University of Kentucky UKnowledge Theses and Dissertations--Medical Sciences Medical Sciences 2020 Involvement of the Sigma-1 Receptor in Methamphetamine- Mediated Changes to Astrocyte Structure and Function Richik Neogi University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0002-8716-8812 Digital Object Identifier: https://doi.org/10.13023/etd.2020.363 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Neogi, Richik, "Involvement of the Sigma-1 Receptor in Methamphetamine-Mediated Changes to Astrocyte Structure and Function" (2020). Theses and Dissertations--Medical Sciences. 12. https://uknowledge.uky.edu/medsci_etds/12 This Master's Thesis is brought to you for free and open access by the Medical Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Medical Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Richik Neogi, Student Dr. Pavel Ortinski, Major Professor Dr. Melinda Wilson, Director of Graduate Studies INVOLVEMENT OF THE SIGMA-1 RECEPTOR IN METHAMPHETAMINE- MEDIATED CHANGES TO ASTROCYTE STRUCTURE AND FUNCTION ________________________________________ THESIS ________________________________________ A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Medicine at the University of Kentucky By Richik Neogi Lexington, Kentucky Co-Directors: Dr. Pavel Ortinski, Assistant Professor of Neuroscience and Dr. Chris Richards, Associate Professor of Chemistry Lexington, Kentucky 2020 Copyright © Richik Neogi 2020 https://orcid.org/0000-0002-8716-8812 ABSTRACT OF THESIS Involvement of the Sigma-1 Receptor in Methamphetamine Mediated Changes to Astrocyte Structure and Function Methamphetamine is a highly addictive psychostimulant drug. There are currently no FDA-approved pharmacological treatments for methamphetamine addiction. Pharmacologically, (+)-methamphetamine is a dopamine releasing agent and dopamine transporter substrate that redistributes dopamine from intracellular vesicles via the vesicular monoamine transporter 2 (VMAT2) and reverses the direction of dopamine transport in the dopamine transporter. Methamphetamine, cocaine, and other drugs of abuse are also sigma-1 receptor ligands, and previous studies have demonstrated the role of the sigma-1 receptor in modulating the behavioral and cellular effects of these drugs. However, almost all these studies were conducted in cell culture systems or with emphasis on the neuronal effects of sigma-1 receptor binding. In the literature, the sigma-1 receptor localizes robustly within neurons throughout the brain. However, there are conflicting results regarding localization in astrocytes. Here, immunohistochemical experiments were conducted on rat brain tissue containing either the nucleus accumbens or the prelimbic cortex, two brain regions critical in mediating drug- seeking behavior, in order to confirm expression of the sigma-1 receptor in astrocytes. It was determined that the sigma-1 receptor is ubiquitously expressed among neurons and modestly expressed among glial fibrillary acidic protein (GFAP)-positive astrocytes. There were no noted statistically significant differences between the proportion of cell types expressing the sigma-1 receptor in the prelimbic cortex and the nucleus accumbens. Next, the effect of methamphetamine and the role of the sigma-1 receptor on astrocytic morphology and sigma-1 receptor distribution were probed by incubating slices of striatal tissue in methamphetamine solution with or without the sigma-1 receptor antagonist BD- 1063 and the sigma-1 receptor agonist PRE-084. Only the methamphetamine-treated slice exhibited a significantly different amount of stellations than the other slices. The sigma-1 receptor antagonist BD-1063 and PRE-084 abolished the redistribution of sigma-1 receptor to the processes of the astrocytes induced by methamphetamine. Thus, the data confirms that astrocytes in both the prelimbic cortex and nucleus accumbens express the sigma-1 receptor. Moreover, methamphetamine induces statistically significant redistribution of the sigma-1 receptor that is abolished by BD-1063 and PRE-084. KEYWORDS: methamphetamine, astrocytes, sigma-1 receptor Richik Neogi (Name of Student) 08/10/2020 Date Involvement of the Sigma-1 Receptor in Methamphetamine-Mediated Changes to Astrocyte Structure and Function By Richik Neogi Dr. Pavel Ortinski Co-Director of Thesis Dr. Chris Richards Co-Director of Thesis Dr. Melinda Wilson Director of Graduate Studies 08/10/2020 Date DEDICATION To my parents, my PI, and my coworkers for supporting me throughout graduate school. ACKNOWLEDGMENTS The following thesis, an individual work, would not be possible without support and training from the Light Microscopy Core at the University of Kentucky. I will always fondly remember the long hours sitting at the confocal microscope and receiving support from the staff at the Light Microscopy Core. I would also like to acknowledge my coworkers for providing critical assistance when I was beginning to learn the technique of immunohistochemistry and when I was planning the experiments entailed in this manuscript. Finally, I would like to thank Lindsey Hammerslag for her assistance with perfusion of animals and her guidance on the immunohistochemistry protocol. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................................. iii LIST OF TABLES ........................................................................................................................................ vi LIST OF FIGURES ..................................................................................................................................... vii CHAPTER 1. MECHANISMS OF METHAMPHETAMINE ACTION .................................................................. 1 1.1 Methamphetamine as Drug of Abuse ................................................................................................... 1 1.2 Mechanism of AMP/METH-Mediated DA Release ............................................................................ 1 1.2.1 Actions at DAT .............................................................................................................................. 1 1.2.2 Vesicular Actions of AMP/METH ............................................................................................. 4 1.2.3 Hypotheses of AMP/METH-Mediated DA Release .................................................................. 6 1.2.4 Effects on DA Synthesis and Metabolism .............................................................................. 13 1.3 Astrocytes ........................................................................................................................................... 15 1.3.1 General Role of Astrocytes in the CNS ........................................................................................ 15 1.3.1.1 History of Astrocytes ........................................................................................................... 15 1.3.1.2 Astrocyte Development ...................................................................................................... 16 1.3.1.3 Potassium Buffering Roles of Astrocytes ........................................................................... 17 1.3.1.4 Role of Astrocytes in Glutamate Homeostasis ................................................................... 18 1.3.1.5 Astrocyte Morphology ........................................................................................................ 20 1.3.2 Astrocytic Calcium ...................................................................................................................... 23 CHAPTER 2. THE SIGMA-1 RECEPTOR ....................................................................................................