UNIVERSITY OF CALIFORNIA, SAN DIEGO The Role of Alpha4 Containing Nicotinic Acetylcholine Receptors in Dopamine Neurons A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Neurosciences by Tresa Michelle McGranahan Committee in charge: Professor Stephen Heinemann, Chair Professor Darwin Berg, Co-Chair Professor Stephan Anagnostaras Professor Athina Markou Professor Martin Paulus 2011 Copyright Tresa Michelle McGranahan, 2011 All rights reserved. The Dissertation of Tresa Michelle McGranahan is approved, and it is acceptable in quality and form for publication on microfilm: Co-Chair Chair University of California, San Diego 2011 iii DEDICATION I would like to dedicate this thesis to my family and my friend family. Without your love and support, I would not have the strength to reach for my dreams. iv TABLE OF CONTENTS Signature Page ........................................................................................................... iii Dedication .................................................................................................................. iv Table of Contents ...................................................................................................... v List of Figures ............................................................................................................ vii List of Abbreviations ................................................................................................. viii Acknowledgements ................................................................................................... ix Vita, Publications, and Fields of Study ..................................................................... xii Abstract ...................................................................................................................... xii Chapter I Introduction ............................................................................................ 1 Introduction ....................................................................................................... 2 Chapter II Generating the Mouse Model .............................................................. 14 Introduction ....................................................................................................... 15 Deletion of α4 Subunit from Dopaminergic Neurons ....................................... 16 Functional Deletion of α4*-nAChRs from Dopaminergic Neurons and Preservation of α6(non-α4)-nAChRs ............................................................. 17 Quantification of α4β2*-nAChRs and α6β2*-nAChRs .................................... 19 Conclusions ...................................................................................................... 20 Methods............................................................................................................. 21 Chapter III General Characterization and Acute Nicotine Behaviors ............... 34 Introduction ....................................................................................................... 35 v Learning and Memory ....................................................................................... 35 Locomotor Activity ........................................................................................... 38 Sensitivity to Nicotine-Mediated Behaviors .................................................... 40 Nicotine Induced Locomotor Changes.............................................................. 41 Nicotine Induced Changes in Body Temperature ............................................. 43 Methods............................................................................................................. 45 Chapter IV The Role of α4*-nAChRs in Reward ................................................. 53 Introduction ....................................................................................................... 54 Nicotine Reward................................................................................................ 60 Cocaine Reward ................................................................................................ 61 Discussion ......................................................................................................... 63 Methods............................................................................................................. 66 Chapter V The Role of α4*-nAChRs in Anxiety .................................................. 70 Introduction ....................................................................................................... 71 Basal Anxiety .................................................................................................... 75 Anxiolytic Effects of Nicotine .......................................................................... 76 Discussion ......................................................................................................... 77 Methods............................................................................................................. 78 Chapter V Conclusions and Future Directions ..................................................... 81 Conclusions ....................................................................................................... 82 Future Directions............................................................................................... 90 References ................................................................................................................ 91 vi LIST OF FIGURES Chapter II Figure 1.1 Generation of α4 lox-only mouse............................................................ 30 Figure 1.2 Deletion of α4 in dopaminergic regions in α4-DA mice ........................ 31 Figure 1.3 Selective deletion of α4 in dopaminergic neurons ................................. 32 Figure 1.4 Functional confirmation of deletion of α4 from dopaminergic neurons 33 Chapter III Figure 2.1 Mutations do not alter bodyweight ......................................................... 47 Figure 2.2 Fear conditioning .................................................................................... 48 Figure 2.3 Locomotor activity ................................................................................. 49 Figure 2.4 Locomotor habituation (30 minutes) ...................................................... 50 Figure 2.5 Locomotor habituation (2 hours)............................................................. 50 Figure 2.6 Locomotor suppression effects of nicotine.............................................. 51 Figure 2.7 Thermoregulatory effects of nicotine. .................................................... 52 Chapter IV Figure 3.1 Conditioned place preference methods ................................................... 67 Figure 3.2 α4-containing nAChRs mediate nicotine reward .................................... 68 Figure 3.3 α4-containing nAChRs do not mediate cocaine reward.......................... 69 Chapter V Figure 4.1 Basal anxiety .......................................................................................... 79 Figure 4.2 α4-containing nAChRs mediate the anxiolytic effects of nicotine.......... 80 vii LIST OF ABBREVIATIONS Abbreviation Definition α alpha β beta μ micro Ach acetylcholine VTA ventral tegmental area bp base pairs CPP conditioned place preference Cre P1 gene causes recombination CS conditioned stimulus CX cortex DAT dopamine transporter EC50 half maximal effective concentration EPM elevated plus maze ES embryonic stem cells fMRI functional magnetic resonance imaging g gram GABA gamma-aminobutyric acid GAD67 glutamate decarboxylase Hz Hertz i.p. intraperitoneal Kb kilo base pairs LTP long term potentiation M molar ml milliliter mM milimolar mPFC medial prefrontal cortex NAc nucleus accumbens nAChRs nicotinic acetylcholine receptors NMDA N-methyl-D-aspartate neor neomysin resistance gene OT olfactory tubercle PCR polymerase chain reaction pM picomolar SN substantia nigra ST striatum TH tyrosine hydroxlase WT wild type viii ACKNOWLEDGEMENTS I would like to thank Dr. Stephen Heinemann for providing me with the extraordinary opportunity to work in his prestigious laboratory. His direction and advice has allowed me to focus not only on science, but also my future as a scientist. His mentorship has personalized many of the critical findings that first attracted me to science. In addition to serving as a scientific mentor, Dr. Heinemann has been an extraordinary role model for how to live life fully. I would also like to thank my committee members. Dr. Stephan Anagnostaras, his patience with me in discussing reward paradigms and interpretations of my findings were instrumental in my success. Dr. Darwin Berg, your suggestions to further examine various parts of the literature greatly improved my understanding and this thesis. I also greatly appreciate your advice on the limits to study one graduate student can undertake. I would like to thank Dr. Athina Markou, for encouraging me to think about and organize projects in relation to publications, although things did not always work out as planned, it is advise I will carry with me for all of my future studies. Dr. Martin Paulus, you are truly a role model for translational science and your synthesis of basic science findings with knowledge of psychiatry has served, and continues to serve, as an extraordinary example to me. I also owe a huge debt of gratitude to all of my great colleagues in the Heinemann