THE ROLE OF GASTRIN-RELEASING PEPTIDE IN PHOTIC ENTRAINMENT

Transcription

1 THE ROLE OF GASTRIN-RELEASING PEPTIDE IN PHOTIC ENTRAINMENT A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by George J. Kallingal May 2008

2 Dissertation written by George J. Kallingal B.S. Santa Clara University, 2002 Ph.D., Kent State University, 2008 Approved by Dr. E. Mintz, Chair, Doctoral Dissertation Committee Dr. J. Glass, Member, Doctoral Dissertation Committee Dr. S. Veney, Member, Doctoral Dissertation Committee Dr. J. Marcinkiewicz, Member, Doctoral Dissertation Committee Dr. R. Salisbury, Member, Doctoral Dissertation Committee Accepted by Dr. R. Dorman, Director, School of Biomedical Sciences Dr. J. Stalvey, Dean, College of Arts and Sciences ii

3 TABLE OF CONTENTS Page LIST OF FIGURES..viii ACKNOWLEDGEMENTS..xiv CHAPTER I. Introduction.. 1 General Background on Circadian Rhythms... 1 Introduction to the Suprachiasmatic Nucleus SCN Inputs... 4 SCN Outputs 7 Photic vs. Non-photic Stimuli Organization of Neuropeptides in the SCN The Role of Glutamate in Circadian Rhythms The Role of GABA in Circadian Rhythms The Role of Gastrin-releasing Peptide in Circadian Rhythms The Role of Neuropeptide Y in Circadian Rhythms The Role of Serotonin in Circadian Rhythms Current Model of Circadian Shifts Cellular and Molecular Events of Circadian Shifts The Use of c-fos to Understand the Mechanisms of Photic Entrainment.. 29 Overall Aims.. 32 iii

4 Reference List CHAPTER II. Glutamatergic Activity Modulates the Phase-shifting Effects of Gastrinreleasing Peptide and Light Abstract.. 45 Introduction Materials and Methods...48 Subjects Surgical Procedure Microinjection Histology Data Analysis Results Discussion Acknowledgements Reference List CHAPTER III. Gastrin-releasing Peptide and Neuropeptide Y Exert Opposing Actions on Circadian Phase Abstract Introduction Materials and Methods iv

5 Subjects Immunohistochemistry Surgical Procedures Microinjection Histology for Behavioral Experiments Data Analysis Results Discussion Acknowledgements Reference List CHAPTER IV. The Influence of Gastrin-releasing Peptide on the Suprachiasmatic Nucleus Is Regulated by Glutamate, Serotonin, and the Supraoptic Nucleus Abstract..86 Introduction Materials and Methods...89 Subjects.. 89 Surgical Procedure. 89 Microinjection 90 Experiments 1 and Experiment Experiment v

6 Experiments 5 and Perfusion...92 Immunohistochemistry..92 Histology for Behavioral Experiments..94 Data Analysis of Phase Shifts Quantification of c-fos Immunoreactivity. 94 Quantification of p-erk Immunoreactivity..95 Results 96 Discussion 116 Reference List CHAPTER V. An NMDA Antagonist Inhibits Light But Not GRP-induced Phase Shifts When Administered After the Phase-shifting Stimulus Abstract 128 Introduction Materials and Methods. 131 Subjects 131 Surgical Procedure Microinjection Experiments 1 and Experiment Histology for Behavioral Experiments 134 vi

7 Data Analysis of Phase Shifts Results Discussion 142 Reference List..147 CHAPTER VI. Global Discussion Behavioral Endpoints Cellular Endpoints SCN Outputs 158 Future Directions.159 Reference List vii

8 LIST OF FIGURES Page CHAPTER I. Introduction Fig. 1A. SCN Inputs Fig. 1B. Effects of Phase Shifting Stimuli on Circadian Phase Fig. 1C. Photic and Non-Photic Phase Response Curves Fig. 1D. Distribution of Neuropeptides in Hamster SCN Fig. 1E. Gastrin-releasing Peptide Immunoreactivity in Hamster SCN Fig. 1F. Neuropeptide Y Immunoreactivity in Hamster SCN Fig. 1G. Serotonin Immunoreactivity in Hamster SCN Fig. 1H. Current Model of Circadian Shifts Fig. 1I. Cellular/Molecular Model of Circadian Shifts CHAPTER II. Glutamatergic Activity Modulates the Phase-shifting Effects of Gastrinreleasing Peptide and Light Fig. 2A. Representative actograms of circadian activity rhythms before and after microinjection of gastrin-releasing peptide, GRP and (±)-2-amino-5- phosphopentanoic acid, L-trans-pyrrolidine-2,4-dicarboxylic acid, and saline..52 Fig. 2B. Phase delays in hours induced by microinjection of gastrin-releasing peptide, GRP and (±)-2-amino-5-phosphopentanoic acid, L-trans-pyrrolidine-2,4- viii

9 dicarboxylic acid, or saline or by administration of a light pulse (LP) on the phase of free-running activity rhythms...53 Fig. 2C. Representative actograms of circadian activity rhythms before and after microinjection of gastrin-releasing peptide, GRP and L-trans-pyrrolidine-2,4- dicarboxylic acid, PDC and a light pulse, and saline and a light pulse.55 Fig. 2D. Actogram showing an unusual response to microinjection of gastrin-releasing peptide...61 CHAPTER III. Gastrin-releasing Peptide and Neuropeptide Y Exert Opposing Actions on Circadian Phase Fig. 3A. Representative actograms of circadian activity rhythms before and after microinjection of GRP, NPY, GRP/NPY cocktail, and SAL at CT Fig. 3B. Phase shift in hours induced by microinjection of GRP, NPY, GRP/NPY, and SAL near the SCN at CT 6 and CT Fig. 3C. Representative actograms of circadian activity rhythms before and after microinjection of NPY, GRP, NPY/GRP cocktail, and SAL at CT 6.77 Fig. 3D. Photomicrographs showing overlap of GRP and NPY immunoreactivity in the hamster SCN...81 CHAPTER IV. The Influence of Gastrin-releasing Peptide on the Suprachiasmatic Nucleus Is Regulated by Glutamate, Serotonin, and the Supraoptic Nucleus Fig. 4A. Representative photomicrographs of the hamster SCN at anterior, medial, and ix

10 posterior levels following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL 97 Fig. 4B. c-fos counts in the ventral 1/3 and dorsal 2/3 of the medial SCN induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL.98 Fig. 4C. Representative photomicrographs of the hamster SON and PVN following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL Fig. 4D. c-fos counts in a small section of the cortex induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL 101 Fig. 4E. Representative photomicrographs of the hamster SCN at anterior, medial, and posterior levels following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and p-erk optical density induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL.102 Fig. 4F. p-erk optical density in the ventral 1/3 and dorsal 2/3 of the medial SCN induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL..104 Fig. 4G. Representative photomicrographs of the hamster SON and PVN following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and p-erk optical density induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL x

11 Fig. 4H. Photomicrographs showing overlap of GRP and serotonin immunoreactivity in the hamster SCN. 106 Fig. 4I. Representative actograms of circadian activity rhythms before and after microinjection of GRP, DPAT, GRP and DPAT, and SAL and phase shift in hours induced by microinjection of GRP, GRP and DPAT, DPAT and SAL Fig. 4J. Representative photomicrographs of the hamster SCN at anterior, medial, and posterior levels following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into a region just dorsal to the SON 109 Fig. 4K. c-fos counts in the ventral 1/3 and dorsal 2/3 of medial SCN induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL near the SON 111 Fig. 4L. Representative photomicrographs of the hamster ipsilateral SON and contralateral SON following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into a region just dorsal to the SON 112 Fig. 4M. Representative photomicrographs of the hamster PVN following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL and c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into a region just dorsal to the SON Fig. 4N. Representative actograms of circadian activity rhythms before and after xi

12 microinjection of GRP and SAL and phase shift in hours induced by microinjection of GRP and SAL into a region just dorsal to the SON. 115 Fig. 4O. Representative actogram of circadian activity rhythms before and after microinjection of BIC and SAL and phase shift in hours induced by microinjection of BIC and SAL into a region just dorsal to the SON.117 CHAPTER V. An NMDA Antagonist Inhibits Light But Not GRP-induced Phase Shifts When Administered After the Phase-shifting Stimulus Fig. 5A. Representative actograms of circadian activity rhythms before and after a 15 min light pulse (2400 lux) and microinjection of AP5 before, SAL before, AP5 after, and SAL after light pulse Fig. 5B. Representative actograms of circadian activity rhythms before and after a 15 min light pulse (2400 lux) and microinjection of AP5 15 min after, SAL 15 min after, AP5 30 min after, and SAL 30 min after light pulse.137 Fig. 5C. Phase shift in hours induced by microinjection of AP5 or SAL near the SCN before, after, 15 min after, and 30 min after a 15 min light pulse (2400 lux) 138 Fig. 5D. Representative actograms of circadian activity rhythms before and after a 15 min light pulse (300 lux) and microinjection of AP5 before, SAL before, AP5 after, and SAL after light pulse 139 Fig. 5E. Representative actograms of circadian activity rhythms before and after a 15 xii

13 min light pulse (300 lux) and microinjection of AP5 15 min after, SAL 15 min after, AP5 30 min after, and SAL 30 min after light pulse Fig. 5F. Phase shift in hours induced by microinjection of AP5 or SAL near the SCN before, after, 15 min after, and 30 min after a 15 min light pulse (300 lux) Fig. 5G. Representative actograms of circadian activity rhythms before and after microinjection of GRP and microinjection of AP5 or SAL 15 min after GRP administration.143 Fig. 5H. Phase shift in hours induced by microinjection of AP5 or SAL near the SCN 15 min after the administration of GRP..144 xiii

14 ACKNOWLEDGEMENTS I would initially like to thank my advisor, Dr. Eric Mintz for his guidance, support, and friendship. His unique advising style unless needed was a perfect match for me. He allowed the members of his lab to work at their own pace, at times to his frustration. If he felt we were working hard, he would send a quick acknowledging our work. If he felt that more work could be done, he would make a comment or two, delicately crafted, yet sufficient to get our goals accomplished. It is difficult to imagine spending the last four years in a different lab. As is almost universally known within the department, my slight obsessive tendencies were not ridiculed but welcomed by the lab. In filing, (or if I am being honest refilling) my Kent Sate University general binder, I came upon my application to this institution. In that application, we were instructed to look up the research interests of departmental professors and to select five with whom we wished to work. Many students often get paired with advisors in a random fashion. I did not even remember until recently that Dr. Eric Mintz was one of my top choices. In addition to the professional relationship that we have acquired over the years, I have additionally developed a personal respect for Dr. Mintz. I am certain that we will continue to foster a relationship, both professional and personal alike, in the years to come. I wish him and his family all of the success they deserve and I am proud to have been a member of the Mintz lab. I would additionally like to thank the members of my committee. I appreciate the guidance of Drs. Glass, Marcinkiewicz, Veney, and Salisbury throughout my tenure at xiv

15 Kent State University. They have offered me incredible insight into the various fields of biology in the classroom and in their respective offices. I also am happy to have gotten to know them outside of the department, whether it be at social outings or conferences. Their suggestions and yes, even their tough questions during the defense of my prospectus have positively guided me throughout the last four years. I of course am grateful for the love, affection, and the financial support of my family. My parents, Drs. George and Matilda Kallingal, have been an incredible source of personal pride and I will never forget that they have stuck by me through the good times and the bad. Their unconditional love and support have helped me get through the years passed. An additional special thank you goes to my sister, Rosemary, my brotherin-law, Nuku, and their beautiful daughter, Sophia. As they have wished for me, I wish them all of the happiness and success that they deserve. Finally, I would like to extend a thank you to my friends and extended family, individuals too numerous to name. You have each affected me in unique ways that have helped to make me a better person. A special thank you is in order for the members of my lab, both past and present, including Colleen Campbell, Erin Herrman, Veronica Porterfield, and Erin Gilbert. For better or for worse, ours truly is a lab that will live in infamy. I would also like to take the opportunity to thank the departmental secretaries, especially Judith Wearden, for all of their help in registering me for classes and for research hours and being available to answer all of my questions, the pertinent and the not-so-pertinent alike. xv

16 CHAPTER I Introduction General Background on Circadian Rhythms Throughout the ages, the notion of time has always been significant in terms of evolutionary adaptation and Darwinian fitness. Plants, animals, and even bacteria have evolved cellular, molecular, and behavioral mechanisms to maximize the effectiveness of their daily activities. Plants, whose sustenance is largely dependent on the availability of their photosynthetic machinery to respond to sunlight, have evolved internal timing cues to maximize exposure to light (Myneni et al., 2007). Nocturnal animals can avoid increased rates of predation by being active at a time when there is little to no light. Certain kinds of bacteria have also learned to respond to environmental lighting conditions to minimize exposure to ultraviolet radiation during the replication phase of the cell cycle (McCready et al., 2005). In order to effectively predict optimal times for certain physiological and behavioral activities, living creatures have relied on the daily rotation of the earth, and the resulting light/dark cycles of the environment. This seemingly simple idea formed the basis for what is now known as chronobiology. Chronobiology can best be understood by breaking down the term into its roots. Chronos, bios, and logos are Greek terms that mean time, life, and study respectively. By combining these ideas, chronobiology comprises the scientific study of living timing 1

