MECHANISMS OF AUTORECEPTOR-MEDIATED INHIBITION IN CENTRAL MONOAMINE NEURONS By NICHOLAS A. COURTNEY Submitted in partial fulfillment for the requirements For the degree of Doctor of Philosophy Thesis Advisor: Christopher P. Ford, Ph.D. Department by Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2016 CASE WESTERN RESERVE UNIVERISTY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Nicholas A. Courtney candidate for the degree of Doctor of Philosophy. Thesis Advisor………………………………. Dr. Christopher Ford Committee Chair……………………..………………Dr. Corey Smith Committee Member………………..…………… Dr. Stephen Jones Committee Member………………...…….… Dr. Ben Strowbridge Committee Member…………………..………… Dr. Roberto Galán Defense Date: October 30, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. ii TABLE OF CONTENTS LIST OF FIGURES ………………………………………………………………………………………….....…. vi LIST OF ABBREVIATIONS …………………………………….…………………………………………… vii ACKNOWLEDGEMENTS ……………………………………………………………..……………………… ix ABSTRACT …………………………………………..………………….….……………………………………… xi CHAPTER 1 Introduction………………………………………………………………..……..…………..….. 1 Foreword………………………….……………...…………………………………..…….….………. 2 Monoamine life cycles…………….……………...………………………...……………….….… 5 G-protein coupled receptor signaling……………………………………………………. 12 Dopamine receptors………………………………………………………...…..……....……… 16 Noradrenaline receptors……………………………………………..…………..…………… 18 Serotonin receptors…………………………………………….…………………..…………… 20 G-protein coupled, inwardly rectifying potassium channels……………...…… 22 Central monoamine systems…………………..…………………………..………………… 25 Mechanisms of neurotransmission…………………………………….………….……… 37 Mechanisms of monoamine feedback inhibition.…………………..…..…………… 42 Rationale………………………………………………………………………………….………..… 45 Statement of the problem …………………………………………………….………….…… 46 CHAPTER 2 iii Species differences in somatodendritic dopamine transmission determine D2-autoreceptor mediated inhibition of ventral tegmental area neuron firing ……………………………………………………………. 51 Foreword……………………………………………………………………………………….……… 52 Abstract……………………………………………………………………………………..…….......... 53 Introduction……………………………………………………………………………………..……. 54 Materials and methods………………………………………………………………………....... 56 Results…………………………………………………………………………………………….…..… 60 Discussion……………………………………………………………………………………….…….. 68 Figures and figure legends…………………………………………..…………………...…..… 74 CHAPTER 3 The timing of dopamine- and noradrenaline-mediated transmission reflects underlying differences in the extent of spillover and pooling. …………………………………………………………………… 84 Abstract……………………………………………………………………………………..…............. 85 Introduction…………………………………………………………………………………..………. 86 Materials and methods……………………………………………………………………..…….. 88 Results……………………………………………………………………………………………...….... 94 Discussion……………………………………………………………………………………….…... 108 Figures and figure legends…..…………………………………………………………...…... 114 iv CHAPTER 4 Synaptic activation of 5-HT1A receptors in dorsal raphe serotonin neurons. …………………………………………………………………………………………. 130 Abstract……………………………………………………………………………………..…........... 131 Introduction……………………………………………………………………………………….... 132 Materials and methods…………..………….….……………………………………………..... 134 Results…………………………………..…………..…………………………………………….…... 137 Discussion………………………………..………….………………………………………….…… 145 Figures and figure legends…………..……….…………………………………………...…... 150 CHAPTER 5 Discussion.……………………………………………………………………………..…..…….162 Inhibitory timing and monoamine activity………………………………………..….. 164 Synchronous verse asynchronous neuronal activity……………...………….…….. 167 Reuptake transporters actively shape monoamine transmission ……….…... 170 Implications for monoamine transmission in terminal regions …………….…. 172 Future directions…………………………………….………………………………………..…... 175 REFERENCES…………………………………………………………………………………………….... 179 v LIST OF FIGURES FIGURE 1.1 ……………………………………………………………………………………………………… 47 FIGURE 1.2 ……………………………………………………………………………………………………… 49 FIGURE 2.1 ……………………………………………………………………………………………………… 74 FIGURE 2.2 ……………………………………………………………………………………………………… 76 FIGURE 2.3 ……………………………………………………………………………………………………… 78 FIGURE 2.4 ……………………………………………………………………………………………………… 80 FIGURE 2.5 ……………………………………………………………………………………………………… 82 FIGURE 3.1 …………………………………………………………………………………….……………… 114 FIGURE 3.2 …………………………………………………………………………………….……………… 116 FIGURE 3.3 …………………………………………………………………………………….……………… 118 FIGURE 3.4 …………………………………………………………………………………….……………… 121 FIGURE 3.5 …………………………………………………………………………………….……………… 123 FIGURE 3.6 …………………………………………………………………………………….……………… 125 FIGURE 3.7 …………………………………………………………………………………….……………… 127 FIGURE 4.1 …………………………………………………………………………………….……………… 150 FIGURE 4.2 …………………………………………………………………………………….……………… 152 FIGURE 4.3 …………………………………………………………………………………….