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ABSTRACT Gloria E. Malpass. INTERACTION of MEMANTINE with ETHANOL CONSUMPTION and DOPAMINERGIC FUNCTION in HIGH ETHANOL PREFERR

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ABSTRACT Gloria E. Malpass. INTERACTION of MEMANTINE with ETHANOL CONSUMPTION and DOPAMINERGIC FUNCTION in HIGH ETHANOL PREFERR ABSTRACT Gloria E. Malpass. INTERACTION OF MEMANTINE WITH ETHANOL CONSUMPTION AND DOPAMINERGIC FUNCTION IN HIGH ETHANOL PREFERRING RATS. (Under the supervision of Dr. Brian A. McMillen) Department of Pharmacology and Toxicology, November 2009. N-methyl-D-aspartate (NMDA) receptor antagonists have been reported to decrease ethanol consumption in rodents, but these drugs often produce adverse side effects. Memantine is a neuroprotective and low-affinity, noncompetitive NMDA receptor antagonist shown to be an effective treatment for Alzheimer’s disease with a favorable clinical profile. This study investigated effects of memantine on volitional ethanol consumption in the Myers’ high ethanol preferring (mHEP) rat using a two-choice 24 hour access paradigm. Memantine was found to reduce ethanol consumption in an apparent dose-dependent manner. Behavioral experiments indicated that memantine, at a dose shown to decrease ethanol consumption, did not adversely affect locomotor ability and activity, induce sedation, or add to ethanol-induced hypothermia. Previously, ethanol has been shown to alter levels of dopamine metabolism (DA) in brain regions receiving DA input in the DA reward pathway. Therefore, high performance liquid chromatrography (HPLC) was used to compare levels of DA metabolism in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and striatum (STR) of rats treated with saline, memantine (10.0 mg/kg, i.p.) and/or ethanol (1.0 or 2.5 g/kg, i.p.). No significant treatment effects were detected in levels of DA or its metabolite, 3,4- dihydroxyphenylacetic acid (DOPAC). Ethanol (2.5 g/kg) increased striatal DOPAC to levels bordering on significance, but mematine clearly produced no effect. To determine if memantine alters ethanol intake via signaling downstream of activation of the DA D1 receptor, Western blots were used to compare effects of the same treatments on levels of DARPP-32, a protein implicated as an intracellular regulator in ethanol reward, and its phosphorylation at Thr34 and Thr75 sites in the mPFC, NAc, and STR. Bands for phospho-DARPP-32 (Thr34) in the mPFC were undetectable. All other blots indicated no significant treatment effects on levels of DARPP-32 or its phosphorylation at Thr34 and Thr75 sites. Together, these results suggest that mechanisms which do not involve glutamate and the NMDA receptor may also activate ethanol reward in the mHEP rat, and a mechanism not downstream of the DA D1 receptor is involved. The effect of memantine on consumption of ethanol does not involve modification of the DA/DARPP- 32 signaling system. ©Copyright 2009 Gloria Elaine Malpass INTERACTION OF MEMANTINE WITH ETHANOL CONSUMPTION AND DOPAMINERGIC FUNCTION IN HIGH ETHANOL PREFERRING RATS A Dissertation Presented To The Faculty of the Department of Pharmacology and Toxicology The Brody School of Medicine at East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Pharmacology and Toxicology by Gloria Elaine Malpass November, 2009 INTERACTION OF MEMANTINE WITH ETHANOL CONSUMPTION AND DOPAMINERGIC FUNCTION IN HIGH ETHANOL PREFERRING RATS by Gloria Elaine Malpass APPROVED BY: DIRECTOR OF DISSERTATION: _________________________________ Brian A. McMillen, Ph.D. COMMITTEE MEMBER AND: _________________________________ David A. Taylor, Ph.D. COMMITTEE MEMBER: _________________________________ M. Saeed Dar, Ph.D. COMMITTEE MEMBER: _________________________________ Kori L. Brewer, Ph.D. COMMITTEE MEMBER: _________________________________ Tuan Tran, Ph.D. CHAIR OF THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY: _________________________________ David A. Taylor, Ph.D. DEAN OF THE GRADUATE SCHOOL: _________________________________ Paul J. Gemperline, Ph.D. Acknowledgements Many people have generously provided guidance and support throughout my graduate experience. I wish to thank my thesis advisor Dr. Brian A. McMillen for providing direction and encouragement throughout this project. I am also grateful to Helen L. Williams for the assistance and support that she has kindly provided both as a laboratory technician and as a friend. Completion of this project required numerous resources ranging from technical assistance to the use of equipment and reagents from multiple laboratories. I am grateful to everyone who contributed to this project, especially Dr. David A. Taylor, Chair of the Department of Pharmacology and Toxicology, for his guidance and support. Many thanks to my parents, Benny E. and Joyce L. Malpass, and the countless family and friends who have graciously given their love, support, and prayers as I complete this chapter in my life. Above all, I thank God for His blessings. To My Parents With Love and Gratitude TABLE OF CONTENTS LIST OF FIGURES……………..