17 2 processes in plants and animals. Instead of passively responding to their environmental conditions, chronobiology teaches us that living beings can actively alter their behaviors by correctly predicting changes in the environment. The mechanisms by which these circadian activities occur have been the subject of intense scientific inquiry. There are several criteria that have been established to define certain activities as being circadian in nature (for review see (Miller, 1998)). The first is that the activity in question persists in constant conditions, such as constant dark, with a period of about 24 hours. If this can be shown to be true, then it logically follows that external conditions have been imprinted on some internal time-keeping mechanism. The second criterion is known as temperature compensation. When animals are exposed to different environmental temperatures, their free running period is very similar, despite changes in ambient temperatures. Finally, the third criterion is that any given rhythm can be reset by the simple exposure to an external stimulus. This will allow for the opportunity to reset internal timing mechanisms when they are not perfectly synchronized with the 24 hour daily schedule. The Webster s dictionary defines circadian as noting or pertaining to rhythmic biological cycles that recur at approximately 24-hour intervals. In this definition, the operative word is approximately. Most animals have periods that are close to, but not quite 24-hours. For example, Syrian hamsters have periods that are slightly shorter or slightly longer than 24-hours (Refinetti, 2006), while humans have periods that are closer to 25-hours (Czeisler and Klerman, 1999). For an animal exposed to environmental conditions, which generate a 24-hour daily cycle, slight changes to their period must

18 3 occur every day so that their physiological and behavioral activities are synchronized with the external environment. This necessity is in support of the third criterion for circadian activities mentioned above. Because internal timing mechanisms generate rhythms that are longer or shorter than 24 hours, the endogenous clock must rely on various time keepers (zeitgebers), to entrain to a strict 24-hour light dark schedule. Such time keepers include photic stimuli such as the light-dark cycle and non-photic stimuli such as food availability, temperature, or social interactions. Introduction to the Suprachiasmatic Nucleus Before exploring how these zeitgebers can affect internal time-keeping, it is essential to examine the neuroanatomical structures that regulate circadian activities. Through surgical and/or electrolytic destruction techniques, scientists identified a region of the anterior hypothalamus that drives rhythmic physiological and behavioral activities (Kafka et al., 1985;Moore and Eichler, 1972;Stephan and Zucker, 1972). This paired region of approximately 10,000 neurons was named the suprachiasmatic nucleus (SCN) because it was situated just dorsal to the optic chiasm and lateral to the third ventricle (Inouye and Kawamura, 1979). It was also discovered that the SCN contains an endogenous clock that can respond to photic and non-photic signals and can express rhythms that occur in roughly 24 hour cycles (Moore, 1983). Interestingly, tumors or lesions of the anterior hypothalamus result in the cessation of rhythms (Schwartz et al., 1986;Cohen and Albers, 1991). Transplantation studies have additionally provided significant support for the anatomical significance of the SCN. Animals that become

19 4 arrhythmic following the electrolytic lesion of the SCN can have their rhythms restored by transplanting SCN tissue from a donor animal (Lehman et al., 1987;Romero et al., 1993). Furthermore, the resulting rhythms of the recipient following transplant match those of the donor rather than the rhythms of the recipient prior to the electrolytic lesion (Lehman et al., 1987;Romero et al., 1993;Kaufman and Menaker, 1993;Ralph et al., 1990). The evidence presented herein provides strong support for the notion that the suprachiasmatic nucleus is the master clock that drives rhythms in both physiology and behavior. SCN Inputs Since the SCN has been identified as the endogenous clock that drives both behavioral and physiological rhythms, it is essential to examine the efferent projections that synapse on this nucleus. There are three primary projections that communicate both photic and non-photic information to the SCN (Fig. 1A). The first projection is the retinohypothalamic tract (RHT) which is a monosynaptic projection from the retina to the SCN and is therefore believed to convey environmental photic information to the hypothalamus (Hendrickson et al., 1972;Pickard, 1982;Pickard and Silverman, 1981). The RHT is believed to heavily innervate the ventral portion of the SCN in rodents (Morin, 1994). Outside of these retinorecipient regions, the RHT less densely innervates the dorsal portion of the SCN (Levine et al., 1991). It is believed that glutamate is the primary signal of the RHT (Castel et al., 1993), as direct stimulation of the optic nerve or administration of light to animals kept in constant darkness directly increases the activity

20 5 Third Ventricle Retinohypothalamic Tract Serotonin RAPHE NUCLEUS Median Dorsal Glutamate (PACAP) Suprachiasmatic Nucleus Intergeniculate NPY Leaflet Geniculohypothalamic Tract Fig. 1A: Schematic showing three major inputs that synapse on the SCN. The retinohypothalamic tract (RHT) utilizes glutamate (PACAP and aspartate) and has dense innervations (thick arrow) of the ventral region of the hamster SCN, while only having sparse innervations (thin, dotted arrow) of the dorsal region. The median raphe nucleus of the midbrain utilizes serotonin and projects to the ventral region of the SCN, while the dorsal component of the raphe projects to the intergeniculate leaflet. The geniculohypothalamic tract (GHT) comprises the third input that originates in the intergeniculate leaflet and utilizes neuropeptide Y (NPY) whose fibers extend to the ventral region of the hamster SCN.

21 6 of SCN neurons and such increases can be attenuated with the subsequent administration of glutamatergic antagonists (Colwell and Menaker, 1992). However, other signals such as pituitary adenylyl cyclase activating polypeptide (PACAP) and aspartate are also localized in the RHT (Castel et al., 1993;De Vries et al., 1993;Ebling, 1996;Hannibal, 2002). This projection is essential for photic entrainment, for the surgical destruction of the RHT leads to the inability to entrain to environmental light-dark cycles (Johnson et al., 1988). Finally, when the SCN is isolated and the optic nerves remain intact, electrical stimulation of the optic nerves elicits a phase response curve similar in shape and magnitude to the phase response curve of light (Shibata and Moore, 1993). These data provide significant support for the notion that a neuronal projection exists between the retina and the SCN and that this projection is essential for photic entrainment. The second major projection to the SCN, the geniculohypothalamic tract (GHT), comes from the intergeniculate leaflet of the thalamus (Ribak and Peters, 1975). Through the GHT, neuropeptide Y (NPY), synthesized in the intergeniculate leaflet, is released in the SCN (Card and Moore, 1982) where it can effect changes in circadian phase. NPY was initially believed to be a mediator of non-photic signals for a variety of reasons, including the fact that the phase response curve following NPY administration (Huhman and Albers, 1994) resembles that of other non-photic signals, like novelty-induced activity (Reebs and Mrosovsky, 1989) and social interactions (Mrosovsky, 1988). However, the GHT can affect photic signaling in the brain as well (Mason et al., 1987). The third major projection to the SCN originates in the median raphe nucleus located in the medial portion of the midbrain. Through this projection, serotonin is

22 7 released in the SCN to regulate circadian rhythms. Like the GHT, the projection from the raphe nucleus was initially believed to be solely a carrier of non-photic signals to the hypothalamus. The phase response curve elicited from the administration of serotonin (Yannielli and Harrington, 2004) to the SCN in vitro is similar in shape to other nonphotic signals (Mrosovsky, 1988;Reebs and Mrosovsky, 1989). An abundance of data however, indicates a potential role for serotonin in photic signaling as well (Weber et al., 1998;Hayashi et al., 2001). These three inputs do not constitute the only inputs to the SCN. There are additional inputs from other regions of the hypothalamus, such as the pre-optic area (POA), dorsomedial hypothalamus (DMH), arcuate nucleus (ARC), and the paraventricular nucleus (PVN) (Kriegsfeld et al., 2004;Watts et al., 1987;Watts and Swanson, 1987;Moga and Moore, 1997;Saeb-Parsy et al., 2000). However, the RHT, GHT, and the projection from the raphe to the SCN constitute three major inputs that synapse within the SCN to facilitate the entrainment of the circadian clock to an environmental light-dark schedule. SCN Outputs The combined use of retrograde and anterograde tracers has been enormously useful in identifying SCN inputs as well as SCN outputs. There are numerous outputs that project from the SCN to regions throughout the brain. These target regions include, but are not limited to, the bed nucleus of the stria terminalis (BST), medial pre-optic area (MPO), sub-paraventricular zone (SPVZ), anterior hypothalamic area (AHA),

23 8 paraventricular nucleus (PVN), and supraoptic nucleus (SON), each with varying densities of SCN-originating innervations (Kriegsfeld et al., 2004;Watts et al., 1987;Watts and Swanson, 1987;Moga and Moore, 1997;Cui et al., 1997;Hermes et al., 1996;Saeb-Parsy et al., 2000). These efferents allow for the coordination of circadian activities at distinct sites of the brain, which regulate physiological functions like urine production, hormone release, and body temperature. Just as the suprachiasmatic nucleus has been identified as the endogenous clock of mammals, these regions of the brain have specific functions. For example, the PVN and SON of the hypothalamus synthesize the neuropeptides arginine-vasopressin (AVP) and oxytocin (Windle et al., 1992). These peptides are believed to play important roles in the maintenance of water balance and milk ejection respectively (Windle et al., 1992) and day-night rhythms of the magnocellular neurons that synthesize these neuropeptides are evident (Cui et al., 1997). Photic vs. Nonphotic Stimuli The major inputs described above facilitate the entrainment of behavioral activity to the normal light-dark schedule. A useful nomenclature has been established to evaluate these rhythms. Circadian time (CT) is an indicator of the circadian phase and CT 12 is defined as the onset of activity within a 24-hour cycle. CT 6 and CT 18 are defined as six circadian hours before and after activity onset, with a circadian hour being equivalent to 1/24 of a circadian cycle. Photic stimuli presented in the early subjective night cause delays of circadian rhythms, while light exposure during the late subjective night phase advances the clock (DeCoursey, 1960). During the subjective day, however,

24 9 photic signals have little to no effect on circadian timing. Non-photic stimuli and certain signals, like serotonin or NPY, presented during the subjective day phase advance the clock, and presentation of these signals in the subjective night has little to no effect on clock phase (Fig. 1B). The responses to these various types of signals can be plotted on a phase response curve (PRC), which can be used to display the response of the circadian clock to phase-resetting cues (Fig. 1C). Organization of Neuropeptides in the SCN There now exists in the literature a consensus that the SCN is heterogeneous in structure and function (Antle and Silver, 2005;Morin et al., 2006). The cells that comprise the SCN have notable differences in morphological appearance, electrophysiological firing rates, and patterns of gene expression. These differences have led to the identification of two regions of the SCN that differ in their cellular, molecular, and neurochemical properties (Abrahamson and Moore, 2001;Card and Moore, 1984;Moore et al., 2002) (Fig. 1D). The shell is comprised of arginine-vasopressin (AVP) neurons that lack photic input (Abrahamson and Moore, 2001;Card and Moore, 1984;Moore et al., 2002). These cells are believed to be rhythmic, for oscillations of clock genes Per1 and Per2 mrna are noted in this region. In contrast, the core shows high levels of immunoreactivity for gastrin-releasing peptide (GRP) and vasoactive intestinal polypeptide (VIP) (Abrahamson and Moore, 2001;Card and Moore, 1984;Karatsoreos et al., 2004;Moore et al., 2002;Morin et al., 2006;Tanaka et al., 1997). It should be noted that the core/shell model, albeit a useful tool, is an oversimplification,

25 10 Early Night Photic Stimulus Late Night Photic Stimulus Day Photic Stimulus Early Night Non-Photic Stimulus Late Night Non-Photic Stimulus Day Non-Photic Stimulus Fig. 1B: Schematic of actograms showing the various responses to photic (top) and nonphotic (bottom) signals presented to animals kept in constant conditions at different times throughout the day. Red oval indicates the time in which the stimulus is presented. Dark bars indicate a measure of behavioral activity. Photic stimuli presented in the early and late night elicit phase delays and advances respectively, while having little effect on circadian phase when presented during the day. Non-photic stimuli presented in the middle of the day elicit phase advances, while having little effect on the circadian activity rhythm during the night.

26 Phase Shift (min.) Circadian Time (hrs.) Photic Signal Non-photic Signal Fig. 1C: Sample phase response curve (PRC) showing the behavioral response to both photic (blue diamonds) and non-photic (pink boxes) stimuli presented at various time points throughout the day. Circadian time (CT) is depicted on the x-axis with the subjective day ranging from CT 0-12 and the subjective night ranging from CT The y-axis depicts the magnitude of the phase shifts with the positive and negative values indicating a phase advance and phase delay respectively.

27 12 3 rd Ventricle Cap Cells AVP GRP VIP cell bodies & Serotonin & NPY Immunoreactivity Fig. 1D: Schematic showing the distribution of neuropeptides in the hamster SCN. The SCN shell, where cells rhythmically express arginine-vasopressin (AVP; red) is localized in the dorsomedial region of the hamster SCN. The SCN core includes various cell groups that express vasoactive intestinal polypeptide (VIP; yellow) and gastrin-releasing peptide (GRP; blue). The ventral region of the hamster SCN is also immunoreactive for serotonergic and NPY-ergic immunoreactivity (yellow). The cap cells (green) are situated dorsal to GRP-containing cells.

