PHASE REGULATION OF THE SCN CIRCADIAN CLOCK: SEROTONERGIC AND NEUROPEPTIDERGIC MECHANISMS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Gagandeep Kaur December 2009 Dissertation written by Gagandeep Kaur B.V.Sc.&A.H., Punjab Agricultural University, 2005 Ph.D., Kent State University, 2009 Approved by Dr. J.David Glass , Chair, Doctoral Dissertation Committee Dr. Eric M. Mintz , Member, Doctoral Dissertation Committee Dr. Sean L. Veney , Member, Doctoral Dissertation Committee Dr. Mary Ann Raghanti , Member, Doctoral Dissertation Committee Dr, Stephen B. Fountain , Member, Doctoral Dissertation Committee Accepted by Dr. James L. Blank , Chair, Department of Biological Sciences Dr. John R.D. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS TITLE PAGE……….……………………………………………………………………...i APPROVAL PAGE…….………………………………………………………………... ii TABLE OF CONTENTS……………………………….………………………………. iii LIST OF ABBREVIATIONS……….……………………………………………………iv LIST OF FIGURES……………………………………………………………………...vii ACKNOWLEDGEMENTS……………………………………………………………...ix DEDICATION……….…………………………………………………………………...xi INTRODUCTION………………………………………………………………………...1 SIGNIFICANCE AND SPECIFIC AIMS……………………………………………….32 MATERIALS AND METHODS………………………………………………………...38 RESULTS…......................................................................................................................50 DISCUSSION……………………………………………………………………………67 REFERENCES……………………………......................................................................91 iii LIST OF ABBREVIATIONS 5-HIAA…………………………………………………......….5-hydroxyindoleacetic acid 5-HT………………………………………………………………….5-hydroxytryptamine 8-OH-DPAT………………………………………8-hydroxy-2-di-m-propylamino tetralin ACSF………………………………………………………...artificial cerebral spinal fluid ACTH………………………………………………….…….adrenocorticotropic hormone AVP…………………………………………………………………...arginine vasopressin BMAL1 …brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 cAMP………………………………………………..3,5-cyclic adenosine monophosphate CRE ……………………………………………..cAMP response element binding protein CRH………………………………………………………corticotropin releasing hormone CSF……………………………………………………………………..cerebrospinal fluid CT……………………………………………………………………………circadian time DD……………………………………………………………………….constant darkness DRN…………………………………………………………………..dorsal raphe nucleus GABA…………………………………………………………………γ-aminobutyric acid GHT……………………………………………………………geniculohypothalamic tract GRP…………………………………………………………….…gastrin releasing peptide IGL…………………………………………………………………..intergeniculate leaflet i.p……………………………………………………………………………intraperitoneal iv LD………………………………………………………………………………...light:dark LL…………………………………………………………………………….constant light LLb…………………………………………………………………...…brief constant light MRN………………………………………………………………....median raphe nucleus NMDA……………………………………………………………….N-methy-D-aspartate NPY………………………………………………………………………..neuropeptide Y PACAP……………………………………..pituitary adenylate cyclase activating peptide PC ……………………………………....polycarbonate-polyether copolymeric membrane PCPA……………………………………………………………..parachlorophenylalanine PK2 ………………………………………………………………….............prokineticin 2 PRC…………………………………………………………………..phase response curve PVN…………………………………………………………….….paraventricular nucleus RIA...…………………………………………………………………..radioimmunoassay RHT………………………………………………………………retinohypothalamic tract SCN…………………………………………………………….…suprachiasmatic nucleus SERT……………………………............................................serotonin transporter protein SON………………………………………………………………….….supraoptic nucleus SSRI…………………………………………………selective serotonin reuptake inhibitor VIP……………………………………………………….vasoactive intestinal polypeptide V1a ………………………………………………………………..vasopressin receptor 1a V1b ………………………………………………………………..vasopressin receptor 1b V2 …………………………………………………………………..vasopressin receptor 2 v ZT……………………………………………………………………………zeitgeber vi LIST OF FIGURES Figure 1 ...............................................................................................................................4 Figure 2 ...............................................................................................................................5 Figure 3 ...............................................................................................................................6 Figure 4 ...............................................................................................................................8 Figure 5 .............................................................................................................................11 Figure 6 .............................................................................................................................22 Figure 7 .............................................................................................................................41 Figure 8 .............................................................................................................................43 Figure 9 .............................................................................................................................48 Figure 10 ...........................................................................................................................51 Figure 11 ...........................................................................................................................52 Figure 12 ...........................................................................................................................54 Figure 13 ...........................................................................................................................56 Figure 14 ...........................................................................................................................57 Figure 15 ...........................................................................................................................58 Figure 16 ...........................................................................................................................59 Figure 17 ...........................................................................................................................61 Figure 18 ...........................................................................................................................62 Figure 19 ...........................................................................................................................63 vii Figure 20 ...........................................................................................................................65 Figure 21 ...........................................................................................................................66 viii ACKNOWLEDGEMENTS First of all, I would like to thank my doctoral advisor Dr. J. David Glass for his guidance and support. I feel honored to have been mentored by a great scientist like him. Science has ups and downs, but his determination, consistently positive attitude and excellent writing skills are lessons to be followed. Not to forget, his time devoted towards improving my writing skills will serve as a strong foundation in my future career. My sincere gratitude to Dr. Glass for believing in me as a graduate student. Next, I would like to thank the members of my advisory committee, Dr. Eric Mintz, Dr. Sean Veney and Dr. Maryann Raghanti, for their valuable time and guidance. Special thanks to Dr. Mintz for answering all my important as well as trivial queries instantly and making complicated things simpler. I would like to thank all the Glass lab members, Jessie Francl, Jessie Guinn, Marc Depaul, Steve Hammer, Allison Brager and Christina Ruby for being wonderful friends and for all the fun and support. I treasure the time we spent together in lab and outside. Sharing a project with Jessie Francl was a great experience. I am thankful to Amelie Cornill for her help with animal handling during my early days in the lab and to Marc Depaul, for his great help with computer programming and softwares. A special thanks to Dr. Douglas Kline for his encouragement and direction to bring out the best in me as a teacher. Additionally thanks to Pat Williams and Donna Warner for their help with registering classes and getting official paperwork done. ix Finally, a special thanks to my soul mate and husband Raja Thind, for his love, support and confidence in me. For always putting me before himself in career and comfort. Throughout these four years, there were ups and downs, but I have had so much love and support from him, that this journey always seemed smooth. And thanks to my elder sister Sukhdeep and brother Japjit for their unconditional love and support. x DEDICATION I dedicate this dissertation to my wonderful, loving and caring parents, Mr. and Mrs. Iqbal Singh. To their hard work, sacrifices, blessings and never ending support. They have and will always be my strength and inspiration. Their encouragement and belief in me had made all this possible, it is their hard work that will shine as a gem in my career. xi INTRODUCTION The Suprachiasmatic Nucleus The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the master circadian clock in mammals (Inouye and Kawamura, 1979; Klein et al., 1991; Stephan and Zucker, 1972). It acts as body’s endogenous pacemaker and generates nearly 24 hour (i.e. circadian) rhythms of physiologic, metabolic and electrical activities. Several studies provide credible evidence for the SCN