AMYLIN MEDIATES BRAINSTEM CONTROL OF HEART RATE IN THE DIVING REFLEX A Dissertation Submitted to The Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy By Fan Yang May, 2012 Examination committee members: Dr. Nae J Dun (advisor), Dept. of Pharmacology, Temple University Dr. Alan Cowan, Dept. of Pharmacology, Temple University Dr. Lee-Yuan Liu-Chen, Dept. of Pharmacology, Temple University Dr. Gabriela Cristina Brailoiu, Dept. of Pharmacology, Temple University Dr. Parkson Lee-Gau Chong, Dept. of Biochemistry, Temple University Dr. Hreday Sapru (external examiner), Depts. of Neurosciences, Neurosurgery & Pharmacology/Physiology, UMDNJ-NJMS. i © 2012 By Fan Yang All Rights Reserved ii ABSTRACT AMYLIN’S ROLE AS A NEUROPEPTIDE IN THE BRAINSTEM Fan Yang Doctor of Philosophy Temple University, 2012 Doctoral Advisory Committee Chair: Nae J Dun, Ph.D. Amylin, or islet amyloid polypeptide is a 37-amino acid member of the calcitonin peptide family. Amylin role in the brainstem and its function in regulating heart rates is unknown. The diving reflex is a powerful autonomic reflex, however no neuropeptides have been described to modulate its function. In this thesis study, amylin expression in the brainstem involving pathways between the trigeminal ganglion and the nucleus ambiguus was visualized and characterized using immunohistochemistry. Its functional role in slowing heart rate and also its involvement in the diving reflex were elucidated using stereotaxic microinjection, whole-cel patch-clamp, and a rat diving model. Immunohistochemical and tract tracing studies in rats revealed amylin expression in trigeminal ganglion cells, which also contained vesicular glutamate transporter 2 positive. With respect to the brainstem, amylin containing fibers were discovered in spinal trigeminal tracts. These fibers curved dorsally toward choline acetyltransferase immunoreactive neurons of the nucleus ambiguus, suggesting that amylin may synapse to parasympathetic preganglionic neurons in the nucleus ambiguus. Microinjection of fluorogold to the nucleus ambiguus retrogradely labeled a population of trigeminal ganglion neurons; some of which also contained amylin. In urethane-anesthetized rats, stereotaxic microinjections of amylin to the nucleus ambiguus caused a dose-dependent iii bradycardia that was reversibly attenuated by microinjections of the selective amylin receptor antagonist, salmon calcitonin (8-32) (sCT (8-32)) or AC187, and abolished by bilateral vagotomy. In an anesthetized rat diving model, diving bradycardia was attenuated by glutamate receptor antagonists CNQX and AP5, and was further suppressed by AC187. Whole-cel patch-clamp recordings from cardiac preganglionic vagal neurons revealed that amylin depolarizes neurons while decreasing conductance. Amylin also resulted in a reduction in whole cell currents, consistent with the decrease in conductance. Amylin is also found to increase excitability of neurons. In the presence of TTX, spontaneous currents in cardiac preganglionic vagal neurons were observed to decrease in frequency in response to amylin while amplitude remained constant, signifying that amylin reduces presynaptic activity at cardiac preganglionic vagal neurons. Finally, evoked synaptic currents revealed that amylin decreases evoked currents, further demonstrating that amylin depolarization and increase in excitability of cardiac preganglionic vagal neurons is also associated with simultaneous inhibition of presynaptic transmission. Our study has demonstrated for the first time that the bradycardia elicited by the diving reflex is mediated by amylin from trigeminal ganglion cells projecting to cardiac preganglionic neurons in the nucleus ambiguus. Additionally, amylin results in the depolarization and increased excitability of cardiac preganglionic vagal neurons while inhibiting presynaptic transmission. iv DEDICATION I would like to dedicate this thesis to my family, who without their endless love and support, this day would not be possible. To my loving wife and best friend Irene Hwa Yang, thank you for supporting me and walking by my side through this journey. To my parents, who I have the highest respect for, thank you for the never ending love and enthusiasm. To my brother, in whom I see myself, you have taught me more about myself and about life than anyone else. Thank you. v ACKNOWLEDGEMENTS I would like to thank everyone who has made helped make my thesis work possible through supporting my professional development and personal growth. I would like to thank my PhD mentor Dr. Nae Dun. Through his thoughtful guidance in my thesis work, he has helped open my mind to science. Dr. Dun has exposed me to a depth of scientific thinking that has made a lasting impact in my life. His unwavering support in my work has given me the ability to develop my intellectual capability immensely. Most of all, as a mentor, Dr. Dun has helped me think out of the box and apply what I have learned not only to furthering scientific knowledge, but also to all areas of life. The members of the Dun lab have provided a nurturing and supportive environment in which I have spent some of the most memorable years of my life. I want to thank Mrs. Siok Le Dun for the patience in teaching me immunohistochemistry and helping me navigate through the immense resources available in the Dun Lab. Without her patience, guidance, and support, none of my experiments would be possible. I also want to thank Dr. Cristina Brailoiu, for serving my PhD committee, and also helping me learn electrophysiology. It is the most technically challenging scientific tool I have ever mastered and it would not have been possible without her help. I want to thank my fellow PhD students in the Dun Lab whom have taken the quest for science together- Saadet Inan, Xiaofang Huang, and Elena Deliu. Through countless failed experiments we persevered while building lifelong friendships. I want to thank my thesis committee members for providing me with thoughtful feedback over the years and helping nurture my love for science. vi TABLE OF CONTENTS Pages ABSTRACT .................................................................................................................................... iii DEDICATION ................................................................................................................................. v ACKNOWLEDGEMENTS ............................................................................................................ vi LIST OF FIGURES: ........................................................................................................................ x LIST OF ABBREVIATIONS ................................................................................................................. xi CHAPTER 1: INTRODUCTION ............................................................................................................ 1 1.1 Importance of neural control of heart rate: .......................................................................... 1 1.2 Organization of the nervous system: ..................................................................................... 2 1.3 Autonomic Nervous System ................................................................................................... 3 1.3.1 Somatic Reflexes ............................................................................................................. 4 1.3.2 Autonomic Reflexes ........................................................................................................ 4 1.3.3 Baroreceptor reflex ......................................................................................................... 5 1.4 Information transmission and processing ............................................................................. 7 1.4.1 Excitable membranes ...................................................................................................... 7 1.4.2 Membrane permeability ................................................................................................. 8 1.5 Synapse ................................................................................................................................ 12 1.6 Neurotransmitters ............................................................................................................... 13 1.7 Ionotropic and Metabotropic receptors .............................................................................. 15 1.8 Neuropeptides ..................................................................................................................... 18 1.9 Neuropeptide synthesis and processing .............................................................................. 21 1.10 Neuropeptide Receptors .................................................................................................... 23 1.11 Amylin: ............................................................................................................................... 24 1.11.1 Circulating amylin ....................................................................................................... 24 1.11.2 Amylin the neuropeptide ............................................................................................ 27 Amylin action on neurons ...................................................................................................... 29 1.11.3 Amylin Genetics .........................................................................................................