28 13 for these regions possess different functional subregions as well. GRP and VIP cells receive direct retinal input, indicating for these peptides a potential role for the integration and synchronization of environmental lighting conditions (Abrahamson and Moore, 2001;Karatsoreos et al., 2004;Morin et al., 2006;Tanaka et al., 1997). Dorsal to GRP cell bodies in the hamster SCN there is a group of cells that rhythmically express the phosphorylated form of extracellular related kinase (p-erk) and whose activities are driven by the retina (Lee et al., 2003b). These cells have come to be known as cap cells due to their appearance (Antle and Silver, 2005) and location in the hamster SCN and contain significant overlap with gastrin-releasing cell bodies (Lee et al., 2003a). While the exact roles of cap cells are unclear, it remains a possibility that these cells receive and integrate information coming from GRP and/or VIP cell bodies and utilize the projections that exist between the cap cells and AVP cells of the dorsomedial SCN to effect light-induced changes in circadian phase. The spatial relationship between the peptides in the core and the AVP-ir oscillator cells of the shell highlight the heterogeneity of the SCN (Antle and Silver, 2005) and form the basis of the current model used to understand the signaling events involved in photic entrainment. The Role of Glutamate in Circadian Rhythms Glutamate is extremely abundant not just throughout the hypothalamus, but throughout the entire central nervous system as well. Immunohistochemical studies have indicated that glutamate and its various receptors, both ionotropic and metabotropic, are found in the SCN and that most cells of the hypothalamus are responsive to glutamate

29 14 levels manipulated by the presence or absence of glutamatergic agonists or antagonists (Van Den Pol, 1991). Glutamate, along with aspartate and pituitary adenylyl cyclase activating polypeptide (PACAP), is localized in the retinohypothalamic tract and is therefore involved in the visual pathway essential for photic entrainment (for review see (Ebling, 1996)). While less is known about the role of aspartate in the regulation of circadian function, it is believed that PACAP works in concert with glutamate during entrainment (Hannibal, 2006). Glutamate immunoreactivity has been localized to presynaptic cells of the SCN (Van Den Pol, 1991;Castel et al., 1993) and tracer experiments using cholera toxin and horseradish peroxidase injected into the eye lead to the presence of these tracers in cells that express glutamate immunoreactivity as well (Castel et al., 1993;De Vries et al., 1993), adding further support to the notion that glutamate is present in the RHT itself. Furthermore, in situ hybridization studies have indicated that mrna for NMDAR1 (N-methyl-D-aspartate receptor subtype 1), a receptor that glutamate acts on, is present in cells throughout the SCN (Mikkelsen et al., 1993). Electrophysiological, but not behavioral, studies have also indicated that glutamate plays a significant role in mediating photic entrainment. Glutamate applied to the hypothalamic slice preparation in the early subjective night can phase delay the circadian phase (Ding et al., 1994); however, analysis of wheel running behavior indicates that in vivo, glutamate, is unable to elicit shifts (Eric Mintz, personal communication). The reason for this finding remains unclear. If glutamate is present in the RHT and its release is a significant step in mediating photic entrainment, then there is an expectation that the increase in available glutamatergic neurotransmission in the SCN

30 15 would be able to elicit behavioral shifts. Two potential explanations for this unexpected finding include an insufficient dose utilized or the rapid glutamate re-uptake mechanisms that exist, both of which may attenuate the response of SCN cells to exogenous glutamate. Finally, studies utilizing the immunoreactivity of immediate-early genes, like c-fos, have additionally provided overwhelming support indicating a significant role for glutamate in the maintenance of clock function. The administration of NMDA to the ventricles increases the expression of c-fos in the SCN (Ebling et al., 1991) and the administration of glutamatergic antagonists attenuates the number of cells expressing c- fos following exposure to light (Rea et al., 1993). These data strongly support the theory that glutamate plays an active role in mediating the response of the SCN to environmental photic cues. The Role of GABA in Circadian Rhythms All SCN neurons can respond to GABA and express a combination of GABA A or GABA B receptors (Van Den Pol, 1986;Francois-Bellan et al., 1989;Gao et al., 1995). While many potential roles of GABA in the regulation of circadian phase have been suggested, the primary role of this neuromodulator remains unclear. Of the many possibilities suggested, a likely role for this neurotransmitter is to modulate the responsiveness of SCN neurons (Trachsel et al., 1996). GABA is widely believed to be an inhibitory neurotransmitter and therefore its presence or absence can either inhibit or potentiate a cellular response to an excitatory signal. Behavioral studies using pharmacological agents that affect GABAergic neurotransmission in the SCN have

31 16 additionally provided some insight into the role that GABA may play in photic entrainment. The administration of GABA A or GABA B receptor agonists near the SCN can attenuate light-induced (Gillespie et al., 1996) and NMDA-induced (Mintz et al., 2002) shifts during the subjective night. Furthermore, the administration of GABA antagonists to the SCN region can potentiate the magnitude of phase shifts elicited by neuropeptides (Gillespie et al., 1996). Finally, the blockade or excitation of the GABA receptor can alter the expression of c-fos in the SCN following a light pulse (Gillespie et al., 1999). These data suggest that GABA may be acting, in part, at the interface of the various efferent projections to the SCN. There is evidence that GABA can reduce glutamatergic neurotransmission at the RHT-SCN junction, thereby inhibiting an integral step in photic entrainment (Gillespie et al., 1997). Other possible junctions could include the GHT-SCN or the raphe-scn interface, where NPY and serotonin are respectively known to act to affect circadian phase. These data suggest that the activation of GABAergic signaling within the SCN can affect clock function by regulating glutamatergic, NPY-ergic, or serotonergic neurotransmission in the SCN. The Role of Gastrin-Releasing Peptide in Circadian Rhythms Rodent studies have consistently reported that gastrin-releasing peptide plays an integral role in photic entrainment. In rats and mice, GRP cell bodies are localized to the ventral region of the SCN and in hamster, GRP immunoreactivity is noted in the central

32 17 region of the nucleus (Abrahamson and Moore, 2001;Karatsoreos et al., 2006;Karatsoreos et al., 2004;LeSauter et al., 2002;Morin et al., 2006;Silver et al., 1996;Tanaka et al., 1997) (Fig. 1E). In all rodent species examined, the GRP receptor and the bombesin receptors, both of which GRP act on, are localized more dorsally (Karatsoreos et al., 2004;Karatsoreos et al., 2006;Abrahamson and Moore, 2001). Studies that examine the expression of the immediate early gene c-fos have also been utilized to show a role of GRP in the regulation of clock function. When animals are kept in constant conditions and presented with a light pulse in the early night, the expression of c-fos is found in the retinorecipient region of the respective species that are in part, localized to GRP-immunoreactive cells (Earnest et al., 1993;Karatsoreos et al., 2004;Romijn et al., 1996). Both behavioral and electrophysiological recording studies have been used to show the significance of GRP in photic entrainment. Microinjection of GRP in the early subjective night into the third ventricle (Antle et al., 2005) or near the SCN (Albers et al., 1991;Piggins et al., 1995) causes phase delays, while the administration of GRP in the middle of the subjective day has little to no effect on the circadian phase (Albers et al., 1991;Piggins et al., 1995). The effects of GRP administration on circadian rhythms are therefore similar to the phase response curve generated by the exposure of animals kept in constant darkness to brief light pulses, indicating that GRP activation may be an important step in the signaling required for photic entrainment. Studies using electrophysiological recordings have also shown that the behavioral response to GRP in vivo can be replicated in the hypothalamic slice preparation in vitro (McArthur et al., 2000). Finally, GRP alters the expression of

33 Fig. 1E: Confocal laser photomicrographs of coronal images showing single fluorescent label (Cy-3) identifying gastrin-releasing peptide (GRP) immunoreactivity at different levels (anterior, medial, and posterior) of the hamster SCN. Adjacent images were taken from the same animal. 18

34 19 immediate-early genes in a manner similar to light (Kornhauser et al., 1990;Piggins et al., 2005). The ventricular administration of GRP in the early subjective night increases c- fos (Antle et al., 2005) and the phosphorylated form of extracellular-regulated kinase (p- ERK) in a region just dorsal to the localization of GRP cell bodies (Antle et al., 2005). These data suggest that GRP plays an active role in mediating the effects of light on the circadian clock. The Role of Neuropeptide Y in Circadian Rhythms Recent studies have shown that NPY is an important component of the rodent circadian clock. NPY is synthesized in the intergeniculate leaflet of the thalamus and is released in the SCN through the geniculo-hypothalamic tract (Card and Moore, 1982) (Fig. 1F). It is believed that while NPY can convey both photic and non-photic signals to the endogenous clock, it is primarily used to transmit non-photic cues (Mason et al., 1987). The phase response curve following the administration of NPY to the hypothalamus is similar to other signals that are primarily non-photic. Microinjection of NPY into the SCN region during the middle of the subjective day phase advances the circadian clock, while the subsequent administration of NPY in the subjective night has little effect on the circadian phase (Huhman and Albers, 1994). NPY receptors, including Y1 and Y5 in hamsters (Cutler et al., 1998) and Y5 receptors in both mice (Porterfield et al., 2007) and rats (Gribkoff et al., 1998), are heavily expressed in the SCN. Furthermore, there is evidence that NPY can block light-induced phase shifts. The use of single-unit recording techniques and behavioral wheel running analyses have shown that

35 Fig. 1F: Confocal laser photomicrographs of coronal images showing single fluorescent label (Alexa-633) identifying neuropeptide Y (NPY) immunoreactivity at different levels (anterior, medial, and posterior) of the hamster SCN. Adjacent images were taken from the same animal. 20

36 21 NPY can inhibit NMDA-induced phase delays (Yannielli and Harrington, 2001;Soscia and Harrington, 2004) and microinjection of a Y5 receptor agonist can also inhibit lightinduced advances (Gamble et al., 2005). Reciprocally, the microinjection of NMDA can additionally inhibit NPY induced phase shifts in the subjective day (Soscia and Harrington, 2004). Also, while light has a depolarizing effect on the membrane potential (Meijer et al., 1993), NPY is believed to be hyperpolarizing (Van Den Pol et al., 1996). It is clear that the effect of NPY on ionic conductance and the membrane potential of SCN neurons can attenuate the excitatory effects observed by light. These data indicate that while NPY may act as a non-photic signal to the SCN, it is capable, at least in part, of modulating, or fine-tuning photic signaling within the SCN. The effects of NPY on clock genes have additionally been described. NPY can suppress light-induced Per1 and Per2 mrna in the SCN of golden hamsters (Brewer et al., 2002). These data suggest that NPY can affect photic entrainment by modulating the effects of photic signals on the circadian clock. The Role of Serotonin in Circadian Rhythms Like NPY, previous data have indicated that serotonin plays important roles in the establishment of circadian rhythms in rodents. Serotonin is synthesized in the raphe nuclei and fibers projecting from the median raphe nucleus to the ventral region of the SCN in the hamster brain mediate both photic and non-photic signals (Meyer-Bernstein and Morin, 1999;Morin, 1999;Pickard and Rea, 1997;Weber et al., 1998;Mintz et al., 1997) (Fig. 1G). The effects of serotonin on circadian phase are believed to be mediated,

37 Fig. 1G: Brightfield photomicrographs of coronal images showing diamino benzadine (DAB) identifying serotonin (5-HT) immunoreactivity at different levels (anterior, medial, and posterior) of the hamster SCN. Adjacent images were taken from the same animal. After altering the contrast and brightness of the original images, inverted images are shown here for the sake of clarity. 22

38 23 in part, by the serotonin 1 A,7 and 1 B receptor subtypes (Pickard et al., 1996), both of which show immunoreactivity within the SCN. Phase shifting analysis indicates that, like NPY, the administration of serotonin agonists in the middle of the subjective day can phase advance the clock (Medanic and Gillette, 1992;Shibata et al., 1992;Prosser et al., 1993). The subsequent administration of serotonin in the subjective night has little effect on the circadian phase. These effects appear to resemble the phase response curve of other non-photic signals that can affect circadian phase. As seen with other non-photic signals however, there is an abundance of data that indicate that serotonin can also affect photic signaling. Interestingly, the application of serotonin can inhibit the phase shifting effects of photic stimuli in the early subjective night, indicating that while serotonin may primarily be a mediator of non-photic signals to the SCN, it can also modulate photic signals at a time when photic signals are known to be able to shift the circadian clock (Weber et al., 1998). This assertion is further supported by the fact that the inhibition of serotonergic signaling in the SCN can potentiate the effect of photic signaling in the early subjective night (Weber et al., 1998). It is also notable that retrograde tracer experiments using cholera toxin injected into the eye has shown that retinal innervation and serotonin immunoreactivity overlap in the ventral SCN (Hayashi et al., 2001;Meyer-Bernstein and Morin, 1999;Pickard and Rea, 1997;Weber et al., 1998). Moreover, an additional tract tracing study in rats has indicated that the raphe nuclei are innervated by the retina (Hay- Schmidt et al., 2003), which may provide the basis for the regulation of serotonergic activity within the circadian visual system. Finally, experiments that examined the induction of light-induced c-fos in the SCN indicate a potential role of serotonin in photic

39 24 entrainment. Animals presented with a light pulse in the subjective night show increased immunoreactivity for c-fos in the cells of the ventral SCN which are proximal to serotonergic fibers (Amir et al., 1998). These numerous reports illustrate that serotonin is an integral component of the rodent circadian clock. Current Model of Circadian Shifts In order to expand upon our understanding of the mechanisms involved in phase shifts, a current model has been described (for review see (Meijer and Schwartz, 2003;Antle and Silver, 2005)(Fig. 1H). According to this model, photic entrainment begins with the light-induced activation of specialized retinal ganglion cells in the eye which then utilize glutamate (Castel et al., 1993) in the retinohypothalamic tract to synapse on retinorecipient neurons of the SCN. These neurons show immunoreactivity for GRP and/or vasoactive intestinal polypeptide (VIP), both of which can additionally respond to carriers of non-photic signals, like serotonin and NPY, to mediate or fine-tune a photic response. The release of GRP and/or VIP then acts on specific receptors that are located dorsal to GRP- and VIP-containing cells. These light-driven signal cascades terminate in the dorsomedial region of the SCN where circadian oscillations are driven. Because this model may be an oversimplification, many experiments, including those described in this dissertation, seek to further expand upon this model by focusing on the interaction of various neurotransmission systems at the level of the SCN. These interactions may be directly related to the cellular and molecular events that lead to the resetting of the circadian clock.

