University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2012-01-01 The ffecE ts Of Extended Access To Methamphetamine Self-Administration On Dopaminergic Markers In The trS iatum Joe Luevano University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Behavioral Neurobiology Commons, Biological Psychology Commons, Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Luevano, Joe, "The Effects Of Extended Access To Methamphetamine Self-Administration On Dopaminergic Markers In The Striatum" (2012). Open Access Theses & Dissertations. 2129. https://digitalcommons.utep.edu/open_etd/2129 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. THE EFFECTS OF EXTENDED ACCESS TO METHAMPHETAMINE SELF-ADMINISTRATION ON DOPAMINERGIC MARKERS IN THE STRIATUM JOE LUEVANO Department of Biological Sciences APPROVED: Manuel Miranda Ph.D., Chair Laura O’Dell, Ph.D. Kristin Gosselink, Ph.D. Benjamin C. Flores, Ph.D. Dean of the Graduate School Copyright © By Joe Luevano 2012 THE EFFECTS OF EXTENDED ACCESS TO METHAMPHETAMINE SELF-ADMINISTRATION ON DOPAMINERGIC MARKERS IN THE STRIATUM by Joe Luevano, B.S. THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Biological Sciences THE UNIVERSITY OF TEXAS AT EL PASO December 2012 ACKNOWELDGEMENTS The research described herein would not have been possible without the support of many people across discipline lines. First and foremost I would like to thank my mentor Dr. Manuel Miranda for his diligent support and patience. I was fortunate enough to have joined his lab as an undergraduate student more than four years ago. I am extremely grateful for the opportunity that Manuel has given me at a time when so many were quick to turn me away. Since then we have worked through the best and worst of times. For his commitment with me, I have much admiration and respect for him as an instructor, mentor and person. I only hope that someday when I become an independent investigator I will have half the amount of dedication and patience for my students as Manuel has for his. He is a major part of my journey and his compassion, understanding and kindness in and outside the lab are things I will never forget. I would next like to thank the support of the Miranda, Khan, O’Dell and Gosselink labs. Without the help and co-operation of the many individuals from these labs the work in this project would have been incomplete. I would like to thank Oscar Torres and Evelyn Escalante for teaching myself and Jonathan Jackson the jugular catheter implant surgeries. I also would like to thank Luis Natividad for teaching myself and Jonathan the operation of the animal behavioral IVSA. A special thanks to Dr. Gosselink and Chris D’Arcy for teaching me how to perfuse and section the animal brain tissue. Thanks to Dr. Arshad Khan for training me on immunohistochemistry design and staining optimization. I would also like to thank the Miranda lab postdocs Dr. Vincent Castecon and Dr. Luciana Gentil for sharing their expert tips and western blotting troubleshooting experiences with me. A sincere thank you to Jonathan Jackson for all his assistance and support, without his help this project would not have been feasible. Thank you to Dr. Christina Sobin for all of the very beneficial writing suggestions and feedback for my Thesis document. I am grateful for everyone’s valuable assistance, without which I would not have been as productive. iv Thank you to the Vulnerability Issues in Drug Abuse (VIDA) program for their financial support on this fascinating research project. Without the hard work and dedication of the many involved in the writing of this exceptional grant this research opportunity would not have been possible. Thank you to VIDA secretary Rosemary Landa-Medlin for all her help with reagent orders, travel arrangements and event organization. Thank you to VIDA director Dr. Edward Castaneda for all his extremely beneficial workshops and support. I wish to acknowledge all of my thesis committee members: Dr. Miranda, Dr. O’Dell and Dr. Gosselink. Your guidance has helped me to complete my work and develop my thesis in a method that would clearly communicate an interesting story. Thank you all for your feedback and cooperation during all the committee meetings we had. I am grateful for the time we spent working on the project and my skills as a young investigator, orator and writer. Thank you to all whom provided me with recommendation letters. Lastly, I would like to thank my parents for being present with me at every step in my academic journey. Without their support I would not have been able to make it as far as I have. Their love, support and encouragement has helped me to gain prospective on the most important aspects on life and thus aided me to finish my graduate studies. v ABSTRACT Methamphetamine (METH) abuse is a persistent problem in the U.S. and abroad. Escalation of METH use among independent users occurs for a variety of physiological and psychological reasons. Methamphetamine dependence may be attributed to the rewarding effect of this drug via the dopaminergic systems of the central nervous system (CNS). The presence of METH in the CNS increases synaptic release of dopamine. This increase in dopaminergic neurotransmission is thought to be directly attributed to the rewarding effects of METH. Following METH use, compensatory changes have been found to occur in the dopaminergic system during various periods of abstinence. It is proposed that these molecular compensatory responses during periods of abstinence following stimulant usage may in part be responsible for facilitating addiction behavior and drug relapse. Previous studies have shown that rats given extended access to methamphetamine self-administration display an escalation of drug intake as compared to animals given limited access to this drug. Many other studies using both self-administration operant conditioning and non-contingent delivery of METH in rodents have found various changes in key dopaminergic proteins following access to high-dose METH under limited durations. However, dopaminergic protein changes which may occur shortly following an extended access (20 days or more) METH paradigm are not well-characterized. The goal of this study was to compare changes in dopaminergic systems in rats allowed extended versus limited access to methamphetamine self-administration following an extended access paradigm. Adult Wistar rats (n=10 extended, n=7 limited, n=8 naïve) were given either extended (6 hr/day) or limited (1 hr/day) access to intravenous self-administration (IVSA) of METH (0.025 mg/infusion) for 28 days. Control rats received surgical implantation of intravenous catheters, but were not given access to METH IVSA. The rats were sacrificed 72 hours after the final self- vi administration session. All groups were compared with respect to striatal dopaminergic protein expression (Tyrosine hydroxylase, TH, dopamine transporter, DAT, and dopamine receptor 2, D2R) using western-blot and immunocytochemistry procedures. The results revealed that rats given extended access to METH displayed an escalation of METH intake during the first hour of each session, and this behavior was not observed in animals given limited access. Seventy-two hours after termination of extended access paradigm the largest changes in striatal dopaminergic markers were observed between drug naïve controls versus rats that self-administered METH for the extended access (6hrs) duration. Western-blot analyses of striatal tissue revealed no significant difference in immunoreactivity for TH between treatment groups and naïve animals. By contrast, a significant increase in total striatal DAT was found in extended access self-administration rats as compared to naive controls. Preliminary immunocytochemistry analysis was consistent with the latter results, suggesting an increase in the levels of DAT throughout the corpus striatum. On the other hand, no significant changes were found between METH IVSA and naïve controls for D2R striatal immunoreactivity. Taken together, our results revealed that rats allotted 6 hours access to METH IVSA showed an escalation of intake compared to rats allotted 1 hour of access. In addition, it was found that expression of dopaminergic markers in the striatum were altered after extended methamphetamine self-administration access. These findings cast new insight into the molecular mechanism of long-term METH use and subsequent escalation, which may evoke cravings for sustained use and relapse during initial periods of withdrawal. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………………………….....iv ABSTRACT……………………………………………………………………………………………….vi TABLE OF CONTENTS………………………………………………………………………………..viii LIST OF TABLES………………………………………………………………………………………....x LIST OF FIGURES……………………………………………………………………………………….xi Chapter 1 CHAPTER 1: INTRODUCTION………………………………………………………………..1 1.1 Stimulants of Abuse………………………………………………………………………..1 1.2 Molecular Characterization…………………………………………………………………3 1.3 The Reward Pathway……………………………………………………………………….5 1.4 Addiction Behavior and Neural Plasticity………………………………………………..8