(Title of the Thesis)*

THE PHYSIOLOGICAL ACTIONS OF ADIPONECTIN IN CENTRAL AUTONOMIC NUCLEI: IMPLICATIONS FOR THE INTEGRATIVE CONTROL OF ENERGY HOMEOSTASIS by Ted Donald Hoyda A thesis submitted to the Department of Physiology In conformity with the requirements for the degree of Doctor of Philosophy Queen‟s University Kingston, Ontario, Canada (September, 2009) Copyright © Ted Donald Hoyda, 2009 ABSTRACT Adiponectin regulates feeding behavior, energy expenditure and autonomic function through the activation of two receptors present in nuclei throughout the central nervous system, however much remains unknown about the mechanisms mediating these effects. Here I investigate the actions of adiponectin in autonomic centers of the hypothalamus (the paraventricular nucleus) and brainstem (the nucleus of the solitary tract) through examining molecular, electrical, hormonal and physiological consequences of peptidergic signalling. RT-PCR and in situ hybridization experiments demonstrate the presence of AdipoR1 and AdipoR2 mRNA in the paraventricular nucleus. Investigation of the electrical consequences following receptor activation in the paraventricular nucleus indicates that magnocellular-oxytocin cells are homogeneously inhibited while magnocellular-vasopressin neurons display mixed responses. Single cell RT-PCR analysis shows oxytocin neurons express both receptors while vasopressin neurons express either both receptors or one receptor. Co-expressing oxytocin and vasopressin neurons express neither receptor and are not affected by adiponectin. Median eminence projecting corticotropin releasing hormone neurons, brainstem projecting oxytocin neurons, and thyrotropin releasing hormone neurons are all depolarized by adiponectin. Plasma adrenocorticotropin hormone concentration is increased following intracerebroventricular injections of adiponectin. I demonstrate that the nucleus of the solitary tract, the primary cardiovascular regulation site of the medulla, expresses mRNA for AdipoR1 and AdipoR2 and mediates adiponectin induced hypotension. Adiponectin has electrical effects on a majority of medial solitary tract neurons and depolarizes those expressing mRNA for the hypotensive neuropeptide Y, revealing a central mechanism to modulate blood pressure. ii Finally, I show that adiponectin controls paraventricular nucleus neuron excitability by either inhibiting a tetraethyl ammonium-sensitive potassium current thereby depolarizing neurons or activating a glibenclamide-sensitive voltage independent potassium current hyperpolarizing neurons. Therefore, adiponectin differentially modulates potassium current to confer its central effects. These results are the first to show the physiological and electrical actions of adiponectin on individual neurons in blood brain barrier protected central autonomic nuclei. This thesis provides a framework for how adiponectin acts centrally to coordinate whole body energy homeostasis and feeding behavior in the rat. iii CO-AUTHORSHIP Chapter 2: RT-PCR experiments were performed with assistance from Dr. Mark Fry. The in situ hybridization study was performed by Dr. Rexford S. Ahima. In all other instances, including conception, design, collection and analysis were performed by Ted D. Hoyda. The manuscript was written by Ted Hoyda with the assistance of Dr. Alastair V. Ferguson. Chapter 3: Single Cell RT-PCR experiments were performed with assistance from Ms. Christie Hopf, while icv injections of adiponectin followed by plasma assays for ACTH and TSH were performed by Dr. Willis K. Samson. Design, collection and analysis of all other experiments were performed by Ted D. Hoyda. The manuscript was composed by Ted D. Hoyda with assistance from Dr. Alastair V. Ferguson. Chapter 4: Experiments were designed by Ted D. Hoyda. Microinjection of adiponectin in the nucleus of the solitary tract and analysis of blood pressure and heart rate was performed by Ms. Pauline M. Smith. Single cell RT-PCR experiments were performed with Ms. Christie Hopf‟s assistance. All other data was collected and analyzed by Ted D. Hoyda. The manuscript was written by Ted D. Hoyda with the assistance of Dr. Alastair V. Ferguson. Chapter 5: All experiments were designed, collected and analyzed by Ted D. Hoyda. The manuscript was written by Ted Hoyda with assistance from Dr. Alastair V. Ferguson. iv ACKNOWLEDGEMENTS First and foremost I acknowledge and thank the „Boss‟, Al Ferguson for the important contributions you made to my development as a student and human-being. On my first „serious‟ day as a graduate student I remember you sharing those important words; “the data is what the data is”, a truth which has served me well throughout my studies and will for the rest of my life. Thanks for taking a chance and allowing me to pursue an important step in my education (and for that I will never count a „foot wedge‟ on your scorecard, even if we are playing with Dean). For all of you who I have had the pleasure of sharing a lab with past and present, thank you for making this seem rarely like work and more a time just spent with friends. Tequila Barbara, Jungle Cat Jenny, „Dish‟, Christie (phone: “h., o., p., f..for..”), Nancy („Drew‟ ie solving the mystery of the coed-softball double play), Matty (July 23, 2009, „Fran‟ time: 9:15, just for posterity‟s sake), Fry Guy and Spud (Friday afternoon/night? I know where to find you), PMS (is that Peter Gabriel singing?) and Dr. Price (Jebus…where to begin?) thanks for the great times and interesting conversations. Thanks to Coach G and Coach B who reveal me an important outlet with which to take out my (occasional) PhD frustrations, usually to the point where I just can‟t remember them anymore (hypoglycemia). A deep thanks to my parents, who show me the importance of being a „finisher‟, as they are themselves. Thanks to ES, FD and LT for helping me through the last couple of months. To the one who knows me best, I send to you my deepest thanks, appreciation and respect. I know I‟m complicated and difficult; often teetering on the brink in the whimsical fantasyland that I live in, but when I get lost Laura, I know I only have to look for your beacon, start walking and I‟ll be home. v TABLE OF CONTENTS ABSTRACT ------------------------------------------------------------------------------------------------- ii CO-AUTHORSHIP --------------------------------------------------------------------------------------- iv ACKNOWLEDGEMENTS ------------------------------------------------------------------------------- v TABLE OF CONTENTS -------------------------------------------------------------------------------------- vi LIST OF FIGURES --------------------------------------------------------------------------------------------- ix LIST OF TABLES ----------------------------------------------------------------------------------------------- xi LIST OF ABBREVIATIONS -------------------------------------------------------------------------------- xii Chapter 1 : GENERAL INTRODUCTION ----------------------------------------------------------- 1 ADIPOSE TISSUE AS AN ENDOCRINE ORGAN -------------------------------------------------- 3 ADIPONECTIN: STRUCTURE AND FUNCTION -------------------------------------------------- 3 The generation of adiponectin knockout mice 5 The insulin sensitizing effects of adiponectin 7 ADIPONECTIN RECEPTORS: DISCOVERY ---------------------------------------------------- 8 The generation of adiponectin receptor knockout mice 12 SIGNAL TRANSDUCTION PATHWAYS OF THE ADIPONECTIN RECEPTORS ---- 13 ADIPONECTIN ACTIONS IN THE CENTRAL NERVOUS SYSTEM --------------------- 14 Effects on sympathetic nervous system activity and autonomic function 16 THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS -------------------- 17 Neural inputs to the PVN 18 Projections of the PVN 19 Neuronal Subtypes in the PVN: Morphological and Electrical Characteristics 20 Type 1: Magnocellular Neurons 20 Type II: Pre-autonomic Parvocellular neurons 21 Type III: Parvocellular Neuroendocrine 22 Neurochemical cell phenotypes in the PVN 23 Oxytocin 23 Vasopressin 24 vi Thyrotropin releasing hormone 26 Corticotropin releasing hormone 27 THE NUCLEUS OF THE SOLITARY TRACT -------------------------------------------------- 28 Afferent projections to the NTS 28 Efferent projections of the NTS 29 Electrophysiology of the NTS 29 Neurochemical expression in the NTS 30 Neuropeptide Y 31 STATEMENT OF PROBLEM ----------------------------------------------------------------------- 33 Chapter 2 : ADIPONECTIN SELECTIVELY INHIBITS OXYTOCIN NEURONS OF THE PARAVENCTRICULAR NUCLEUS OF THE HYPOTHALAMUS -------------------- 34 ABSTRACT 35 INTRODUCTION 37 MATERIALS AND METHODS 38 RESULTS 44 DISCUSSION 51 ACKNOWLEDGEMENTS 58 Chapter 3 : ADIPONECTIN DEPOLARIZES PARVOCELLULAR PARAVENTRICULAR NUCLEUS NEURONS CONTROLLING NEUROENDOCRINE AND AUTONOMIC FUNCTION ---------------------------------------------------------------------- 59 ABSTRACT 60 INTRODUCTION 61 MATERIALS AND METHODS 62 RESULTS 68 DISCUSSION 77 ACKNOWLEDGEMENTS 81 Chapter 4 : ADIPONECTIN ACTS IN THE NUCLEUS OF THE SOLITARY TRACT TO DECREASE BLOOD PRESSURE BY MODULATING THE EXCITABILITY OF NEUROPEPTIDE Y NEURONS ----------------------------------------------------------------------- 82 ABSTRACT 83 INTRODUCTION 85 MATERIALS AND METHODS 86 RESULTS 93 DISCUSSION 99 ACKNOWLEDGEMENTS 103 Chapter 5 : IONIC MECHANISMS RESPONSIBLE FOR ADIPONECTIN INDUCED CHANGES TO THE EXCITABILITY OF PARVOCELLULAR PVN NEURONS ------- 104 vii ABSTRACT 105 INTRODUCTION 106 MATERIALS AND METHODS 108 RESULTS 111 DISCUSSION

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