40 25 3 rd Ventricle (3) (?) (4) Cap Cells (?) (4) AVP GRP Retinorecipient Region (2) VIP cell bodies & Serotonin & NPY Immunoreactivity Input from the retina (1) Fig. 1H: Illustration of the current model of shifts of the circadian phase. Light information from the retina reaches the SCN through the retinohypothalamic tract (RHT) which releases glutamate (PACAP) (1). Glutamate activates neurons in the retinorecipient region of the SCN whose cells are immunoreactive for gastrin-releasing peptide (GRP; blue) and vasoactive intestinal polypeptide (VIP; yellow) (2). Retinorecipient cells either act on neurons (cap cells; green) that are located more dorsally (3) which then act on the oscillator cells (AVP; red) of the dorsomedial SCN to drive circadian rhythms (4), or they may be able to by-pass the cap cells and act on the dorsomedial SCN directly (?).

41 26 Cellular and Molecular Events of Circadian Shifts Any examination of behavioral phase shifts would be incomplete without a brief review of signaling cascades. Although many different pathways have been described as essential for circadian entrainment, a special focus has been placed on the role of the ERK/MAP kinase pathway. The expression of immediate-early genes (c-fos, junb, and egr-1) (Aronin et al., 1990;Kornhauser et al., 1990;Rusak et al., 1990;Kornhauser et al., 1992) and circadian clock genes (period 1 and period 2) (Akiyama et al., 1999) can be induced by light during the subjective night, the time in which light can alter circadian phase (Dziema et al., 2003). By using pharmacological agents that inhibit steps of specific signaling cascades and immunohistochemical techniques, research groups have identified the activation of the ERK/MAP kinase pathway as necessary for the induction of these genes (Dziema et al., 2003). Two additional lines of support come from the fact that following photic stimulation, but before the induction of either immediate-early genes or clock genes, MAP kinase pathway activation occurs (Obrietan et al., 1998) and that any disruption of this pathway leads to an attenuation of light-induced shifts of the circadian clock (Butcher et al., 2002). Briefly, the MAP kinase pathway includes three kinases that are sequentially phosphorylated and can be activated by specific signaling molecules like glutamate or GABA (Butcher et al., 2002). Following the activation of the third kinase, often referred to as ERK (extra-cellular regulated kinase), dimerization and translocation into the nucleus occurs, thereby allowing ERK to directly affect the transcriptional machinery, and therefore the translational output, of a cell (for review see (Cobb, 1999)). One experiment described in this dissertation will evaluate the response

42 27 of the SCN and SCN outputs to GRP by analyzing the expression of the phosphorylated form of ERK following GRP administration. Of equal importance is the CREB/CRE transcriptional pathway. CRE is an 8- base pair sequence that is found in the regulatory region of most immediate-early and clock genes (Montminy, 2008;Lonze and Ginty, 2002;Herdegen and Leah, 1998). Since the activation of CRE has been described as a necessary step in the induction of immediate-early genes and since the induction of these genes is immediately preceded by the activation of the MAP kinase pathway described above, it remains a possibility that CRE and MAP kinase activity converge to directly affect circadian phase (Obrietan et al., 1999;Sweatt, 2001). It has been further identified that the MAP kinase signaling pathway is involved in the phosphorylation of CREB, which allows CREB to translocate into the nucleus where it can affect transcriptional activity (Obrietan et al., 1999). Finally, if the MAP kinase pathway becomes disrupted, light is unable to induce CRE-dependent transcription (Dziema et al., 2003). While it is clear that the ERK/MAP kinase and CREB/CRE transcriptional pathways are integral in light-induced entrainment, less is known about how non-photic signals can either mediate entrainment during the day or oppose the action of entraining signals during the night. To take as an example, NPY is a carrier of non-photic signals that can induce large phase advances during the day (Yannielli and Harrington, 2001) and can oppose the action of light during the night (Soscia and Harrington, 2004;Lall and Biello, 2003;Weber and Rea, 1997;Weber et al., 1998). Signaling cascades have been identified that account for these different behavioral responses. Through the use of in vivo

43 28 application of pharmacological agents that block specific pathways, it clear that NPY can act on one of several receptor subtypes, each of which may result in a different behavioral response. For example, the Y2 receptor subtype leads to the activation of protein kinase C (PKC) and can induce large phase advances during the subjective day (for review see (Yannielli and Harrington, 2001)). It is believed that the activation of the Y5 receptor subtype may be responsible, through an unknown mechanism, for blocking light-induced increases of Per1 and Per2, both of which are necessary for phase shifts during the subjective night (for review see (Yannielli and Harrington, 2001)). Both positive and negative feedback loops and the fluctuations in RNA and protein levels of clock components are essential for the optimal resetting capabilities of the circadian clock (for review see (Reppert and Weaver, 2002)). The transcription of two factors, CLOCK and BMAL1 are involved in the maintenance and/or alteration of circadian phase (King et al., 1997;Gekakis et al., 1998;Hogenesch, 1998;Bunger et al., 2000); however, CLOCK, unlike BMAL1, is not essential, for CLOCK-deficient mice continue to show robust circadian rhythms in locomotor activity (DeBruyne et al., 2006) (Fig.1I). These transcription factors can form heterodimers that can bind to specific genes whose protein products are needed for clock function (Gekakis et al., 1998;Hogenesch, 1998). Such protein products include period proteins (Per1, Per2, Per3) and cryptochrome proteins (Cry1 and Cry2). During negative feedback, these protein products can be translocated into the nucleus where they can interact with the CLOCK- BMAL1 heterodimers to effectively act as negative regulators (Kume et al., 1999;Vitaterna et al., 1999;Okamura et al., 1999;Shearman et al., 2000). A positive

44 29 feedback loop has since been identified. In addition to activating Per and Cry proteins, the CLOCK-BMAL1 heterodimer additionally activates the transcription of a nuclear receptor gene known as Rev-Erbα (Preitner et al., 2002) whose protein product then inhibits the transcription of BMAL1 (Preitner et al., 2002;Ueda et al., 2002). As the levels of BMAL1 protein falls, less BMAL1 is available to dimerize with CLOCK and the levels of clock gene protein products are able to increase. This positive feedback loop is turned off by a similar mechanism seen in the negative feedback loop. When Per and Cry re-enter the nucleus to negatively regulate CLOCK-BMAL1, they additionally inhibit Rev-Erbα which would lead to the concomitant increase of BMAL1 (Preitner et al., 2002;Ueda et al., 2002;Yu et al., 2002). It is clear that the fluctuations in the transcription of clock genes are essential in the maintenance of circadian synchrony, for the disruption of these genes causes behavioral arrythmicity (Vitaterna et al., 1999;Bae et al., 2001;Zheng, 2001;van der Horst, 1999). While the experiments of this dissertation do not specifically examine the cellular or molecular events that lead to GRP-induced entrainment, an understanding of these events can be useful in describing the results observed. Use of c-fos to Understand the Mechanisms of Photic Entrainment Measurement of the expression of the immediate-early gene product c-fos has been instrumental in furthering our understanding of the mechanistic steps involved in photic entrainment. In addition to indicating a cellular response to stimuli, the induction

45 Fig. 1I: Illustration showing the molecular regulation of clock function. Light activates ganglion cells in the retina which project directly to the SCN through the retinohypothalamic tract (RHT) where glutamate (PACAP) is released. These ligands can act on the ventral regions of the SCN. Various signal transduction events lead to the activation of kinases like PKA and mitogen-activating protein kinase (MAPK), both of which can phosphorylate factors that affect transcription, including camp response element binding protein (CREB). Once phosphorylated, CREB becomes activated and can enter the nucleus where it will bind camp response element (CRE), leading to the transcription of clock components like Per1 and Per2. It should be noted that Per1 and Per2 may exist on different genes, but a single gene is illustrated here for simplicity. The cells of the ventral SCN, once activated, can release various neuropeptides, one of which is gastrin-releasing peptide (G). These neuropeptides then act on neurons in dorsomedial SCN where clock proteins are regulated by a transcriptional/translational feedback loop. CLOCK (C) and BMAL (B) dimerize, enter the nucleus, and bind E-boxes in the promoter region of specific genes whose protein products include Period (Per), Cryptochrome (Cry), and Rev-Erbα. Once again, these clock components may be transcribed from different genes, but a single gene is illustrated here for clarity. The negative and positive feedback loops that regulate the expression of these clock components are not illustrated here (Science Slides, Viviscience Corporation, 2006). 30

46 31

47 32 of c-fos also designates long term changes in gene expression (Morgan and Curran, 1991). Following exposure to light during times in which light can result in a shift of circadian phase, c-fos is quickly induced in the SCN (Kornhauser et al., 1990;Rea, 1989;Rusak et al., 1990). Furthermore, pharmacological agents that either block NMDA/AMPA receptors (Abe et al., 1992;Colwell et al., 1991;Ebling et al., 1996) or activate serotonergic receptors (Ginty et al., 1993;Moriya et al., 1996;Pickard et al., 1996) can attenuate the amount of c-fos induced in the SCN following exposure to light. By identifying individual cells that express c-fos after exposure to light, and localizing specific neuropeptides and receptor subtypes in those cells, the varying elements involved in entrainment can be characterized. Overall Aims The experiments outlined in this manuscript will examine the role that gastrinreleasing peptide plays in mediating photic entrainment. The following experiments will cover four specific aims. The first aim is the examination of the behavioral response to both microinjection of GRP into the third ventricle or the presentation of a light pulse to animals kept in constant conditions and to determine whether such responses are dependent on the availability of glutamatergic signaling. The second aim is to examine the integration of signals from GRP and NPY by determining whether the administration of each of these neuropeptides near the SCN can oppose the expected action on circadian phase of the other. The third aim of this dissertation is to examine the behavioral and the cellular response in the SCN to microinjection of neuropeptides near the SCN or into the

48 33 third ventricle. This third aim additionally seeks to identify regions of the brain that are activated by the exogenous application of GRP and to determine if GRP-induced activity in these regions is inhibited by glutamatergic antagonists or serotonergic agonists. The final aim of this manuscript expands upon our understanding of the first aim by examining whether the microinjection of a glutamate receptor antagonist near the SCN can inhibit shifts of the circadian phase elicited by either GRP or light following the administration of these phase-shifting stimuli. These data will greatly increase our knowledge of the role of GRP in mediating photic entrainment. Reference List Abe H, Rusak B, Robertson HA (1992) NMDA and non-nmda receptor antagonists inhibit photic induction of Fos protein in the hamster suprachiasmatic nucleus. Brain Res Bull 28: Abrahamson EE, Moore RY (2001) Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections. Brain Res 916: Akiyama M, Kouzu Y, Takahashi S, Wakamatsu H, Moriya T, Maetani M, Watanabe S, Tei H, Sakaki Y, Shibata S (1999) Inhibition of light- or glutamate-induced mper1 expression represses the phase shifts into the mouse circadian locomotor and suprachiasmatic firing rhythms. J Neurosci 19: Albers HE, Liou SY, Stopa EG, Zoeller RT (1991) Interaction of colocalized neuropeptides: functional significance in the circadian timing system. J Neurosci 11: Amir S, Robinson B, Ratovitski T, Rea MA, Stewart J, Simantov R (1998) A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced Fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet. Neuroscience 84: Antle MC, Kriegsfeld LJ, Silver R (2005) Signaling within the master clock of the brain: localized activation of mitogen-activated protein kinase by gastrin-releasing peptide. J Neurosci 25:

