THE ROLE OF CARBONIC ANHYDRASE IN THE MODULATION OF CENTRAL RESPIRATORY-RELATED PH/CO2 CHEMORECEPTOR-STIMULATED BREATHING IN THE LEOPARD FROG (RANA PIPIENS) FOLLOWING CHRONIC HYPOXIA AND CHRONIC HYPERCAPNIA by Kajapiratha Srivaratharajah A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology (Zoology) University of Toronto © Copyright by Kajapiratha Srivaratharajah (2008) The Role of Carbonic Anhydrase in the Modulation of Central Respiratory-Related pH/CO2 Chemoreceptor-Stimulated Breathing in the Leopard Frog (Rana pipiens) Following Chronic Hypoxia and Chronic Hypercapnia Kajapiratha Srivaratharajah Master of Science Graduate Department of Cell and Systems Biology University of Toronto 2008 ABSTRACT The aim of this thesis was to elucidate the role of carbonic anhydrase (CA) in the modulation of central pH/CO2-sensitive fictive breathing (measured using in vitro brainstem-spinal cord preparations) in leopard frogs (Rana pipiens) following exposure to chronic hypercapnia (CHC) and chronic hypoxia (CH). CHC caused an augmentation in fictive breathing compared to the controls (normoxic normocapnic). Addition of acetazolamide (ACTZ), a cell-permeant CA inhibitor, to the superfusate reduced fictive breathing in the controls and abolished the CHC- induced augmentation of fictive breathing. ACTZ had no effect on preparations taken from frogs exposed to CH. Addition of bovine CA to the superfusate did not alter fictive breathing in any group, suggesting that the effects of ACTZ were due to inhibition of intracellular CA. Taken together, these results indicate that CA is involved in central pH/CO2 chemoreception and the CHC-induced increase in fictive breathing in the leopard frog. ii ACKNOWLEDGMENTS First and foremost, I would like to thank my supervisor, Dr. Stephen Reid, without whom this thesis project would not be possible. Dr. Reid, thank you for encouraging me to pursue this accelerated Masters program and for your support and guidance throughout the course of this project. I also wish to thank Dr. Herbert Kronzucker, Dr. Rene Harrison and Dr. Les Buck for their input and suggestions. I am indebted to many student colleagues for their assistance at various stages of this thesis project and they are: Jessica McAneney for her technical expertise with the in vitro brainstem-spinal cord preparation and histological analysis of amphibian brain sections; Balinda Phe for her knowledge on gelatin-chrome-alum coating of slides and tissue staining protocols as well as her assistance with the analysis of stained brain tissue and finally, Sherri Thiele for her input with regards to histochemical analysis of stained brain tissue. In addition, I wish to thank the entire Reid lab (Balinda, Jeff, Jessica and Alex) for providing me with an enjoyable and stimulating environment to conduct my thesis in. I would also like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this thesis project. Finally, to my family and friends, thanks for putting up with my long hours in the laboratory and I am ever grateful for your love and support throughout the years. Permission has been obtained from the copyright owners (Elsevier and Permissions Department of Annual Reviews) and or authors (Dr. Stephen Reid) for the inclusion of figures (Figures 2 and 3 in Chapter 1) from published manuscripts and for the use of this thesis by the National Library. iii TABLE OF CONTENTS ABSTRACT....................................................................................................................................ii ACKNOWLEDGEMENTS............................................................................................................iii TABLE OF CONTENTS................................................................................................................iv LIST OF FIGURES......................................................................................................................viii LIST OF ABBREVIATIONS..........................................................................................................x CHAPTER 1: GENERAL INTRODUCTION................................................................................1 Preamble..............................................................................................................................2 Breathing in Anuran Amphibians........................................................................................3 The Mechanics of Breathing....................................................................................3 Bimodal Breathing...................................................................................................9 Discontinous Breathing..........................................................................................10 Respiratory Control Systems in Anuran Amphibians........................................................12 Central Control of Breathing.................................................................................12 Olfactory Chemoreceptors.....................................................................................14 Pulmonary Stretch Receptors................................................................................15 Peripheral (Arterial) Chemoreceptors....................................................................16 iv Central Chemoreceptors.........................................................................................17 The In Vitro Brainstem-Spinal Cord Preparation..............................................................18 Environmental Hypoxia and Hypercapnia.........................................................................22 Chronic Hypercapnia Studies............................................................................................24 Chronic Hypoxia Studies...................................................................................................30 Carbonic anhydrase............................................................................................................34 Discovery and Kinetics..........................................................................................34 Role of Carbonic Anhydrase in CO2 Chemoreception..........................................36 Hypothesis & Goals of the thesis.......................................................................................41 CHAPTER 2: EFFECTS OF CARBONIC ANHYDRASE INHIBITION WITH ACETAZOLAMIDE ON FICTIVE BREATHING IN CHRONICALLY HYPOXIC AND HYPERCAPNIC LEOPARD FROGS (RANA PIPIENS)...................................................................43 Introduction........................................................................................................................44 Materials and Methods.......................................................................................................46 Experimental Animals...........................................................................................46 Exposure to Chronic Hypoxia and Hypercapnia...................................................46 In Vitro Brainstem Spinal Cord Preparations........................................................47 Acetazolamide........................................................................................................49 Experimental Protocol...........................................................................................50 v Data and Statistical Analysis.................................................................................51 Results................................................................................................................................53 Discussion..........................................................................................................................76 Conclusion.........................................................................................................................83 CHAPTER 3: EFFECTS OF EXOGENOUS CARBONIC ANHYDRASE APPLICATION ON FICTIVE BREATHING IN ISOLATED IN VITRO BRAINSTEM-SPINAL CORD PREPARATIONS TAKEN FROM CHRONICALLY HYPOXIC AND HYPERCAPNIC LEOPARD FROGS (RANA PIPIENS)......................................84 Introduction........................................................................................................................85 Materials and Methods.......................................................................................................87 Experimental Animals...........................................................................................87 Exposure to Chronic Hypoxia and Hypercapnia...................................................87 In Vitro Brainstem Spinal Cord Preparations........................................................87 Carbonic Anhydrase...............................................................................................87 Experimental Protocol...........................................................................................88 Data and Statistical Analysis.................................................................................88 Results................................................................................................................................89 Discussion........................................................................................................................110 Conclusion.......................................................................................................................114