……………… 154 FIGURE 4.4 …………………………………………………………………………………….……………… 156 FIGURE 4.5 …………………………………………………………………………………….……………… 158 FIGURE 4.6 …………………………………………………………………………………….……………… 160 vi LIST OF ABBREVIATIONS 5-HT – Serotonin ACSF – Artificial Cerebral Spinal Fluid COMT - Catechol-O-Methyl Transferase DA – Dopamine DAT – Dopamine Reuptake Transporter DBH – Dopamine β Hydroxylase DRN – Dorsal Raphe Nucleus FSCV – Fast Scan Cyclic Voltammetry GIRK – G-protein Coupled, Inwardly Rectifying Potassium Channel GPCR – G-protein Coupled Receptor IPSC – Inhibitory, Post-synaptic Current LC – Locus Coeruleus LDCV – Large Dense Core Vesicle MAO – Monoamine Oxidase NA – Noradrenaline NET – Noradrenaline Reuptake Transporter N.S. – No Statistical Significance SERT – Serotonin Reuptake Transporter SSV – Small Synaptic Vesicle TPH – Tryptophan Hydroxylase TH – Tyrosine Hydroxylase vii VMAT – Vesicular Monoamine Transporter VTA – Ventral Tegmental Area viii ACKNOWLEDGEMENTS This work would not have been possible without the support that I have received from many people for which I am extremely grateful. First and foremost, I would like to thank my advisor, Dr. Chris Ford. He has provided me sound guidance in both my graduate studies and my pursuit of a research career. For all that Dr. Ford has taught me about science, it is his passion for his research that I most hope to emulate. Thank you for your guidance, wisdom, and at times patience. I have enjoy my time in the Ford lab and will look back fondly on my Ph.D. experience. I would like to thank the members of my thesis committee: Dr. Corey Smith, Dr. Stephen Jones, Dr. Ben Strowbridge, and Dr. Roberto Galán, as well as a previous member Dr. Diana Kunze. This committee has played instrumental part in my development as a scientist. Thank you very much for dedicating time and effort to assisting me throughout my graduate studies. I would like to thank the members of the Ford lab: Didi Mamaligas and Pam Marcott. I cannot count the number of times you have helped me think through experiments, evaluate drafts, practice talks and posters, and generally be supportive during my graduate studies. Thank you very much and I wish you the best of luck with your own studies. ix I would like to thank Dr. Joe LaManna and the Neurodegenerative Training Grant staff here at Case Western. Thank you for the support demonstrated by funding my research, and more importantly, my training and future career. I would like to thank the entire Department of Physiology and Biophysics, especially the front office staff and fellow graduate trainees. I would like to thank my family members and friends that have supported me through my graduate career. To my parents, thank you for patience and encouragement while I was searching for a career that I could be happy pursuing. Finally, I would especially like to thank my wife and fellow graduate student, Becki. Your unwavering support has been instrumental in my Ph.D. journey. Thank you for everything. x Mechanisms of Autoreceptor-Mediated Inhibition in Central Monoamine Neurons Abstract By NICHOLAS A. COURTNEY This thesis examined the mechanisms of neurotransmission underlying feedback inhibition mediated by somatodendritic G-protein coupled dopamine D2- autoreceptors in the ventral tegmental area (VTA), noradrenaline α2-autoreceptors in the locus coeruleus (LC), and serotonin 5-HT1A autoreceptors in the dorsal raphe nucleus (DRN). Local feedback inhibition mediated by these GI-coupled monoamine receptors had been speculated to ubiquitously occur via extended transmitter spillover and obligatory extracellular transmitter pooling. Collectively termed volume transmission, these mechanisms of transmission are presumed to result in a tonic, inhibitory tone that modulates the firing rates of monoaminergic neurons in response to slowly changing extracellular concentrations of monoamines. However, evidence for the volume transmission hypothesis is indirect and relies on measuring bulk extracellular monoamine concentrations and predicting receptor activation based on steady-state affinities and mathematical diffusion modeling. In monoamine neurons, autoreceptors couple to G-protein coupled, inwardly rectifying potassium channels (GIRKs) through the βγ-subunits of trimetric G- proteins. Activation of somatodendritic autoreceptors by locally released monoamines generates potassium currents that inhibit neuronal excitability. In this xi thesis, I directly investigated the synaptic mechanisms controlling autoreceptor activity by using electrophysiological methods to measure GIRK-mediated currents in response to evoked transmitter release in rodent brain slices. After characterizing the calcium dependence and clearance of midbrain dopamine