REGULATION OF NEUROPEPTIDE RELEASE IN THE SCN CIRCADIAN CLOCK: IN VIVO ASSESSMENTS OF NPY, VIP, AND GRP A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Jessica M. Francl December 2010 i ii Dissertation written by Jessica M. Francl B.S., Kent State University, 2003 M.S., The University of Akron, 2005 Ph.D., Kent State University, 2010 Approved by Dr. J.David Glass , Chair, Doctoral Dissertation Committee Dr. Eric M. Mintz , Member, Doctoral Dissertation Committee Dr. Robert V. Dorman , Member, Doctoral Dissertation Committee Dr. Brian P. Bagatto , Member, Doctoral Dissertation Committee Dr. William Lynch , Member, Doctoral Dissertation Committee Accepted by Dr. James L. Blank , Chair, Department of Biology Dr. John R. D. Stalvey , Dean, College of Arts and Sciences ii iii 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 SPECIFIC AIMS...............................................................................................................22 MATERIALS AND METHODS......................................................................................27 RESULTS..........................................................................................................................39 DISCUSSION....................................................................................................................61 REFERENCES..................................................................................................................82 iii iv LIST OF ABBREVIATIONS 5-HT.....................................................................................................5-hydroxytryptamine 8-OH-DPAT...............................(±)8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide ACSF.......................................................................................artificial cerebral spinal fluid AP5.............................................................................(2R)-amino-5-phosphonovaleric acid AVP.......................................................................................................arginine vasopressin BB2........................................................................................................bombesin receptor 2 BSA....................................................................................................bovine serum albumin CalB........................................................................................................................calbindin cAMP..................................................................................................................cyclic AMP CNS.....................................................................................................central nervous sytem CSF........................................................................................................cerebral spinal fluid CT....................................................................................................................circadian time DD.............................................................................................................constant darkness DMH...........................................................................................dorsomedial hypothalamus DR.........................................................................................................dorsal raphe nucleus GABA....................................................................................................γ-aminobutyric acid GHT............................................................................................geniculohypothalamic tract GRP................................................................................................gastrin-releasing peptide iv IGL......................................................................................................intergeniculate leaflet LD...........................................................................................................................light:dark MAPK................................................................................mitogen-activated protein kinase MR......................................................................................................median raphe nucleus mRNA........................................................................................messenger ribonucleic acid NMDA................................................................................................N-methyl-D-aspartate NPY...............................................................................................................neuropeptide Y p-ERK..........................................................................extracellular signal-regulated kinase PAC1.............................................pituitary adenylate cyclase-activating peptide receptor 1 PACAP..........................................................pituitary adenylate cyclase-activating peptide PCPA............................................................................................para-chlorophenylalanine Per1..................................................................................................................period gene 1 Per2..................................................................................................................period gene 2 PHI............................................................................................peptide histodine isoleucine PKA............................................................................................................protein kinase A PRC......................................................................................................phase response curve PVN................................................................paraventricular nucleus of the hypothalamus PVT.........................................................................paraventricular nucleus of the thalamus RGC.......................................................................................................retinal ganglion cell RHT................................................................................................retinohypothalamic tract RIA..........................................................................................................radioimmunoassay SCN.................................................................................................suprachiasmatic nucleus v vi SP........................................................................................................................substance P SS.......................................................................................................................somatostatin TBS.........................................................................................................Tris-buffered saline VIP.................................................................................... vasoactive intestinal polypeptide VPAC2..............................................................vasoactive intestinal polypeptide receptor 2 Y1.....................................................................................................NPY receptor subtype 1 Y2.....................................................................................................NPY receptor subtype 2 Y5.....................................................................................................NYP receptor subtype 5 ZT....................................................................................................................zeitgeber time vi vii LIST OF FIGURES Figure 1 ...............................................................................................................................6 Figure 2 ...............................................................................................................................7 Figure 3 .............................................................................................................................12 Figure 4 .............................................................................................................................29 Figure 5 .............................................................................................................................32 Figure 6 .............................................................................................................................36 Figure 7 .............................................................................................................................37