Nitric Oxide-Dependent Mechanisms of Cocaine-Induced Place Preference and Mu Opioid Receptor Expression in the Nucleus Accumbens by Rachel-Karson Thériault A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Biomedical Sciences and Neuroscience Guelph, Ontario, Canada © Rachel-Karson Thériault, July, 2017 ABSTRACT THE INVESTIGATION OF NITRIC OXIDE-DEPENDENT MECHANISMS OF COCAINE-INDUCED PLACE PREFERENCE AND MU OPIOID RECEPTOR EXPRESSION Rachel-Karson Thériault Advisors: Dr. Bettina Kalisch and University of Guelph, 2017 Dr. Francesco Leri Cocaine administration increases both mu opioid receptor (MOR) expression and nitric oxide (NO) production in brain areas associated with reward. This thesis explores the effect of acute and ‘subchronic’ cocaine administration on MOR and neuronal NO synthase (nNOS) expression, and the role of NO in cocaine reward and the cocaine-mediated expression of MOR and nNOS in rats. Acute cocaine (20 mg/kg) administration increased MOR and nNOS mRNA and protein expression in the nucleus accumbens (NAc), within 72 hours. Following 4 days of conditioning, cocaine (20 mg/kg) produced a conditioned place preference (CPP) and an increase in MOR mRNA in the rat NAc. Both effects were attenuated in rats pre-treated with the nNOS inhibitor, 7-nitroindazole (7-NI) (25 mg/kg and 50 mg/kg). Taken together, these results indicate that acute cocaine administration modulates MOR and nNOS expression at the transcriptional and translational level, and that nNOS plays a role in both cocaine CPP and the cocaine-induced MOR mRNA expression. ACKNOWLEDGMENTS First and foremost, I would like to thank my advisors Dr. Bettina Kalisch and Dr. Francesco Leri for their constant support, guidance, and encouragement in pursuing a Master’s degree. Thank you for always challenging me and reminding me that patience is a virtue in research. You have both solidified my love of learning, research, and academia, and for that I will always be grateful. Thank you to all the lab members and staff without whom this work would not have been possible: Carolyn Creighton and Faisal Alibhai for their guidance with qPCR and RNA work, Thomas Horman for training me on animal/behavioural work, and Cheryl Limebeer for teaching me how to complete tissue extractions. Last but not least, a big shout out to Ari Mendell for constantly answering my endless number of questions for 3 years. I would also like to give a big thank you to all the members of the Leri lab, as well as the graduate students and faculty members in the department of Biomedical Sciences, for making this an incredible and memorable two years. To my family, friends, and cats: thank you for all your love, support, and confidence in me. Finally, I would like to acknowledge the funding provided by the Canadian Institute of Health Research (CIHR) and Ontario Veterinary College (OVC). III DECLARATION OF WORK PERFORMED I declare that all the work reported in this thesis was performed by me. IV TABLE OF CONTENTS ABSTRACT .................................................................................................................................. II ACKNOWLEDGMENTS .......................................................................................................... III DECLARATION OF WORK PERFORMED ......................................................................... IV TABLE OF CONTENTS ............................................................................................................ V LIST OF FIGURES .................................................................................................................. VII LIST OF ABBREVIATIONS ................................................................................................. VIII Introduction ................................................................................................................................... 1 Review of the Literature ............................................................................................................... 3 1.0. Drug abuse and addiction ................................................................................................. 3 1.1. Cocaine addiction ............................................................................................................. 4 1.2. The neuropharmacology of cocaine and the reward system ............................................ 6 1.3. Cocaine-induced neuroadaptations ................................................................................ 11 2.0. The endogenous opioid system ........................................................................................ 13 2.1. The endogenous opioid system and cocaine addiction .................................................. 15 2.2. Regulation of the mu opioid receptor by cocaine: Biochemical evidence ..................... 17 2.3. The role of the mu opioid receptor in cocaine addiction: Behavioural evidence .......... 20 3.0. The nitrergic system ........................................................................................................ 22 3.1. Nitric oxide signalling in the brain ................................................................................ 23 3.2. The effect of cocaine on the nitrergic system ................................................................ 25 3.3. The role nitric oxide in cocaine-motivated behaviours .................................................. 27 3.4. The link between the cocaine-induced nitric oxide efflux and the endogenous opioid system ................................................................................................................................... 30 Rationale ...................................................................................................................................... 32 Hypothesis .................................................................................................................................... 35 Objectives ..................................................................................................................................... 35 Materials and Methods ............................................................................................................... 36 Materials .................................................................................................................................. 36 Subjects .................................................................................................................................... 36 Drugs ........................................................................................................................................ 37 Testing Apparatus ................................................................................................................... 37 Activity Chambers/Conditioned Place Preference ................................................................ 37 Biochemical Assays ................................................................................................................. 38 Quantitative Polymerase Chain Reaction (qPCR) analysis .................................................. 38 Immunoblot analysis ............................................................................................................. 41 Procedures ............................................................................................................................... 44 Experiment 1: Cocaine-induced locomotor activity ............................................................. 44 Experiment 2: Effect of nNOS inhibition on the development of cocaine CPP ................... 44 Data and Statistical Analyses ................................................................................................. 45 Results .......................................................................................................................................... 46 V Experiment 1: The effect of acute cocaine administration on locomotion, and MOR and nNOS mRNA and protein expression in the NAc ................................................................ 46 Cocaine-induced locomotor activity ..................................................................................... 46 Qualitative detection and quantification of MOR and nNOS mRNA .................................. 46 MOR and nNOS protein expression ..................................................................................... 47 Experiment 2: The role of nNOS in cocaine-induced locomotion, CPP, and MOR and nNOS mRNA and protein expression in the NAc ................................................................ 48 The effect of nNOS inhibition on cocaine-induced locomotor activity ................................ 48 The effect of nNOS inhibition on the development of cocaine CPP .................................... 49 Cocaine-induced MOR and nNOS mRNA expression ......................................................... 49 Cocaine-induced MOR and nNOS protein expression ......................................................... 50 Figure Legends ............................................................................................................................ 51 Figures .......................................................................................................................................... 53 Discussion ...................................................................................................................................