49 34 Antle MC, Silver R (2005) Orchestrating time: arrangements of the brain circadian clock. Trends Neurosci 28: Aronin N, Sagar SM, Sharp FR, Schwartz WJ (1990) Light regulates expression of a Fosrelated protein in rat suprachiasmatic nuclei. Proc Natl Acad Sci U S A 87: Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mper1, mper2, and mper3 in the SCN circadian clock. Neuron 30: Brewer JM, Yannielli PC, Harrington ME (2002) Neuropeptide Y differentially suppresses per1 and per2 mrna induced by light in the suprachiasmatic nuclei of the golden hamster. J Biol Rhythms 17: Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, Hogenesch JB, Simon MC, Takahashi JS, Bradfield CA (2000) Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103: Butcher GQ, Dziema H, Collamore M, Burgoon PW, Obrietan K (2002) The p42/44 mitogen-activated protein kinase pathway couples photic input to circadian clock entrainment. J Biol Chem 277: Card JP, Moore RY (1982) Ventral lateral geniculate nucleus efferents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity. J Comp Neurol 206: Card JP, Moore RY (1984) The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution. Neuroscience 13: Castel M, Belenky M, Cohen S, Ottersen OP, Storm-Mathisen J (1993) Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus. Eur J Neurosci 5: Cobb MH (1999) MAP kinase pathways. Prog Biophys Mol Biol 71: Cohen RA, Albers HE (1991) Disruption of human circadian and cognitive regulation following a discrete hypothalamic lesion: a case study. Neurology 41: Colwell CS, Foster RG, Menaker M (1991) NMDA receptor antagonists block the effects of light on circadian behavior in the mouse. Brain Res 554: Colwell CS, Menaker M (1992) NMDA as well as non-nmda receptor antagonists can prevent the phase-shifting effects of light on the circadian system of the golden hamster. J Biol Rhythms 7:

50 35 Cui LN, Saeb-Parsy K, Dyball RE (1997) Neurones in the supraoptic nucleus of the rat are regulated by a projection from the suprachiasmatic nucleus. J Physiol 502 ( Pt 1): Cutler DJ, Piggins HD, Selbie LA, Mason R (1998) Responses to neuropeptide Y in adult hamster suprachiasmatic nucleus neurones in vitro. Eur J Pharmacol 345: Czeisler CA, Klerman EB (1999) Circadian and sleep-dependent regulation of hormone release in humans. Recent Prog Horm Res 54: De Vries MJ, Nunes CB, van der WJ, de Wolf A, Meijer JH (1993) Glutamate immunoreactivity in terminals of the retinohypothalamic tract of the brown Norwegian rat. Brain Res 612: DeBruyne JP, Noton E, Lambert CM, Maywood ES, Weaver DR, Reppert SM (2006) A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron 50: DeCoursey PJ (1960) Daily light senstivity rhythm in a rodent. Science 131: Ding JM, Chen D, Weber ET, Faiman LE, Rea MA, Gillette MU (1994) Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO. Science 266: Dziema H, Oatis B, Butcher GQ, Yates R, Hoyt KR, Obrietan K (2003) The ERK/MAP kinase pathway couples light to immediate-early gene expression in the suprachiasmatic nucleus. Eur J Neurosci 17: Earnest DJ, DiGiorgio S, Olschowka JA (1993) Light induces expression of fos-related proteins within gastrin-releasing peptide neurons in the rat suprachiasmatic nucleus. Brain Res 627: Ebling FJ (1996) The role of glutamate in the photic regulation of the suprachiasmatic nucleus. Prog Neurobiol 50: Ebling FJ, Maywood ES, Mehta M, Hancock DC, McNulty S, De Bono J, Bray SJ, Hastings MH (1996) FosB in the suprachiasmatic nucleus of the Syrian and Siberian hamster. Brain Res Bull 41: Ebling FJ, Staley K, Maywood ES, Humby T, Hancock DC, Waters CM, Evan GI, Hastings MH (1991) The role of NMDA-type glutamatergic neurotransmission in the photic induction of immediate-early gene expression in the suprachiasmatic nucleus of the Syrian hamster. J Neuroendocrinol 3:

51 36 Francois-Bellan AM, Segu L, Hery M (1989) Regulation by estradiol of GABAA and GABAB binding sites in the diencephalon of the rat: an autoradiographic study. Brain Res 503: Gamble KL, Ehlen JC, Albers HE (2005) Circadian control during the day and night: Role of neuropeptide Y Y5 receptors in the suprachiasmatic nucleus. Brain Res Bull 65: Gao B, Fritschy JM, Moore RY (1995) GABAA-receptor subunit composition in the circadian timing system. Brain Res 700: Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science 280: Gillespie CF, Huhman KL, Babagbemi TO, Albers HE (1996) Bicuculline increases and muscimol reduces the phase-delaying effects of light and VIP/PHI/GRP in the suprachiasmatic region. J Biol Rhythms 11: Gillespie CF, Mintz EM, Marvel CL, Huhman KL, Albers HE (1997) GABA(A) and GABA(B) agonists and antagonists alter the phase-shifting effects of light when microinjected into the suprachiasmatic region. Brain Res 759: Gillespie CF, van der Beek EM, Mintz EM, Mickley NC, Jasnow AM, Huhman KL, Albers HE (1999) GABAergic regulation of light-induced c-fos immunoreactivity within the suprachiasmatic nucleus. J Comp Neurol 411: Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME (1993) Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260: Gribkoff VK, Pieschl RL, Wisialowski TA, Van Den Pol AN, Yocca FD (1998) Phase shifting of circadian rhythms and depression of neuronal activity in the rat suprachiasmatic nucleus by neuropeptide Y: mediation by different receptor subtypes. J Neurosci 18: Hannibal J (2002) Neurotransmitters of the retino-hypothalamic tract. Cell Tissue Res 309: Hannibal J (2006) Roles of PACAP-containing retinal ganglion cells in circadian timing. Int Rev Cytol 251: Hay-Schmidt A, Vrang N, Larsen PJ, Mikkelsen JD (2003) Projections from the raphe nuclei to the suprachiasmatic nucleus of the rat. J Chem Neuroanat 25:

52 37 Hayashi S, Ueda M, Amaya F, Matusda T, Tamada Y, Ibata Y, Tanaka M (2001) Serotonin modulates expression of VIP and GRP mrna via the 5-HT(1B) receptor in the suprachiasmatic nucleus of the rat. Exp Neurol 171: Hendrickson AE, Wagoner N, Cowan WM (1972) An autoradiographic and electron microscopic study of retino-hypothalamic connections. Z Zellforsch Mikrosk Anat 135: Herdegen T, Leah JD (1998) Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev 28: Hermes ML, Buijs RM, Renaud LP (1996) Electrophysiology of suprachiasmatic nucleus projections to hypothalamic paraventricular nucleus neurons. Prog Brain Res 111: Hogenesch JB (1998) The basic helix-loop-helix-pas orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci U S A 95: Huhman KL, Albers HE (1994) Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness. Peptides 15: Inouye ST, Kawamura H (1979) Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus. Proc Natl Acad Sci U S A 76: Johnson RF, Moore RY, Morin LP (1988) Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract. Brain Res 460: Kafka MS, Marangos PJ, Moore RY (1985) Suprachiasmatic nucleus ablation abolishes circadian rhythms in rat brain neurotransmitter receptors. Brain Res 327: Karatsoreos IN, Romeo RD, McEwen BS, Silver R (2006) Diurnal regulation of the gastrin-releasing peptide receptor in the mouse circadian clock. Eur J Neurosci 23: Karatsoreos IN, Yan L, LeSauter J, Silver R (2004) Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus. J Neurosci 24: Kaufman CM, Menaker M (1993) Effect of transplanting suprachiasmatic nuclei from donors of different ages into completely SCN lesioned hamsters. J Neural Transplant Plast 4:

53 38 King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, Takahashi JS (1997) Positional cloning of the mouse circadian clock gene. Cell 89: Kornhauser JM, Nelson DE, Mayo KE, Takahashi JS (1990) Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus. Neuron 5: Kornhauser JM, Nelson DE, Mayo KE, Takahashi JS (1992) Regulation of jun-b messenger RNA and AP-1 activity by light and a circadian clock. Science 255: Kriegsfeld LJ, Leak RK, Yackulic CB, LeSauter J, Silver R (2004) Organization of suprachiasmatic nucleus projections in Syrian hamsters (Mesocricetus auratus): an anterograde and retrograde analysis. J Comp Neurol 468: Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH, Reppert SM (1999) mcry1 and mcry2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98: Lall GS, Biello SM (2003) Attenuation of circadian light induced phase advances and delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist. Neuroscience 119: Lee HS, Billings HJ, Lehman MN (2003a) The suprachiasmatic nucleus: a clock of multiple components. J Biol Rhythms 18: Lee HS, Nelms JL, Nguyen M, Silver R, Lehman MN (2003b) The eye is necessary for a circadian rhythm in the suprachiasmatic nucleus. Nat Neurosci 6: Lehman MN, Silver R, Gladstone WR, Kahn RM, Gibson M, Bittman EL (1987) Circadian rhythmicity restored by neural transplant. Immunocytochemical characterization of the graft and its integration with the host brain. J Neurosci 7: LeSauter J, Kriegsfeld LJ, Hon J, Silver R (2002) Calbindin-D(28K) cells selectively contact intra-scn neurons. Neuroscience 111: Levine JD, Weiss ML, Rosenwasser AM, Miselis RR (1991) Retinohypothalamic tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin. J Comp Neurol 306: Lonze BE, Ginty DD (2002) Function and regulation of CREB family transcription factors in the nervous system. Neuron 35:

54 39 Mason R, Harrington ME, Rusak B (1987) Electrophysiological responses of hamster suprachiasmatic neurones to neuropeptide Y in the hypothalamic slice preparation. Neurosci Lett 80: McArthur AJ, Coogan AN, Ajpru S, Sugden D, Biello SM, Piggins HD (2000) Gastrinreleasing peptide phase-shifts suprachiasmatic nuclei neuronal rhythms in vitro. J Neurosci 20: McCready S, Muller JA, Boubriak I, Berquist BR, Ng WL, Dassarma S (2005) UV irradiation induces homologous recombination genes in the model archaeon Halobacterium sp. NRC-1. Saline Systems 4: 1-3. Medanic M, Gillette MU (1992) Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day. J Physiol 450: Meijer JH, Albus H, Weidema F, Ravesloot JH (1993) The effects of glutamate on membrane potential and discharge rate of suprachiasmatic neurons. Brain Res 603: Meijer JH, Schwartz WJ (2003) In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus. J Biol Rhythms 18: Meyer-Bernstein EL, Morin LP (1999) Electrical stimulation of the median or dorsal raphe nuclei reduces light-induced FOS protein in the suprachiasmatic nucleus and causes circadian activity rhythm phase shifts. Neuroscience 92: Mikkelsen JD, Larsen PJ, Ebling FJ (1993) Distribution of N-methyl D-aspartate (NMDA) receptor mrnas in the rat suprachiasmatic nucleus. Brain Res 632: Miller JD (1998) The SCN is comprised of a population of coupled oscillators. Chronobiol Int 15: Mintz EM, Gillespie CF, Marvel CL, Huhman KL, Albers HE (1997) Serotonergic regulation of circadian rhythms in Syrian hamsters. Neuroscience 79: Mintz EM, Jasnow AM, Gillespie CF, Huhman KL, Albers HE (2002) GABA interacts with photic signaling in the suprachiasmatic nucleus to regulate circadian phase shifts. Neuroscience 109: Moga MM, Moore RY (1997) Organization of neural inputs to the suprachiasmatic nucleus in the rat. J Comp Neurol 389: Montminy M (2008) Transcriptional regulation bt cyclic AMP. Annu Rev Biochem 66:

55 40 Moore RY (1983) Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus. Fed Proc 42: Moore RY, Eichler VB (1972) Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res 42: Moore RY, Speh JC, Leak RK (2002) Suprachiasmatic nucleus organization. Cell Tissue Res 309: Morgan JI, Curran T (1991) Proto-oncogene transcription factors and epilepsy. Trends Pharmacol Sci 9: Morin LP (1994) The circadian visual system. Brain Res Brain Res Rev 19: Morin LP (1999) Serotonin and the regulation of mammalian circadian rhythmicity. Ann Med 31: Morin LP, Shivers KY, Blanchard JH, Muscat L (2006) Complex organization of mouse and rat suprachiasmatic nucleus. Neuroscience 137: Moriya T, Yamanouchi S, Fukushima T, Shimazoe T, Shibata S, Watanabe S (1996) Involvement of 5-HT1A receptor mechanisms in the inhibitory effects of methamphetamine on photic responses in the rodent suprachiasmatic nucleus. Brain Res 740: Mrosovsky N (1988) Phase response curves for social entrainment. J Comp Physiol [A] 162: Myneni RB, Yang W, Nemani RR, Huete AR, Dickins RE, Knazikhin Y, Dida K, Negron Juarez RI, Saatchi SS, Hashimoto H, Ichii K, Shabanoy NV, Tan B, Ratana P, Privette JL, Morisette JT, Vermote EF, Roy DP, Wolfe RE, Freidl MA, Running SW, Votava P, El-Saleous N, Devadiga S, Su Y, Salomonson VV (2007) Large seasonal swings in leaf area of Amazon rainforests. Proc Natl Acad Sci USA 104: Obrietan K, Impey S, Smith D, Athos J, Storm DR (1999) Circadian regulation of camp response element-mediated gene expression in the suprachiasmatic nuclei. J Biol Chem 274: Obrietan K, Impey S, Storm DR (1998) Light and circadian rhythmicity regulate MAP kinase activation in the suprachiasmatic nuclei. Nat Neurosci 1: Okamura H, Miyake S, Sumi Y, Yamaguchi S, Yasui A, Muijtjens M, Hoeijmakers JH, van der Horst GT (1999) Photic induction of mper1 and mper2 in cry-deficient mice lacking a biological clock. Science 286:

56 41 Pickard GE (1982) The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection. J Comp Neurol 211: Pickard GE, Rea MA (1997) Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms. Biol Cell 89: Pickard GE, Silverman AJ (1981) Direct retinal projections to the hypothalamus, piriform cortex, and accessory optic nuclei in the golden hamster as demonstrated by a sensitive anterograde horseradish peroxidase technique. J Comp Neurol 196: Pickard GE, Weber ET, Scott PA, Riberdy AF, Rea MA (1996) 5HT1B receptor agonists inhibit light-induced phase shifts of behavioral circadian rhythms and expression of the immediate-early gene c-fos in the suprachiasmatic nucleus. J Neurosci 16: Piggins HD, Antle MC, Rusak B (1995) Neuropeptides phase shift the mammalian circadian pacemaker. J Neurosci 15: Piggins HD, Goguen D, Rusak B (2005) Gastrin-releasing peptide induces c-fos in the hamster suprachiasmatic nucleus. Neurosci Lett 384: Porterfield, V.M., Pointkivska H, Mintz EM (2007) Identification of novel light-induced genes in the suprachiasmatic nucleus. BMC Neurosci 19. Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, Albrecht U, Schibler U (2002) The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110: Prosser RA, Dean RR, Edgar DM, Heller HC, Miller JD (1993) Serotonin and the mammalian circadian system: I. In vitro phase shifts by serotonergic agonists and antagonists. J Biol Rhythms 8: Ralph MR, Foster RG, Davis FC, Menaker M (1990) Transplanted suprachiasmatic nucleus determines circadian period. Science 247: Rea MA (1989) Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei. Brain Res Bull 23: Rea MA, Buckley B, Lutton LM (1993) Local administration of EAA antagonists blocks light-induced phase shifts and c-fos expression in hamster SCN. Am J Physiol 265: R1191-R1198. Reebs SG, Mrosovsky N (1989) Effects of induced wheel running on the circadian activity rhythms of Syrian hamsters: entrainment and phase response curve. J Biol Rhythms 4:

57 42 Refinetti R (2006) Circadian Biology. New York: CRC Press. Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418: Ribak CE, Peters A (1975) An autoradiographic study of the projections from the lateral geniculate body of the rat. Brain Res 92: Romero MT, Lehman MN, Silver R (1993) Age of donor influences ability of suprachiasmatic nucleus grafts to restore circadian rhythmicity. Brain Res Dev Brain Res 71: Romijn HJ, Sluiter AA, Pool CW, Wortel J, Buijs RM (1996) Differences in colocalization between Fos and PHI, GRP, VIP and VP in neurons of the rat suprachiasmatic nucleus after a light stimulus during the phase delay versus the phase advance period of the night. J Comp Neurol 372: 1-8. Rusak B, Robertson HA, Wisden W, Hunt SP (1990) Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus. Science 248: Saeb-Parsy K, Lombardelli S, Khan FZ, McDowall K, Au-Yong IT, Dyball RE (2000) Neural connections of hypothalamic neuroendocrine nuclei in the rat. J Neuroendocrinol 12: Schwartz WJ, Busis NA, Hedley-Whyte ET (1986) A discrete lesion of ventral hypothalamus and optic chiasm that disturned the daily temperature rhythm. J Neurol 233: 1-4. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH, Reppert SM (2000) Interacting molecular loops in the mammalian circadian clock. Science 288: Shibata S, Moore RY (1993) Neuropeptide Y and optic chiasm stimulation affect suprachiasmatic nucleus circadian function in vitro. Brain Res 615: Shibata S, Tsuneyoshi A, Hamada T, Tominaga K, Watanabe S (1992) Phase-resetting effect of 8-OH-DPAT, a serotonin1a receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro. Brain Res 582: Silver R, Romero MT, Besmer HR, Leak R, Nunez JM, LeSauter J (1996) Calbindin- D28K cells in the hamster SCN express light-induced Fos. Neuroreport 7: Soscia SJ, Harrington ME (2004) Neuropeptide Y attenuates NMDA-induced phase shifts in the SCN of NPY Y1 receptor knockout mice in vitro. Brain Res 1023:

58 43 Stephan FK, Zucker I (1972) Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc Natl Acad Sci U S A 69: Sweatt JD (2001) The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. J Neurochem 76: Tanaka M, Hayashi S, Tamada Y, Ikeda T, Hisa Y, Takamatsu T, Ibata Y (1997) Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat. Neuroreport 8: Trachsel L, Dodt HU, Zieglgansberger W (1996) The intrinsic optical signal evoked by chiasm stimulation in the rat suprachiasmatic nuclei exhibits GABAergic day-night variation. Eur J Neurosci 8: Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, Nagano M, Nakahama K, Suzuki Y, Sugano S, Iino M, Shigeyoshi Y, Hashimoto S (2002) A transcription factor response element for gene expression during circadian night. Nature 418: Van Den Pol AN (1986) Gamma-aminobutyrate, gastrin releasing peptide, serotonin, somatostatin, and vasopressin: ultrastructural immunocytochemical localization in presynaptic axons in the suprachiasmatic nucleus. Neuroscience 17: Van Den Pol AN (1991) Glutamate and aspartate immunoreactivity in hypothalamic presynaptic axons. J Neurosci 11: Van Den Pol AN, Obrietan K, Chen G, Belousov AB (1996) Neuropeptide Y-mediated long-term depression of excitatory activity in suprachiasmatic nucleus neurons. J Neurosci 16: van der Horst GT (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398: Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, Fruechte EM, Hitomi K, Thresher RJ, Ishikawa T, Miyazaki J, Takahashi JS, Sancar A (1999) Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc Natl Acad Sci U S A 96: Watts AG, Swanson LW (1987) Efferent projections of the suprachiasmatic nucleus: II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat. J Comp Neurol 258:

59 44 Watts AG, Swanson LW, Sanchez-Watts G (1987) Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat. J Comp Neurol 258: Weber ET, Gannon RL, Rea MA (1998) Local administration of serotonin agonists blocks light-induced phase advances of the circadian activity rhythm in the hamster. J Biol Rhythms 13: Weber ET, Rea MA (1997) Neuropeptide Y blocks light-induced phase advances but not delays of the circadian activity rhythm in hamsters. Neurosci Lett 231: Windle RJ, Forsling ML, Guzek JW (1992) Daily rhythms in the hormone content of the neurohypophysial system and release of oxytocin and vasopressin in the male rat: effect of constant light. J Endocrinol 133: Yannielli P, Harrington ME (2004) Let there be "more" light: enhancement of light actions on the circadian system through non-photic pathways. Prog Neurobiol 74: Yannielli PC, Harrington ME (2001) The neuropeptide Y Y5 receptor mediates the blockade of "photic-like" NMDA-induced phase shifts in the golden hamster. J Neurosci 21: Yu W, Nomura M, Ikeda M (2002) Inactivating feedback loops within the mammalian clock: BMAL1 is negatively autoregulated and upregulated by CRY1, CRY2, and PER2. Biochem Biophys Res Commun 290: Zheng B (2001) Nonredundant roles of the mper1 and mper2 genes in the mammalian circadian clock. Cell 105:

60 CHAPTER II Glutamatergic Activity Modulates the Phase-shifting Effects of Gastrin-releasing Peptide and Light 2 Abstract Previous studies have established that microinjection of gastrin releasing peptide (GRP) into the suprachiasmatic nucleus (SCN) region or third ventricle causes circadian phase shifts similar to those produced by light pulses. Activation of N-methyl-D-aspartate (NMDA) receptors in the SCN region also produces light-like phase shifts. This study was designed to test the effects of (±)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist, and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate reuptake inhibitor, on GRP-induced shifts. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the third ventricle were housed in contestant darkness until stable free-running rhythms of wheel-running activity were apparent. Microinjection of GRP into the third ventricle at circadian time (CT) 13 induced large phase delays. These GRP-induced phase delays were completely blocked by coadministration of AP5, suggesting that GRP-induced phase delays require concurrent activation of NMDA receptors. Microinjection of AP5 alone did not induce significant phase shifts. A second set of experiments were designed to test whether GRP-induced shifts would be enhanced by PDC. Co-administration of PDC and GRP elicited 2 Reproduced with permission of Blackwell Publishing, UK, as it appears in European Journal of Neuroscience (10(2006) ) by G. Kallingal & E.M. Mintz. 45

61 46 significantly larger phase delays at CT 13 than GRP alone. However, administration of PDC alone did not induce a significant phase shift. Finally, when administered just prior to a light pulse, PDC elicited significantly larger phase delays than light pulse plus vehicle controls. These data suggest that the effects of GRP on the circadian clock phase are highly dependent on the level of excitation provided by activated NMDA receptors. Introduction The mammalian suprachiasmatic nucleus (SCN) contains an endogenous clock that regulates physiological and behavioral circadian activities, and is located lateral to the third ventricle of the anterior hypothalamus (Moore and Eichler, 1972;Stephan and Zucker, 1972;Moore and Card, 1985;Moore, 1995). In most animals, light is an important stimulus for resetting the circadian clock. Numerous studies have provided evidence that glutamate is present in the retinohypothalamic tract (RHT) and is responsible for transmitting photic information to the SCN (Castel et al., 1993;Ebling, 1996;Mintz et al., 1999;Hannibal, 2002). If glutamate is the signal that transmits photic information to the SCN, then microinjection of glutamate should mimic the effects seen with brief light exposure. Previous studies have indicated that glutamate does not induce significant phase shifts during the subjective night in vivo (Meijer et al., 1988), although numerous studies using in vitro systems have shown a phase-shifting effect of glutamate (Ding et al., 1994;Shibata et al., 1994). However, when N-methyl-D-aspartate (NMDA), a glutamate agonist, is microinjected into the SCN, the phase-response curve (PRC) that

62 47 results is similar in shape to the PRC produced by brief light pulses (Mintz and Albers, 1997;Mintz et al., 1999). The reasons why microinjection of glutamate alone does not induce significant phase shifts are unknown, although it seems likely that glutamate has a very high clearance rate in the SCN and therefore a single microinjection produces an insufficient stimulus. In addition to glutamate, a number of neuropeptides have been implicated in the regulation of photic phase shifts. One candidate for this role is gastrin-releasing peptide (GRP) (Piggins et al., 1994;LeSauter et al., 2002;Moore et al., 2002;Antle and Silver, 2005;Piggins et al., 2005). GRP is not present in the RHT, but is localized in retinorecipient SCN neurons in rodents (Tanaka et al., 1997;Abrahamson and Moore, 2001;Karatsoreos et al., 2004;Morin et al., 2006). Microinjection of GRP into the SCN in the early subjective night causes phase delays, and in the late subjective night, phase advances (Albers et al., 1995;Piggins et al., 1995). The GRP receptor mrna is heavily expressed in the dorsal and medial regions of the SCN (Karatsoreos et al., 2006), and GRP receptor knockout mice show attenuated phase shifts to light as compared with wild-type mice (Aida et al., 2002). Moreover, whereas c-fos expression is induced by light predominately in the ventral SCN (Kornhauser et al., 1990;Abe and Rusak, 1994;Ebling, 1996), c-fos expression induced by injection of GRP near the SCN is found in a more dorsal location (Piggins et al., 2005). Finally, ventricular administration of GRP induces Per1, Per2, c-fos and MAP kinase activity in the dorsal SCN as well (Antle et al., 2005). These data suggest that GRP activation is downstream of photic signaling and that GRP is involved in communication between two distinct regions of the SCN

63 48 (Piggins et al., 2005;Karatsoreos et al., 2006). It is unknown whether GRP-dependent phase shifts occur completely downstream of glutamatergic input to the SCN, or whether GRP-induced phase shifts require concurrent activation of glutamate receptors. In the present study, we test this by examining the phase-shifting effects of GRP when coadministered with an NMDA antagonist. We also test whether a glutamate reuptake inhibitor alters the effects of light and GRP on the circadian clock. Materials and Methods Subjects Seventy-five adult male Syrian hamsters (2-4 months) were either bred from hamsters purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) or purchased directly from Harlan Sprague Dawley and group-housed in a 14:10 h light:dark cycle for a period of 1-3 weeks. Food and water were available ad libitum, and the experimental procedures were in accordance with the Kent State University Animal Care and Use Committee, and the PHS Guide to the Care and Use of Laboratory Animals. Surgical procedure Hamsters were deeply anesthetized with an anesthesia cocktail (110 mg/kg ketamine, 22 mg/kg xylazine and 1.83 mg/kg acepromazine) and stereotaxically implanted with a 4- mm, 26-gauge guide cannula (Plastics One, Roanoke, VA, USA) aimed at the third ventricle. Stereotaxic coordinates were 1.3 mm anterior to bregma and 7.2 mm ventral to