…………………………………….………... ix LIST OF TABLES........................................................................................ xii LIST OF ABBREVIATIONS……………………………………………………. xiii CHAPTER 1: INTRODUCTION………..…………………………...………… 1 Literature Review............…………………………………………………. 2 Alcohol Use: A Historical Perspective........................................... 2 Alcohol Abuse and Alcohol Dependence…………………………. 3 Genetics and the Development of Alcohol Use Disorders………. 4 Costs Associated with Alcohol Abuse and Alcohol Dependence.. 5 Current Trends………………………….......................................... 5 Pharmacological Treatments for Alcohol Dependence………… 6 Disulfiram.................................................................... 7 Naltrexone................................................................... 7 Acamprosate............................................................... 8 Other Proposed Pharmacological Therapies.............. 8 Animal Models and Experimental Paradigms…………………… 9 Addiction……………………………………………………………… 11 Ethanol and Addiction………………………………………………. 11 Dopamine Receptors………………………………………….....…. 12 Mesolimbic and Mesocortical Pathways……………..…………… 13 Ethanol and the Mesolimbic Dopamine System…………….….… 16 Glutamate Receptors…………………………………..………..…... 17 NMDA Receptor as a Potential Target for the Treatment of 19 Alcohol Dependence………………………………………………… Memantine……………………………………………………………. 21 Intracellular Pathways Involved in Alcohol Addiction……………. 22 Purpose of This Study………………………………………………………. 34 CHAPTER 2: EFFECTS OF MEMANTINE ON VOLITIONAL ETHANOL 38 CONSUMPTION IN THE MALE MYERS’ HIGH ETHANOL PREFERRING RAT IN A TWO-CHOICE 24HOUR ACCESS PARADIGM……………………………………………………………………… Abstract............…………………………..………………………………... 39 Introduction………………………………………..……………………….. 40 Materials and Methods......………………………………….....…………. 44 Drugs............................................................................................ 44 Volitional Consumption of Ethanol………………………………..... 44 Subjects and Screening…………………………….….. 44 Experimental Procedures…………………………….… 45 Elevated Plus Maze Test…………………………………………..... 46 Activity Monitor……………………………………………………..… 47 Rectal Temperature………………………………………….………. 48 Results……………………………..…………………………………….…. 50 Effects of Memantine on Volitional Consumption of Ethanol……. 50 Effects of Memantine on Anxiety…………………………………… 56 Effects of Memantine on Locomotor Activity…………………...…. 59 Effects of Memantine on Rectal Temperature…………………….. 62 Discussion………………………………………………………………….. 68 CHAPTER 3: EFFECTS OF DOPAMINERGIC D1 AND D2 DRUGS ON 74 THE VOLITIONAL CONSUMPTION OF ETHANOL BY GENETIC DRINKING MHEP RATS IN A TWO-CHOICE 24 HOUR ACCESS PARADIGM…………………………………………………………………...…. Abstract……………………..……………………………………...………. 75 Introduction………………………………………………………………… 76 Materials and Methods……………………………………………………. 79 Drugs............................................................................................ 79 Subjects and Screening………………………..………………..….. 79 Experimental Procedures………………………………………..….. 80 Results…………………………………………………………………..….. 83 Discussion………………………………………………………………...... 97 CHAPTER 4: EFFECTS OF GROUP II METABOTROPIC GLUTAMATE 101 AGONIST AND ANTAGONIST DRUGS ON VOLITIONAL ETHANOL CONSUMPTION BY GENETIC DRINKING RATS……………………...….. Abstract............…………………………………………..………………... 102 Introduction………………………………………………………………… 104 Materials and Methods......…………………………...………………..…. 107 Drugs............................................................................................ 107 Volitional Ethanol Consumption Experiment……………………… 107 Subjects and Screening………………………… 107 Experimental Procedures……………………………… 108 Rotating Rod Experiment………………………………….………… 108 Results……………………………………….………………………..……. 110 Effects of LY379268 and LY341495 on Volitional Consumption 110 of Ethanol………………………………………………………...…… Effects of LY379268 and LY341495 on Locomotor Activity…….. 116 Discussion………………………………………………………………….. 121 CHAPTER 5: EFFECTS OF MEMANTINE ON DOPAMINE 124 METABOLISM AND PHOSPHORYLATION OF DARPP-32 IN THE DOPAMINERGIC REWARD PATHWAY OF MALE MYERS’ HIGH ETHANOL PREFERRING RATS……………………………………………... Abstract............………………………………………….……………..….. 125 Introduction………………………………………………………………… 127 Materials and Methods......……………………………………………….. 130 Drugs and Reagents…………………………………………………. 130 Subjects……………………………………………………………….. 130 Treatment Groups……………………………………………………. 130 Tissue Samples……………………………………………………… 131 HPLC Analysis……………………………………………………….. 131 Western Blots………………………………………………………… 132 Samples and Blotting…………………………………… 132 Antibody Procedure…………………………………….. 132 Densitometry………………..…………………………… 138 Statistical Analysis..…………………………………….. 138 Results……………………………………………………………………… 139 Effects of Ethanol and/or Memantine on
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