Adenosinergic and GABAergic modulation of neuronal activity in the hypoxia-tolerant pond snail Lymnaea stagnalis by Aqsa Malik A thesis submitted in conformity with the requirements For the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Aqsa Malik (2010) Adenosinergic and GABAergic modulation of neuronal activity in the hypoxia-tolerant pond snail Lymnaea stagnalis Aqsa Malik Master of Science (2010) Department of Cell and Systems Biology University of Toronto ABSTRACT The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABAA receptor because of sensitivity to GABAA receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity. ii ACKNOWLEDGEMENTS This thesis is the result of the wonderful company, guidance, and inspiration that I received from my mentors, collaborators, and friends during the last two years of my graduate education. I would like to extend my gratitude to the following people, without whom the completion of my degree would not have been possible. Firstly, I am indebted to my mentor and supervisor, Dr. Leslie Buck. His enthusiastic disposition and passion for science shaped the development of my positive perspective towards scientific research. His experimental and academic support was instrumental in my success as a graduate student. I am also grateful for his approachable and friendly mentoring approach. I would also like to thank my committee advisors Dr. Melanie Woodin and Dr. Zhong- Ping Feng for their invaluable technical and conceptual help throughout the course of my thesis project. I am thankful for their time, criticism, and attention to my work. I am also very appreciative for having the opportunity to work amongst such bright and welcoming colleagues and peers. I am grateful to Dave Hogg and Matthew Pamenter for their engaging discussions and for their assistance in data analysis. I am thankful to George Zivkovic for his unrelenting technical support and encouragement. I owe special gratitude and recognition to Brooke Acton for being a source of friendship, wisdom, and laughter. I would also like to thank Ian Buglass for helping me navigate through the administrative aspects of graduate life. I am thankful to my brothers and sister for their generosity and kindness and for inspiring me to live a well-balanced fulfilling life. From the three of you I learned the meaning of “If you want to go fast, go alone. If you want to go far, go together.” Finally, and most importantly, I am deeply grateful to my parents for teaching me the value and power of education, encouraging me to have confidence in my abilities, and supporting my academic pursuits and career goals. iii TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii ACNOWLEDGEMENTS................................................................................................ iii TABLE OF CONTENTS ................................................................................................ iv LIST OF TABLES AND FIGURES .............................................................................. vii ABBREVIATIONS ........................................................................................................ viii CHAPTER 1: INTRODUCTION ....................................................................................1 1.1. Anaerobic metabolism and evolution of O2 .........................................................1 1.2. Mammalian neurons—hypoxia-sensitive.............................................................2 1.3. Mechanisms of hypoxia tolerance.........................................................................3 1.3.1. Metabolic Arrest ...........................................................................................3 1.3.2. Channel Arrest ..............................................................................................4 1.4. Adenosine-mediated neuroprotection ..................................................................5 1.4.1. Adenosine structure, receptors & function ...................................................5 1.4.2. Adenosine and ischemic preconditioning .....................................................7 1.4.3. Role of adenosine in hypoxia tolerance—vertebrates .................................8 1.4.4. Role of adenosine in hypoxia tolerance—invertebrates ............................11 1.5. GABA-mediated neuroprotection ......................................................................14 1.5.1. GABA—primitive developmental signal ...................................................14 1.5.2. GABA receptors ..........................................................................................15 1.5.3. Polarity of GABA transmission and cation-chloride cotransporters ..........16 1.5.4. GABA and ischemic preconditioning .........................................................18 1.5.5. Role of GABA in hypoxia tolerance ...........................................................19 1.6. Lymnaea stagnalis as an invertebrate model of hypoxia tolerance ..................20 iv 1.7. Rationale and Hypotheses ...................................................................................22 CHAPTER 2: METHODS ..............................................................................................24 2.1. Animals .................................................................................................................24 2.2. Anoxia tolerance ...................................................................................................24 2.3. Solutions and dissection ......................................................................................24 2.4. Electrophysiology .................................................................................................26 2.5. Fluo-4 intracellular Ca2+ imaging .......................................................................26 2.6. Chemicals ..............................................................................................................27 2.7. Statistical Analysis ...............................................................................................29 CHAPTER 3: RESULTS ................................................................................................30 3.1. Anoxia tolerance ...................................................................................................30 3.2. Adenosinergic modulation of neuronal activity ................................................31 3.3. GABAergic modulation of neuronal activity .....................................................39 CHAPTER 4: DISCUSSION ..........................................................................................46 4.1. Anoxia tolerance ...................................................................................................46 4.2. Modulation of neuronal activity by adenosine ..................................................46 4.2.1. Summary of findings...................................................................................46 4.2.2. Mechanisms of adenosine-mediated depression .........................................47 4.2.3. Seasonal differences in adenosinergic transmission ...................................48 4.3. Modulation of neuronal activity by GABA .......................................................49 4.3.1. Summary of findings...................................................................................49 4.3.2. Regulation of cation-chloride cotransporter function .................................52 v 4.3.3. Seasonal differences in GABAergic neurotransmission .............................54 4.3.4. Physiological significance of excitatory GABA .........................................55 4.3.5. Conclusions and future directions ...............................................................56 CHAPTER 5: REFERENCES ........................................................................................58 vi LIST OF TABLES AND FIGURES CHAPTER 1: INTRODUCTION Table 1-1: Concentration and effects of adenosine on various invertebrate preparations 11 CHAPTER 2: MATERIALS AND METHODS Table 2-1: Cluster F neurons of L. stagnalis pedal ganglia ..............................................25 Figure 2-2: Effects of DMSO on AP frequency in cluster F neurons ...............................28 CHAPTER 3: RESULTS Table 3-1: Anoxic tolerance of L. stagnalis ......................................................................30 Table 3-2: Effect of adenosine on AP frequency measured in summer