Potential Therapeutic Targets
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A Dissertation Titled The role of Glutamate Transporter 1 and Cystine-glutamate exchanger in cocaine and ethanol co-abuse: Potential therapeutic targets by Alaa M. Hammad Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics _________________________________________ Dr. Youssef Sari, Committee Chair _________________________________________ Dr. F. Scott Hall, Committee Member _________________________________________ Dr. Wissam AbouAlaiwi, Committee Member _________________________________________ Dr. Zahoor Shah, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2017 Copyright 2017, Alaa M Hammad This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the express permission of the author. An Abstract of The role of Glutamate Transporter 1 and Cystine-glutamate exchanger in cocaine and ethanol co-abuse: Potential therapeutic targets by Alaa M Hammad Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics The University of Toledo August 2017 One of the most popular combinations of abused drugs among addicts is the concurrent use of cocaine and alcohol (ethanol). Previous studies intensively investigated the role of the dopaminergic system in cocaine and ethanol addiction. In addition, the glutamatergic system is another pathway that emerged as a key player in addiction and relapse and has been investigated intensively for the past decade. Glutamate is the main neurotransmitter in the central nervous system (CNS) that is involved in the process of long-term neuronal potentiation, which can help explain the tendency for relapse and persistence of behavioral characteristics of drugs of abuse even after the acute rewarding effects disappear. One of the main areas of investigation is the role of glial glutamate transporters in maintaining glutamate hemostasis and the effects of drugs of abuse on their expression. Glutamate transporter 1 (GLT-1) is responsible for clearing the majority of the extracellular glutamate from the synapse. Alongside GLT-1, the glutamate-aspartate transporter (GLAST) was found to help in maintaining glutamate homeostasis, albeit to a much lesser extent in the mesocorticolimbic pathway. The iii cystine- glutamine exchanger (xCT) is another glial glutamate transporter that is responsible for modulating basal glutamate concentrations. Importantly, the nucleus accumbens (NAc) and the prefrontal cortex (PFC) are two main brain regions within the mesocorticolimbic circuit that were investigated in regard to drug dependence. To the best of my knowledge, there is less known about the effects of cocaine and ethanol co- exposure on the glutamatergic system, including glial glutamate transporters expression in the mesocorticolimbic pathway, compared to the effects of either drug alone. The aim of this research project is to investigate the effects of cocaine and ethanol co-abuse on different glial glutamate transporters using young adult male alcohol preferring (P) rats in three different models that mimic different co-exposure conditions. We used ampicillin /sulbactam (AMP/SUL), a β-lactam antibiotic known to upregulate the expression of certain glial glutamate transporters, to attenuate relapse-like drug seeking behavior. Repeated cocaine exposure (20 mg/kg, i.p) in male P rats exposed to voluntary home- cage ethanol drinking decreased ethanol intake. Co-exposure of cocaine and ethanol decreased the relative mRNA expression and the expression of GLT-1 protein in the NAc but not in the medial PFC (mPFC). Importantly, co-exposure of cocaine and ethanol decreased the relative mRNA expression of xCT in the NAc but not in the mPFC. Using the alcohol deprivation effect (ADE) model in P rats, repeated cocaine (20 mg/kg, i.p) induced a robust increase in relapse-like ethanol drinking behavior compared to saline- treated subjects, accompanied by a decrease in the expression of GLT-1 and xCT in the NAc core. AMP/SUL (100 mg/kg, i.p.) prevented the robust increase in relapse-like ethanol intake in this model, while AMP/SUL (200 mg/kg, i.p.) decreased the relapse-like iv ethanol drinking behavior and upregulated GLT-1 and xCT in the NAc core, shell and dorsomedial PFC (dmPFC). In a conditioned place preference model (CPP), cocaine- induced reinstatement was investigated in male P rats exposed to home-cage voluntary ethanol drinking. Cocaine and ethanol co-exposure acquired place preference and increased locomotor activity compared to ethanol-exposed rats. GLT-1 and xCT expression were both downregulated after co-exposure to cocaine and ethanol in the NAc core and the shell, but not in the dmPFC. AMP/SUL (200 mg/kg) attenuated cocaine- induced reinstatement, decreased locomotor activity, decreased ethanol intake and preference, and upregulated GLT-1 and xCT expression in the NAc core and shell as well as the dmPFC. GLAST expression was unchanged after ethanol and cocaine co-exposure or after AMP/SUL treatment. We also investigated cocaine-induced reinstatement in ethanol naïve P rats. AMP/SUL treatment reduced cocaine-triggered reinstatement. This effect was associated with a decrease in locomotor activity. Moreover, GLT-1 and xCT were downregulated in the NAc core and shell but not in the dmPFC following cocaine- primed reinstatement. However, cocaine upregulated metabotropic glutamate receptor 1 (mGluR1) expression in the NAc core but not the NAc shell or the dmPFC. Importantly, AMP/SUL treatments normalized GLT-1 and xCT expression in the NAc core and shell; alternatively, the drug normalized mGluR1 expression in the NAc core only. Additionally, AMP/SUL upregulated GLT-1 and xCT in the dmPFC as compared to the untreated group. Taken together, it is evident that ethanol and cocaine co-exposure downregulated GLT-1 and xCT expression in different drugs co-exposure models and that modulating glial glutamate transporters may attenuate relapse-like behavior and craving. Thus, selected glial glutamate transporters could be potential therapeutic targets v for the attenuation of reinstatement to cocaine seeking and relapse-like drinking behavior in polysubstance abusers. vi This dissertation is dedicated to my parents, Mahmoud and Alia, who offered me unconditional love and support and had my back all through my life. I also dedicate this dissertation to my siblings, Amjad, Rasha, Taleb, Ahmad, Mohammad and Rany, who are the light of my life. My sincere gratitude to all of you. Special thanks to my only sister, without you life would have been dull, thank you. vii Acknowledgements I express my deep sense of appreciation to my Advisor, Dr. Youssef Sari, whose help, support and guidance through my PhD work was tremendous. This dissertation would not be possible without his devotion and instruction. I also would like to thank Al- Zaytoonah University for offering me a scholarship that allowed me to pursue my PhD degree. I thank the members of my dissertation committee: Dr. F. Scott Hall, Dr. Zahoor Shah, and Dr. Wissam AbouAlaiwi for their help, support, time and critiques that made me a better researcher and shaped my intellectual thinking. I would like to thank all the wonderful current members of Dr. Sari’s lab: Fawaz Alasmari, Alqassem Hakami, and Fahad Alshehri for helping and encouraging me through my PhD work. Special thanks for the former lab members: Dr. Sujan Das and Dr. Yusuf Althobaiti for their help in the animal work. I thank all the faculty members of College of Pharmacy and Pharmaceutical Sciences for their support. I would like to thank my friends and neighbors in Toledo for making this journey less lonely and more enjoyable. And finally, my deepest thanks to my lovely and wonderful family. You mean everything to me. viii Table of Contents Abstract .............................................................................................................................. iii Acknowledgements .......................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables ...................................................................................................................xx List of Figures .................................................................................................................. xxi List of Abbreviations ..................................................................................................... xxiv List of Symbols .............................................................................................................. xxvi Preface .......................................................................................................................... xxvii 1 Effect of Cocaine and Ethanol on the glutamatergic system ...................................1 Introduction ……………. ......................................................................................3 1.1 The Glutamate homeostasis ...............................................................................6 1.2 Effect of cocaine on the glutamatergic system ..........................................10 1.2.1 Effect of acute cocaine exposure on the glutamatergic system…......12 1.2.1.1 Effect of acute