64 49 dura along the midline. Prior to cannula insertion, the skull was leveled between bregma and lambda. A 32-gauge injection needle (Plastics One) was inserted into the cannula to deliver the treatment to the ventricle. After surgery, hamsters were individually housed in Plexiglass cages (24 x 45.5 x 21 cm) and given 24 hours to recover in their original colony room. Hamsters were then placed in continuously dim environment ( 0.01 lux green light; (Gorman et al., 2005;Gorman et al., 2006;Gorman and Steele, 2006), and were allowed to establish free-running activity rhythms. Hamsters were also equipped with a running wheel (diameter 18 cm) that was monitored by a computer (Dell) using ClockLab software (Actimetrics). Microinjection After 10 days of free-running activity, microinjections were given with a 32-gauge needle that extended beyond the guide cannula by 5.2 mm, attached by polyethylene tubing to a 1 µl Hamilton syringe. The polyethylene tubing was filled with distilled water and an air bubble was introduced to avoid dilution of injected substances. Injections were given in dim red illumination while the hamsters were gently restrained by hand. The final volume of each microinjection was 1 µl, which was administered over a period of 10 s. After each microinjection, the needle was left in place for s to avoid leakage. Microinjections were given at circadian time (CT) 13 ± 15 min. Microinjections included 0.5 nmol GRP (Phoenix Pharmaceuticals, Belmont, CA, USA), 4.94 nmol (±)-2-amino-5- phosphonopentanoic acid (AP5; Research Biochemicals International, Natic, MA, USA) and 4.97 nmol L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; Tocris, Ellisville, MO,

65 50 USA), each dissolved in phosphate buffered saline (PBS), or PBS alone. For the first experiment, each animal received an injection of GRP and GRP/AP5, in the second experiment PDC and saline, and in the third experiment GRP and GRP/PDC. In the final experiment, animals received an injection of PDC and saline, each followed by a 5-min light pulse (300 lux) in their own home cages. In each of the experiments, the order of injections was counterbalanced. A minimum of 10 days separated the two injections. Histology After completion of each experiment, animals were deeply anesthetized with sodium pentobarbital (200 mg/kg) and killed by decapitation. Hamster brains were extracted and postfixed in 4% paraformaldehyde. Coronal sections (100 µm thickness) were sliced on a vibratome and counterstained with hemotoxylin. Injection sites were verified using light microscopy. Only animals with visible needle tracts that penetrated the third ventricle were used in this study. Eleven animals (15% of total) were excluded from this study because the microinjection sites were outside of the third ventricle. Data Analysis Phase shifts in the circadian activity rhythm were quantified using the linear regression method as described (Daan and Pittendrigh, 1976). A line was fitted to activity onsets that occurred on the 10 days preceding the microinjection. A second line was fitted to activity onsets that occurred 4-10 days after microinjection. Days 1-3 postmicroinjection were not used in the data analysis to avoid including transient effects.

66 51 Phase shifts were determined by the difference between the two regression lines. T-tests or the corresponding non-parametric Kruskal-Wallis test were used to evaluate statistical differences between experimental and control groups. The non-parametric test was used in experiments in which the distribution of the data was significantly different from a normal distribution based on the Shapiro-Wilk W-test. Results Experiment 1: an NMDA antagonist blocks GRP-induced phase delays This experiment was designed to test whether microinjection of an NMDA receptor antagonist would inhibit the phase-shifting effects of GRP into the third ventricle. Microinjection of GRP into the third ventricle at CT 13 produced large phase delays of the circadian activity rhythm (-1.83 ± 0.76 h, n=9). Co-administration of AP5 with the GRP at CT 13 resulted in phase shifts that were significantly smaller than those produced by microinjection of GRP alone (-0.07 ± 0.45 h, n=16, two-sample t-test, P=0.012, Figs. 2A and 2B). The phase shifts produced by the GRP/AP5 cocktail were not significantly different from zero (one-sample t-test, P=0.88). Experiment 2: a glutamate reuptake inhibitor does not induce phase delays In order to investigate the effects of increased glutamate neurotransmission on GRPinduced phase shifts, the effects of a glutamate reuptake inhibitor on circadian phase were

67 52 GRP GRP/ AP5 A B PDC SAL C D Fig. 2A. Representative actograms of circadian activity rhythms before and after microinjection of (A) gastrin-releasing peptide (GRP), (B) GRP and (±)-2-amino-5- phosphopentanoic acid (AP5), (C) L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), and (D) saline (SAL). Within each individual actogram, each line represents one 24 h period and depicts the relative amount of wheel-running activity in 5 min bins. Successive days are shown from top to bottom. All injections were given under dim red light illumination at CT 13 (indicated by a white circle).

68 53 Phase Delays (Hours) * GRP GRP/AP5 PDC SAL GRP * GRP/PDC * PDC+LP SAL+LP 0.00 Exp 1 Exp 2 Exp 3 Exp 4 Fig. 2B. Phase delays (mean ± SEM) in hours induced by microinjection of gastrinreleasing peptide (GRP), GRP and (±)-2-amino-5-phosphopentanoic acid (AP5), ), L- trans-pyrrolidine-2,4-dicarboxylic acid (PDC), or saline (SAL) into the third ventricle, or by administration of a light pulse (LP) on the phase of free-running activity rhythms at CT 13. Asterisks denote significant differences (P<0.05) between the two groups within each experiment.

69 54 evaluated. Microinjection of the glutamate reuptake inhibitor PDC into the third ventricle at CT 13 (-0.13 ± 0.19 h, n=10) did not produce phase shifts that were significantly different from those produced by microinjection of vehicle (-0.20 ± 0.09 h, n=18, two-sample t-test, P=0.76, Figs. 2A and 2B). Experiment 3: a glutamate reuptake inhibitor increases the phase-shifting effects of GRP This experiment tested the hypothesis that increases in glutamatergic neurotransmission would enhance the phase-shifting effects of GRP. As in Experiment 1, microinjection of GRP into the third ventricle at CT 13 (-1.25 ± 0.70 h, n=10) induced phase delays. Coadministration of PDC with GRP (-2.52 ± 0.59 h, n=12) significantly increased the magnitude of the observed phase delays (Kruskal-Wallis test, P=0.049, Figs. 2B and 2C). There was no significant difference between the phase-shifting effects of GRP in this experiment vs. those in Experiment 1. Experiment 4: a glutamate reuptake inhibitor increases the phase-shifting effects of light Because PDC significantly enhanced the phase shifts induced by GRP, we tested whether PDC would similarly enhance light-induced phase shifts. PDC or saline was microinjected into the third ventricle at CT 13, followed immediately by a 5-min light pulse. Light-induced phase shifts following microinjection of PDC (-1.34 ± 0.30 h, n=11) were significantly larger than those following injection of vehicle (-0.35 ± 0.22 h, n=7, two-sample t-test, P=0.012, Figs. 2B and 2C).

70 55 GRP GRP/ PDC A B PDC +LP SAL+ LP C D Fig. 2C. Representative actograms of circadian activity rhythms before and after microinjection of (A) gastrin-releasing peptide (GRP), (B) GRP and L-trans-pyrrolidine- 2,4-dicarboxylic acid (PDC), (C) PDC and a light pulse (LP, 300 lux), and (D) saline (SAL) and a LP (300 lux). Within each individual actogram, each line represents one 24 h period and depicts the relative amount of wheel-running activity in 5 min bins. Successive days are shown from top to bottom. All injections were given under dim red light illumination at CT 13 (indicated by a white circle).

71 56 Discussion This study provides evidence in support of the hypothesis that activation of NMDA receptors is a necessary step in the transduction of GRP signaling in the SCN. Previous research had demonstrated that microinjection of GRP near the SCN or into the third ventricle in the early subjective night produces large phase delays, similar to those seen with exposure to light (Piggins et al., 1995;Antle et al., 2005). This effect was also seen in vitro, as the application of GRP to hypothalamic slice preparation can phase shift the SCN neuronal firing rhythm (Albers et al., 1991;Piggins and Rusak, 1993) in a pattern similar to that produced by light (Ding et al., 1994). The results of the present study demonstrate that the level of concurrent glutamatergic signaling in the SCN strongly influences the magnitude of the phase-shifting response to GRP during the early subjective night. There is an increasing body of evidence suggesting that GRP is involved in intracompartmental communication between distinct regions of the SCN: the retinorecipient core and the pacemaker cells of the shell. GRP-positive cells are localized in the SCN core of rodents (Moore et al., 2002), GRP receptors are localized in the SCN shell (Karatsoreos et al., 2006), microinjection of GRP near the SCN causes activation of the immediate-early gene c-fos in the dorsal SCN (Antle et al., 2005;Piggins et al., 2005), microinjection of GRP into the third ventricle causes activation of Per1, Per2 and MAP kinase in the dorsal SCN (Antle et al., 2005), and the administration of GRP causes changes in the expression of clock genes that are similar to those changes seen with

72 57 exposure to light (Dardente et al., 2002). Taken together, these data suggest that GRP is an integral peptide that receives information about photic conditions and transmits that information to a separate population of cells within the SCN. One open question, however, is whether GRP alone is sufficient to shift the circadian clock, or whether glutamate stimulation of the SCN acts though other signaling pathways within the SCN to facilitate the effects of GRP. If GRP were to act solely downstream from glutamatergic, then blocking glutamatergic signaling in the SCN should not alter the ability of GRP to shift the clock. However, if glutamatergic input to the SCN simultaneously activates other necessary routes through the SCN other than the one involving GRP, then blocking the action of glutamate would inhibit GRP-induced phase shifts. The present study finds that the application of AP5, an NMDA antagonist, blocks GRP-induced shifts. The RHT conveys photic information to the brain, and it is well documented that the RHT contains glutamate (Castel et al., 1993;Ebling, 1996;Hannibal, 2002). Glutamate then acts on NMDA receptors to activate signal transduction pathways leading to shifts in circadian clock phase. Application of NMDA to the SCN in vivo or NMDA or glutamate to the SCN in vitro is sufficient to reset the circadian clock and the resulting PRC closely resembles that of light (Ding et al., 1994;Mintz et al., 1999). Because microinjection of AP5 completely blocked GRP-induced shifts, then it can be concluded that NMDA receptor activation is required, either for the initiation of GRPinduced signaling or for the ability of that signal to spread across the SCN and,

73 58 ultimately, shift the phase of the circadian clock. However, note that GRP-induced phase advances were not tested, and the effects of AP5 on these types of shifts may be different. In order to further explore the interaction between glutamate neurotransmission and GRP-induced phase shifts, we employed PDC, a glutamate reuptake inhibitor. Because glutamate is in the RHT and is the neurotransmitter responsible for transmitting photic information to the brain, we wanted to evaluate the effects of increased glutamatergic transmission on GRP-induced shifts. Previous groups have studied the role of glutamate administration on the circadian phase and found that while glutamate can induce shifts in the hypothalamic slice preparation (Ding et al., 1994;Shibata et al., 1994), glutamate was unable to elicit light-like shifts in vivo (Meijer et al., 1988). This discrepancy could be explained by the loss of tonic inhibition from γ-aminobutyric acid (GABA)ergic innervations in the in vitro preparation. Such GABAergic innervations would be intact in vivo and may exert enough of an influence to prevent glutamateinduced shifts. Another possibility is that the uptake of exogenous glutamate in the SCN region is so rapid that a microinjection represents an insufficient stimulus to generate a phaseshifting response. To test this, we examined whether injection of a glutamate reuptake inhibitor would alter circadian phase. Our results demonstrated that PDC does not induce a phase shift when microinjected into the third ventricle at CT 13. However, when injected in a cocktail with GRP, PDC increased GRP-induced shifts. This result further establishes that GRP-induced shifts of the circadian clock are sensitive to the level of glutamatergic input to the SCN. The GRP-induced phase delays in Experiment 3 were

74 59 slightly lower in magnitude than those of Experiment 1; however, the average delays between these two experiments were not statistically different from each other. It is possible that a larger dose of PDC could elicit a phase-shifting response on its own; therefore, we do not conclude that glutamate alone is insufficient to induce a circadian phase shift. Because inhibition of glutamate reuptake increased the phase-shifting effects of GRP, we tested whether the amplitude of light-induced shifts would be similarly affected. The results of Experiment 4 demonstrate that microinjection of PDC into the third ventricle enhances the phase-shifting effects of a brief light pulse (300 lux) during the early subjective night. Combined with the fact that previous studies have failed to demonstrate a light-like effect of glutamate application in vivo, this suggests that exogenous glutamate provides an insufficient stimulus to provoke a phase-resetting response. This may be due to rapid uptake of exogenous glutamate, preventing a single microinjection from providing a sufficient stimulus. This idea is supported by the fact that microinjection of NMDA agonists, for which uptake or degradation is much slower than endogenous glutamate, induces light-like phase shifts (Mintz and Albers, 1997;Mintz et al., 1999). However, as demonstrated in the present study, increases in extracellular glutamate, when coupled with another phase-shifting stimulus such as light or GRP administration, enhance the phase shifting effects of these other stimuli. This demonstrates that GRP-induced phase delays are sensitive to the concentration of extracellular glutamate.

