual memories and reducing the rewarding and relapse potential of morphine-induced contextual memories. The experiments presented in this thesis support the important effects of a T-type calcium channel antagonist in attenuating additive behaviours, thereby complimenting previous findings with TTA-A2 on nicotine self-administration (Uslaner et al., 2010). Despite the fact that no mechanistic experiments were conducted, the breadth of experiments combined with past literature may help to identify the possible mechanisms by which Z944 has its effects. Hence, we hypothesize that Z944 attenuates both DA and glutamate neurotransmission by altering the firing pattern of neurons in the mesocorticolimbic system which underlie addictive behaviours. Furthermore, we propose that the effect on both neurotransmitter systems are concurrent and may be dominated by either DA or glutamate depending on the behaviour. Our results also demonstrate that Z944 attenuates preclinical models of addictive  37 behaviours induced by both amphetamine and morphine. More specifically, the experiments demonstrate that Z944 impairs a series of behaviours that rely on the association of a drug with contextual cues. In humans, these environmental cues often induce the cravings and compulsions experienced by an addict, which in turn serve as the most salient obstacles in treating this disorder (O’Brien et al., 1998). 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