A Thesis Entitled Glutamate Transporter 1 and Cystine-Glutamate Antiporter as Potential Targets for Attenuating Alcohol Consumption in Male P Rats By Munaf Aal-Aaboda Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Master of Science Degree in Pharmaceutical Sciences _____________________________________ Dr. Youssef Sari, Committee Chair _____________________________________ Dr. Ezdihar Hassoun, Committee Member _____________________________________ Dr. Surya Nauli, Committee Member _____________________________________ Dr. Zahoor Shah, Committee Member _____________________________________ Dr. Patricia Komuniecki, Dean College of Graduate Studies The University of Toledo. August-2014 Copyright 2014, Munaf Aal-Aaboda This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Glutamate Transporter 1 and Cystine-Glutamate Antiporter as Potential Targets for Attenuating Alcohol Consumption in Male P Rats By Munaf Aal-Aaboda Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Master of Science Degree in Pharmaceutical Sciences The University of Toledo, OH. August-2014 Alcohol abuse is associated with dysfunction of glutamatergic system along with other neurotransmitter systems in mammalian central nervous system. Studies have shown that both behavioral effects of acutely administered alcohol and neuroadaptation associated with chronic ethanol intake are mediated by glutamatergic neurotransmission in key regions of brain reward circuitry. Extracellular glutamate level has been reported to be elevated following alcohol consumption. The role of several glutamate transporters in restoring glutamate homeostasis has been well established. Among these, glutamate transporter 1 (GLT1) and cystine-glutamate antiporter (xCT) are key players in regulating extracellular glutamate levels. Previous studies from our lab have reported that ceftriaxone, a β-lactam antibiotic known to upregulate GLT1, attenuated ethanol consumption in alcohol preferring (P) rats in chronic ethanol-drinking paradigm. This effect was associated in part with GLT1 upregulation in central brain reward regions. In the present study, we investigated the short and long lasting effects of MS-153, GLT1 activator, on ethanol intake and glutamate transporters’ expression in male P rats. We further examined the effects of MS-153 and Augmentin combination on ethanol iii consumption, body weight and water intake in P rats. P rats were exposed for five weeks to continuous free-choice ethanol drinking paradigm. On the first day of week 6, P rats were injected intraperitoneally with MS-153 (50 mg/kg), or a combination of MS-153 (50 mg/kg) and Augmentin (100 mg/kg), or vehicle for five consecutive days. Interestingly, we found a significant reduction in ethanol intake in P rats treated with MS-153 starting 24 hours after the first injection, which lasted up to ten days after the last injection as compared to vehicle-treated animals. This long lasting effect on ethanol consumption was associated with significant upregulation of GLT1 levels in the nucleus accumbens (NAc) but not in the prefrontal cortex (PFC). Additionally, one day after the last MS-153 injection, xCT levels were significantly upregulated in amygdala and hippocampus. Furthermore, treatment with combination of MS-153 and augmentin resulted in reduction of ethanol consumption as compared to vehicle-treated rats. Our study demonstrates that modulating GLT1 and xCT expression may be a promising therapeutic target for treating alcohol dependence. iv Acknowledgments First of all, I would like to express my sincere gratitude to my advisor, Dr. Youssef Sari, who was extremely helpful and offered invaluable guidance throughout this project. Without his immense knowledge and patience, this thesis would not have been possible. I would also like to thank my committee members Dr. Ezdihar Hassoun, Dr. Surya Nauli and Dr. Zahoor Shah. I thank Dr. Ezdihar Hassoun for her timely advice and encouragement. I would also like to take this opportunity to thank Dr. Surya Nauli and his lab members for their patience and assistance. I also express my greatest appreciation to Dr. Shantanu Rao, Sujan Chandra Das and Sunil Goodwani for their endless support in completion of this project. I would also like to acknowledge the financial support of Higher Committee for Education Development in Iraq (HCED). I also thank the faculty members at the Department of Pharmacology and the University of Toledo-College of Pharmacy and Pharmaceutical Sciences for their support and assistance during my Master’s degree. Last, but by no means the least, I am inordinately thankful to my friends and family for their constant support and understanding. v Contents Abstract ………………………………………………………………………….