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The Regulation of Neuronal Excitability and Nociception by Tonic Gabaergic Inhibition

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The Regulation of Neuronal Excitability and Nociception by Tonic Gabaergic Inhibition THE REGULATION OF NEURONAL EXCITABILITY AND NOCICEPTION BY TONIC GABAERGIC INHIBITION by Robert Paul Bonin A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Robert P. Bonin 2011 The Regulation of Neuronal Excitability and Nociception by Tonic GABAergic Inhibition Robert Paul Bonin Doctor of Philosophy Department of Physiology University of Toronto 2011 Abstract The mammalian central nervous system maintains a delicate balance between neuronal excitation and inhibition. Conventional synaptic inhibition is mediated through the transient activity of postsynaptic γ-aminobutyric acid (GABA) at type A GABA (GABAA) receptors. A subset of GABAA receptors is also located outside of inhibitory synapses. These extrasynaptic receptors generate a tonic inhibitory conductance in response to low concentrations of extracellular GABA. Tonic inhibition broadly suppresses neuronal activity and regulates many vital processes such as sleep, consciousness and memory formation. This thesis examines the physiological effects of tonic inhibition at the cellular level and in the behaving animal. This thesis also explores whether gabapentin, a commonly used sedative, anxiolytic, and analgesic drug, enhances tonic GABAergic inhibition. I hypothesize that: (1) tonic GABAA receptor activity reduces the intrinsic excitability of neurons; (2) the activity of tonically active GABAA receptors in spinal pain pathways attenuates nociception; and (3) tonic inhibition can be upregulated by gabapentin. ii The results show that a tonic inhibitory current generated by α5 subunit-containing GABAA (α5GABAA) receptors reduces the excitability of hippocampal pyramidal neurons excitability by increasing the rheobase, but does not change the gain of action potential firing. A similar shunting inhibition is present in spinal cord lamina II neurons that is generated by δ subunit-containing GABAA receptors. The activity of these receptors in spinal nociceptive pathways reduces acute thermal nociception and may constrain central sensitization in a behavioural model of persistent pain. Finally, gabapentin increases a tonic inhibitory current in cultured hippocampal neurons independent from changes in the expression of α5GABAA receptors or in the concentration of GABAA receptor ligands. The results of this thesis demonstrate that tonically active GABAA receptors play an important role in the regulation of neuronal activity and nociception, and that tonic inhibition represents a novel target of therapeutic drugs. iii Acknowledgments I would like to thank my supervisor, Dr. Beverly Orser for her guidance and mentorship over the years that we have worked together. Her continued support, training and friendship have made me a better scientist and a better person. I also thank my supervisory committee Drs. John MacDonald and Melanie Woodin, who have provided invaluable guidance, and my examination committee members, Drs. Delia Belelli, Colin McCartney, John Dostrovsky, and Richard Horner. I also thank all the members of the Orser lab whom I have been fortunate enough to work with over the years. Your input and effort made this work easier, and your friendship made this fun. I thank Agnieszka Zurek, who was first my student and became a great friend and mentor in both science and life. I will let you know when the time machine is working; Ella Czerwinska, for keeping the lab running and providing a constant supply of neuronal cultures that star so prominently in this thesis, and for her jokes and cheeriness; Loren Martin, who I have always looked up to. Paul Whissell; for his pervasive sanguinity; and Dianshi Wang, our quiet leader. I also thank Dave Eng, Irene Lecker, William To, and Sinziana Avramescu, who all made the lab feel like home. Finally, I reserve a special thank you to my family, who provided unwavering emotional support. I thank my parents, John and Teresa, my brother, Steve, my sister, Amy, and especially my wife, Anna, for their love, encouragement, and understanding. List of Contributions Several investigators contributed to the work presented in this thesis. In Chapter 4, Loren J. Martin completed the electrophysiological experiments in hippocampal slices. In Chapter 5, David G. Eng and Paul D. Whissell assisted with the behavioural experiments and Charalampos Labrakakis assisted with the electrophysiological experiments in spinal slices. In Chapter 6, Victor Y. Cheng completed a subset of the electrophysiological experiments and Mary W. Chiu completed the western blot analysis of GABAA receptor expression. Finally, Gail Rauw conducted the tissue analysis of amino acid and neuroactive steroid levels presented in Chapter 7. This work was completed with the financial support provided to me by the Natural Sciences and Engineering Research Council of Canada, the University of Toronto Department of Physiology, and the University of Toronto Neuroscience Program. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiii List of Abbreviations ................................................................................................................... xiv Chapter 1. Introduction ................................................................................................................... 1 1 Overview .................................................................................................................................... 1 1.1 GABA in the central nervous system .................................................................................. 2 1.2 GABAA receptors ................................................................................................................ 2 1.2.1 Tonically active GABAA receptors ......................................................................... 6 1.2.2 GABAA receptor subtypes generating a tonic current ............................................ 7 1.2.3 Sources of GABA responsible for tonic inhibition ............................................... 13 1.3 Physiological function of tonic inhibition ......................................................................... 14 1.3.1 Regulation of neuronal excitability ....................................................................... 14 1.3.2 Regulation of synaptic plasticity ........................................................................... 19 1.3.3 Behavioural roles of tonic inhibition .................................................................... 20 1.3.4 Interactions between α5GABAA and δGABAA receptors .................................... 22 1.4 GABAA receptors as targets for analgesic drugs .............................................................. 24 1.4.1 Regulation of nociception by GABAA receptors .................................................. 24 1.4.2 Regulation of central sensitization by GABAA receptors ..................................... 25 1.4.3 GABAA receptors expression in pain processing pathways ................................. 27 1.5 Gabapentin and GABAA receptor activity ........................................................................ 29 2+ 1.5.1 α2δ Ca channel subunit ....................................................................................... 30 v 1.5.2 Gabapentin and tonic GABAA receptor activity ................................................... 31 Chapter 2. Hypotheses and Aims .................................................................................................. 32 2 Overview .................................................................................................................................. 32 2.1 General Hypotheses .......................................................................................................... 33 2.2 Specific Aims .................................................................................................................... 33 Chapter 3. Methods ....................................................................................................................... 35 3 Overview .................................................................................................................................. 35 3.1 Animal models .................................................................................................................. 35 3.2 Tissue preparation and analysis ........................................................................................ 36 3.2.1 Cultured neurons ................................................................................................... 36 3.2.2 Acute spinal slices ................................................................................................. 37 3.3 Electrophysiological methods ........................................................................................... 37 3.3.1 Electrophysiological recording equipment ........................................................... 39 3.3.2 Drug delivery in vitro ...........................................................................................
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