Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2016 Studies of nicotinic acetylcholine receptors containing α4 and α6 subunits in nicotine-induced synaptic plasticity in brain reward areas Staci Engle Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Engle, Staci, "Studies of nicotinic acetylcholine receptors containing α4 and α6 subunits in nicotine-induced synaptic plasticity in brain reward areas" (2016). Open Access Dissertations. 1481. https://docs.lib.purdue.edu/open_access_dissertations/1481 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Staci E. Engle Entitled STUDIES OF NICOTINIC ACETYLCHOLINE RECEPTORS CONTAINING 4 AND 6 SUBUNITS IN NICOTINE-INDUCED SYNAPTIC PLASTICITY IN BRAIN REWARD AREAS For the degree of Doctor of Philosophy Is approved by the final examining committee: Ryan Drenan Chair Val J. Watts Greg Hockerman Edward Bartlett To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Ryan Drenan Approved by: Chris Rochet 2/15/2016 Head of the Departmental Graduate Program Date i STUDIES OF NICOTINIC ACETYLCHOLINE RECEPTORS CONTAINING 4 AND 6 SUBUNITS IN NICOTINE-INDUCED SYNAPTIC PLASTICITY IN BRAIN REWARD AREAS A Dissertation Submitted to the Faculty of Purdue University by Staci E. Engle In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy i May 2016 Purdue University West Lafayette, Indiana ii ACKNOWLEDGEMENTS First and foremost I would like to express my sincere gratitude to my major professor, Dr. Ryan Drenan. It has been an honor to be your first Ph.D. student. I would like to thank you for the opportunity to work in your laboratory and for your motivation, patience, support, and guidance, all of which have helped me to grow as a scientist. I am also thankful for the support and guidance provided by the members of my dissertation committee, Dr. Edward Bartlett, Dr. Gregory Hockerman, and Dr. Val Watts. I am grateful for the past and present members of the Drenan Lab, including Hilary Broderick, Dr. Pei-Yu Shih, Dr. Jennifer Berry, and Matthew Arvin who have provided encouragement, insightful conversations, and assistance with experiments. I have indicated in the figure legends where these individuals have contributed to the data collected in that figure. Last but not least, I would like to thank my family and friends because without their support, encouragement, and love, I would not have made it to where I am today. ii iii TABLE OF CONTENTS Page LIST OF ABBREVIATIONS .................................................................................................... vii ABSTRACT ............................................................................................................................. x PUBLICATIONS ................................................................................................................... xiii INTRODUCTION ......................................................................................... 1 1.1 Tobacco Addiction ............................................................................................ 1 1.2 Nicotinic Acetylcholine Receptors .................................................................... 2 1.3 Mesolimbic Reward Pathway ........................................................................... 4 1.4 nAChRs in the VTA ............................................................................................ 7 1.5 Long-Term Potentiation.................................................................................. 10 1.6 Synaptic Plasticity in the VTA ......................................................................... 12 1.7 Mechanisms of Nicotine-Evoked Synaptic Plasticity in the VTA .................... 14 1.8 Nicotine and Ethanol Co-Abuse ...................................................................... 19 1.9 Ethanol and nAChRs ....................................................................................... 20 1.10 Effects of Combined Nicotine and Ethanol Administration ........................... 21 1.11 Ethanol-Mediated Changes in Synaptic Plasticity .......................................... 22 1.12 Objectives ....................................................................................................... 22 iii METHODS ................................................................................................ 23 2.1 Materials ......................................................................................................... 23 2.2 Mice ................................................................................................................ 24 2.3 Stereotaxic Surgery ......................................................................................... 26 2.4 in vivo Drug Administration ............................................................................ 28 2.5 Brain Slice Preparation for Electrophysiology ................................................ 29 iv Page 2.6 Patch-Clamp Electrophysiology ...................................................................... 30 2.7 Single-Cell RT-PCR ........................................................................................... 35 2.8 Immunohistochemistry and Confocal Microscopy. ........................................ 36 2.9 Statistical Analysis. ......................................................................................... 38 462* nAChR ACTIVATION ON VTA DA NEURONS IS SUFFICIENT TO STIMULATE A DEPOLARIZING CONDUCTANCE AND ENHANCE SURFACE AMPA RECEPTOR FUNCTION .................................................................................................. 39 3.1 Electrophysiological Identification of VTA DA Neurons ................................. 39 3.2 Hypersensitive 6* nAChRs in 6L9S mice .................................................... 42 3.3 A Low Concentration of Nicotine is Sufficient to Increase Inward Currents in VTA DA Neurons ............................................................................................. 44 3.4 AMPA-Evoked Current Methodology ............................................................. 47 3.5 Activation of 6* nAChRs is Sufficient to Enhance AMPAR Function on the Surface of VTA DA Neurons ............................................................................ 50 3.6 Time Dependence For Enhancement of AMPA-Evoked Currents in 6L9S VTA DA Neurons ..................................................................................................... 54 3.7 Pharmacology of AMPA-Evoked Current Induction in 6L9S VTA DA Neurons ……………………………………………………………………………………………………………………56 iv 3.8 Enhanced AMPA-Evoked Currents in 6L9S VTA DA Neurons Are Mediated by 4 nAChR Subunits .................................................................................... 59 3.9 6* nAChR Function is Reduced in 4KO Mice ............................................. 61 3.10 NMDA-Evoked Currents Are Not Changed by Nicotine in 6L9S VTA DA Neurons .......................................................................................................... 64 3.11 Systemic Nicotine Acts Through α6-Containing nAChRs to Enhance AMPA Receptor Function in VTA DA Neurons........................................................... 66 3.12 Inhibition of α6-containing nAChRs in VTA Blocks AMPAR Enhancement by Systemic Nicotine. .......................................................................................... 68 v Page NICOTINE AND ETHANOL COOPERATE TO ENHANCE VTA AMPA RECEPTOR FUNCTION VIA α6-CONTAINING NICOTINIC RECEPTORS .......................... 70 4.1 α6* nAChRs Are Involved in Ethanol-Mediated Increases in AMPAR Function in VTA DA Neurons ......................................................................................... 70 4.2 α6* nAChRs Mediate Enhanced AMPAR Function After in vivo Ethanol Administration ................................................................................................ 74 4.3 AMPAR Function is Enhanced by Nicotine Exposure in non-Tg and α6L9S Mice ................................................................................................................ 76 4.4 Nicotine and Ethanol Exposure Enhance Excitatory Synaptic Transmission in non-Tg and α6L9S Slices ................................................................................. 78 4.5 Subthreshold Nicotine and Subthreshold Ethanol Combine to Enhance VTA AMPAR Function in non-Tg Slices ................................................................... 80 4.6 Subthreshold Nicotine and Subthreshold Ethanol Combine to Enhance VTA AMPAR Function in α6L9S Slices .................................................................... 82 REMOVAL OF 4-CONTAINING nAChRs FROM THE VENTRAL MIDBRAIN ALTERS CIRCUITRY WITHIN THE VTA ............................................................................ 84 5.1 Chrna4 vMB cKO Mouse Model ....................................................................