…….... iii Acknowledgments ……………………………….………………………….…..……..... v Contents ………………………………………………………………………………… vi List of Figures.….……………………….………………….……………………………. x List of Abbreviations.…….……….………..……………….…...……….…………….. xii 1. Introduction …………………………………………………………………………...1 1.1 Overview…………………………………………………………………………….1 1.2 Glutamate…………………………………………………………………………....2 1.2.1 Glutamate Transporters…………………………………………………….….4 1.2.1.1 Vesicular Glutamate Transporters………...………………………………...4 1.2.1.2 Excitatory Amino Acid Transporters…………………………….………….4 1.3 Reward Circuitry Involved in Drugs Addiction ……………………………….…....5 1.3.1 Nucleus Accumbens (NAc)…………………...………………………….…....6 1.3.2 Prefrontal Cortex (PFC)………………………………………….………...….7 1.3.3 Amygdala (AMG)……………………………………………………………..7 1.3.4 Hippocampus (Hipp)……………………………………………………….....8 1.4 Glutamate Transporters and Alcoholism……………………………………………9 1.4.1 GLT1 and Alcoholism……………………………………………………...…9 1.4.2 Cystine-glutamate Antiporter and Alcoholism……………………….……...10 vi 1.5 Glutamate Receptors and Alcoholism……………………………….…….………12 1.5.1 Ionotropic Glutamate Receptors………………………………………...…...12 1.5.1.1 NMDA Receptors and Alcoholism………………………………...………12 1.5.1.2 Non-NMDA Receptors and Alcoholism………………………………...…13 1.5.2 Metabotropic Receptors and Alcoholism…………………………...………..14 1.5.2.1 Group I Metabotropic receptors…………………………………...……….14 1.5.2.2 Group II Metabotropic receptors……………………………………..……16 1.5.2.3 Group III Metabotropic receptors……………………………………...…..16 1.6 Other Neurotransmitter Systems and alcoholism……………………………..…...17 1.7 Aims and Objectives…………………………………………………………….....19 2. Materials and Methods ……………………………………………………………...20 2.1. MS-153 .………………………………………………………………………..…20 2.2. Augmentin………………………………………………………..………..……...21 2.3.Animals………………………………………………………………………….....21 2.4.Behavioral Drinking Paradigm………………………………………………….....22 2.4.1. Effect of MS-153 on chronic ethanol drinking paradigm…………….....23 2.4.2. Effect of MS-153 on xCT and pAkt levels in Amygdala and Hippocampus……………………………………………..…………….…24 2.4.3. Effect of Combination of MS-153 and Augmentin on Chronic Ethanol Drinking Paradigm……………………………………………………….24 2.5.Brain Tissue Harvesting……………………………………………………….….25 2.6. Protein Tissue Extraction Protocol…………………………………………….…25 2.6.1. Tissue lysate ………………………………………………………....…..25 vii 2.7.Protein Quantification Assay……………………………………………..…...….26 2.8.Western Blot Procedures………………………………………………………......27 2.8.1. Gels preparation ………………………………………………......…27 2.8.2. Samples Preparation……………………………………………….…27 2.8.3. Running and Transfer of Proteins…………………………………....28 2.8.4. Blocking………………………………………………………..….…28 2.8.5. Incubations with Primary Antibodies……………………………..…29 2.8.6. Incubations with Secondary Antibodies…………………………..…29 2.8.7. Developing Membranes…………………………………………...…30 2.9. Statistical Analyses……………………………………………………………...30 2.9.1. Behavioral Data of MS-153 study……………………………….…..30 2.9.2. Behavioral Data of Augmentin and MS-153 combination study…....30 2.9.3. Western Blot Data……………………………………………………31 3. Results…………………………………………………………………………….…….32 3.1. Effect of MS-153 on Chronic Ethanol Drinking Paradigm………………...….....32 3.1.1. Effect of MS-153 Treatment on Ethanol Consumption in Male P Rats………………………………………………..……………....…32 3.1.2. Effect of MS-153 Treatment on Water Intake in Male P Rats……………………………………………….……………….....34 3.1.3. Effect of MS-153 Treatment on Body Weight in Male P Rats .….….35 3.1.4. Long Lasting Effect of MS-153 on GLT1 Expression in NAc……...37 viii 3.1.5. Long Lasting Effect of MS-153 on GLT1 Expression in PFC………38 3.2. Effect of MS-153 on xCT and pAkt Levels in Amygdala and Hippocampus.......40 3.2.1. Effect of MS-153 on xCT Expression in Amygdala..……………..…40 3.2.2. Effect of MS-153 on pAkt Expression in Amygdala ………..……....41 3.2.3. Effect of MS-153 on xCT Expression in Hippocampus……. ………43 3.2.4. Effect of MS-153 on pAkt Expression in Hippocampus…..………..44 3.3.Effect of Combination of MS-153 and Augmentin on Chronic Ethanol Drinking Paradigm……………………………………………………………….46 3.3.1. Effect of Combination of MS-153 and Augmentin on Ethanol Consumption in Male P rats……………………...…………………..46 3.3.2. Effect of Combination of MS-153 and Augmentin on Water Intake of Male P Rats…………….………………………………………….48 3.3.3. Effect of Combination of MS-153 and Augmentin on Male P rats’ Body Weight………..………………………………………………..50 4. Discussion…………………………………………………………………………...52 4.1.Effect of MS-153 on Chronic Ethanol Drinking Paradigm…………………....54 4.2.Effect of MS-153 on xCT and pAkt levels in Amygdala and Hippocampus.....57 4.3.Effect of Combination of MS-153 and Augmentin on Chronic Ethanol Drinking Paradigm……………………………………………………………..61