75 60 It is important to note that both AP5 and PDC may be altering signaling by the general extracellular pool of glutamate in the SCN, not just the neurotransmission from the RHT. The extracellular concentration of glutamate in the SCN regions shows a circadian rhythm that peaks in the second half of the night, and is independent of action potential-drive glutamate release (Rea et al., 1993). Therefore, we cannot assume that the phase shifting effects of GRP require concurrent synaptic glutamate release, only that there needs to be sufficient extracellular glutamate present to provide the necessary stimulation. It is quite likely that glutamate acts at multiple locations to alter GRP signaling, regulating the excitability of GRP-synthesizing cells and cells receiving GRP signals, at both synaptic as well as non-synaptic receptor sites. Cells in the dorsal SCN that are relatively insensitive to glutamatergic input (Abe et al., 1992) may respond to GRP but be unable to communicate phase-shifting information to the rest of the SCN. Such determinations will require a close examination of temporal and spatial gene expression patterns in the SCN in response to GRP in the presence or absence of glutamatergic input. In analyzing the phase shift data for this experiment, it became clear that there was significant variability in the size of the phase delays induced by GRP, resulting in relatively large standard errors in the groups receiving GRP injections. Further examination of the data revealed that a small number of animals showed an unusual patterns of activity following treatment (Fig. 2D). These animals initially appeared to show large phase delays in response to treatment. The objective measure of phase shift amplitude, the linear regression method, uses Days 4-10 following treatment as one of the

76 Fig. 2D. Actogram showing an unusual response to microinjection of gastrin-releasing peptide (GRP). This animal displayed a large initial phase shift in response to GRP, but after about 8 days, returned to a phase close to that predicted by the original free-running period. The injection was given under dim red light illumination at CT 13 (indicated by a white circle). 61

77 62 variables in the calculation of the phase shift and, by this method, the animal in Fig. 2D had approximately a 6 h phase delay. After Day 10 however, this animal appeared to return to its original phase, as if there had been no shift of the circadian clock. One possible explanation for this effect would be that GRP is shifting the phase of the circadian clock in the cells that directly regulate locomotor output, but not all clock cells in the SCN. Over time, the shifted cells are pulled back into phase with the rest of the SCN via coupling between SCN cells. Regardless of the mechanism, however, these results indicate that a certain amount of subjectivity is necessary in evaluating the size of a circadian phase shift. We also noted that the amplitude of GRP-induced phase shifts appeared to vary between Experiments 1 and 3, with GRP causing larger phase shifts in Experiment 1. Although there was no significant difference between the size of GRP-induced phase shifts between the two experiments, there was a procedural change in timing of cage maintenance that could account for the difference. During Experiment 1, the animals cages were changed between treatments, while in Experiment 3, the cages were changed at the time of treatment. Because a cage change can be considered a non-photic manipulation it would be expected to reduce the size of a photic or photic-like phase shift. This is the direction of the difference in GRP-induced shifts between the two experiments. In summary, microinjection of GRP into the third ventricle produces large phase delays in the early subjective night and these delays can be blocked by the administration of an NMDA antagonist. Further, while a glutamate reuptake inhibitor cannot induce

78 63 phase shifts on its own, it can enhance the resulting phase shifts of both GRP and light. Taken together, the present results show that activation of NMDA receptors is necessary for the effects of GRP on circadian phase, and suggests that a simple linear pathway from the retina to GRP cells to clock-generating cells is insufficient to generate a light-induced phase shift. Acknowledgements The authors would like to thank Tamara J. Scott for her assistance. This research was supported by National Institutes of Health Grant NS to E.M.M. Reference List Abe H, Rusak B (1994) Physiological mechanisms regulating photic induction of Foslike protein in hamster suprachiasmatic nucleus. Neurosci Biobehav Rev 18: Abe H, Rusak B, Robertson HA (1992) NMDA and non-nmda receptor antagonists inhibit photic induction of Fos protein in the hamster suprachiasmatic nucleus. Brain Res Bull 28: Abrahamson EE, Moore RY (2001) Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections. Brain Res 916: Aida R, Moriya T, Araki M, Akiyama M, Wada K, Wada E, Shibata S (2002) Gastrinreleasing peptide mediates photic entrainable signals to dorsal subsets of suprachiasmatic nucleus via induction of Period gene in mice. Mol Pharmacol 61: Albers HE, Gillespie CF, Babagbemi TO, Huhman KL (1995) Analysis of the phase shifting effects of gastrin releasing peptide when microinjected into the suprachiasmatic region. Neurosci Lett 191: Albers HE, Liou SY, Stopa EG, Zoeller RT (1991) Interaction of colocalized neuropeptides: functional significance in the circadian timing system. J Neurosci 11:

79 64 Antle MC, Kriegsfeld LJ, Silver R (2005) Signaling within the master clock of the brain: localized activation of mitogen-activated protein kinase by gastrin-releasing peptide. J Neurosci 25: Antle MC, Silver R (2005) Orchestrating time: arrangements of the brain circadian clock. Trends Neurosci 28: Castel M, Belenky M, Cohen S, Ottersen OP, Storm-Mathisen J (1993) Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus. Eur J Neurosci 5: Daan S, Pittendrigh CS (1976) A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J Comp Physiol 106: Dardente H, Poirel VJ, Klosen P, Pevet P, Masson-Pevet M (2002) Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light. Brain Res 958: Ding JM, Chen D, Weber ET, Faiman LE, Rea MA, Gillette MU (1994) Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO. Science 266: Ebling FJ (1996) The role of glutamate in the photic regulation of the suprachiasmatic nucleus. Prog Neurobiol 50: Gorman MR, Evans HF, Elliot JA (2006) Potent circadian effects of dim illumination at night in hamsters. Chronobiol Int 23: Gorman MR, Kendall AR, Elliot JA (2005) Scotopic illumination enhances entrainment of circadian rhythms to lengthening light: dark cycles. J Biol Rhythms 20: Gorman MR, Steele CT (2006) Phase angle difference alters coupling relations of functionally distinct circadian oscillators revealed by rhythm splitting/. J Biol Rhythms 21: Hannibal J (2002) Neurotransmitters of the retino-hypothalamic tract. Cell Tissue Res 309: Karatsoreos IN, Romeo RD, McEwen BS, Silver R (2006) Diurnal regulation of the gastrin-releasing peptide receptor in the mouse circadian clock. Eur J Neurosci 23: Karatsoreos IN, Yan L, LeSauter J, Silver R (2004) Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus. J Neurosci 24:

80 65 Kornhauser JM, Nelson DE, Mayo KE, Takahashi JS (1990) Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus. Neuron 5: LeSauter J, Kriegsfeld LJ, Hon J, Silver R (2002) Calbindin-D(28K) cells selectively contact intra-scn neurons. Neuroscience 111: Meijer JH, Van der Zee EA, Dietz M (1988) Glutamate phase shifts circadian activity rhythms in hamsters. Neurosci Lett 86: Mintz EM, Albers HE (1997) Microinjection of NMDA into the SCN region mimics the phase shifting effect of light in hamsters. Brain Res 758: Mintz EM, Marvel CL, Gillespie CF, Price KM, Albers HE (1999) Activation of NMDA receptors in the suprachiasmatic nucleus produces light-like phase shifts of the circadian clock in vivo. J Neurosci 19: Moore RY (1995) Organization of the mammalian circadian system. Ciba Found Symp 183: Moore RY, Card JP (1985) Visual pathways and the entrainment of circadian rhythms. Ann N Y Acad Sci 453: Moore RY, Eichler VB (1972) Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res 42: Moore RY, Speh JC, Leak RK (2002) Suprachiasmatic nucleus organization. Cell Tissue Res 309: Morin LP, Shivers KY, Blanchard JH, Muscat L (2006) Complex organization of mouse and rat suprachiasmatic nucleus. Neuroscience 137: Piggins HD, Antle MC, Rusak B (1995) Neuropeptides phase shift the mammalian circadian pacemaker. J Neurosci 15: Piggins HD, Cutler DJ, Rusak B (1994) Effects of ionophoretically applied bombesinlike peptides on hamster suprachiasmatic nucleus neurons in vitro. Eur J Pharmacol 271: Piggins HD, Goguen D, Rusak B (2005) Gastrin-releasing peptide induces c-fos in the hamster suprachiasmatic nucleus. Neurosci Lett 384: Piggins HD, Rusak B (1993) Electrophysiological effects of pressure-ejected bombesinlike peptides on hamster suprachiasmatic nucleus neurons in vitro. J Neuroendocrinol 5:

81 66 Rea MA, Ferriera S, Randolph W, Glass JD (1993) Daily profile of the extracellular concentration of glutamate in the suprachiasmatic region of the Siberian hamster. Proc Soc Exp Biol Med 204: Shibata S, Watanabe A, Hamada T, Ono M, Watanabe S (1994) N-methyl-D-aspartate induces phase shifts in circadian rhythm of neuronal activity of rat SCN in vitro. Am J Physiol 267: R360-R364. Stephan FK, Zucker I (1972) Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc Natl Acad Sci U S A 69: Tanaka M, Hayashi S, Tamada Y, Ikeda T, Hisa Y, Takamatsu T, Ibata Y (1997) Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat. Neuroreport 8:

82 CHAPTER III Gastrin-releasing Peptide and Neuropeptide Y Exert Opposing Actions on Circadian Phase 3 Abstract Microinjection of gastrin-releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other s effect on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other s 3 Reproduced in part with permission from Elsevier Publishing, USA, as appears in Neuroscience Letters (422(2007) 59-63) by G.J. Kallingal & E.M. Mintz 67

83 68 effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells. Introduction The suprachiasmatic nucleus (SCN) drives physiological and behavioral circadian rhythms and responds to both photic and non-photic cues to entrain to a 24 h light-dark cycle (Challet and Pevet, 2003). Photic information is conveyed to the SCN primarily by the retinohypothalamic tract (RHT), a monosynaptic projection from the retina to the SCN (Pickard, 1982). Non-photic information, particularly relating to the activity level of the animal, is communicated to the SCN through a neuropeptide Y (NPY)-containing projection from the intergeniculate leaflet (IGL) (Harrington et al., 1985;Janik et al., 1995) and a serotonergic projection from the median raphe nucleus (Morin and Meyer- Bernstein, 1999). These inputs are integrated by the SCN to regulate the entrainment of circadian rhythms. The SCN is heterogeneous in structure and function (Antle and Silver, 2005;Morin et al., 2006). The cells that comprise the SCN have notable variability in morphological appearance, electrophysiological firing rates, and patterns of gene expression. In Syrian hamsters, one neurochemical marker of the SCN is the presence of cells expressing gastrin-releasing peptide (GRP) in a central region of the nucleus (LeSauter et al., 2002). These cells are thought to be mediators of photic entrainment (Karatsoreos et al., 2004;Tanaka et al., 1997). Animals exposed to a brief light pulse during the subjective night show increased immunoreactivity for c-fos in GRP cell bodies

84 69 (Earnest et al., 1993;Karatsoreos et al., 2004;Romijn et al., 1996). Microinjection of GRP into the third ventricle (Antle et al., 2005), or near the SCN (Kallingal and Mintz, 2006;Piggins et al., 1995), elicits phase shifts in a pattern similar to exposure of light. In mice, the loss of the GRP receptor results in a reduced phase shifting response to bright light (Aida et al., 2002). These data suggest that GRP plays a role in the establishment of circadian phase. NPY, synthesized in the intergeniculate leaflet of the thalamus and released in the SCN, conveys both photic and non-photic signals (Yannielli and Harrington, 2004). Microinjection of NPY into the SCN region during the middle of the subjective day phase advances the circadian clock, while having little effect during the subjective night (Albers and Ferris, 1984;Huhman and Albers, 1994). However, NPY can affect clock function during the subjective night by opposing the ability of light to induce a phase shift (Lall and Biello, 2003a;Weber and Rea, 1997) and induce the expression of Per1 mrna (Yannielli and Harrington, 2004;Gamble et al., 2006). These data suggest that NPY plays an active role in both photic and non-photic effects on the circadian clock. Since light and NPY appear to exert opposing influences on the circadian clock, and GRP is part of the photic signal transduction pathway, we sought to determine whether NPY inhibited the effects of GRP administration on circadian clock phase. In contrast, there is little data regarding a role for GRP in non-photic phase shifting. However, light can inhibit the phase shifting effects of NPY (Biello and Mrosovsky, 1995) and the phase shifting effects of behavioral activation (Mrosovsky, 1991). Therefore, in order to determine whether GRP plays a role in mediating the effects of light on non-photic phase

85 70 shifting, we sought to determine whether GRP could inhibit NPY-induced phase advances. Materials and Methods Subjects Thirty-eight adult male Syrian hamsters (Mesocricetus auratus, 2-4 months) were bred at Kent State University from stock derived from animals purchased from Harlan Sprague Dawley (Indianapolis, IN, USA). Animals were group-housed in a 14:10 h light-dark cycle with food and water available ad libitum. All experimental procedures were approved by the Kent State University Animal Care and Use Committee, and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Immunohistochemistry For GRP (rabbit anti-grp, 1:1,000; Immunostar, Hudson, WI, USA) and NPY IHC (goat anti-5-ht, 1:1,000; Immunostar), untreated hamsters were taken from the colony, deeply anesthetized with sodium pentobarbital (200 mg/kg) and were trans-cardially perfused with 200 ml of Phosphate Buffered Saline (PBS) followed by 200 ml of 4% paraformaldehyde. Coronal sections (40 µm thickness) were sliced on a vibratome and every third section was collected and stored in PBS. In all subsequent steps, sections were rinsed (3 x 5 min) in PBS between incubations. Sections were incubated in blocking solution containing 5% normal donkey serum in antisera diluent with 0.3%

86 71 Triton-X for 1 h, followed by an overnight room-temperature incubation of both primary antibodies. Sections were then incubated in Cy-3 conjugated donkey anti-rabbit (1:500, Jackson Immunoresearch, West Grove, PA, USA) and Alexa 633 donkey anti-goat (1:500; Molecular Probes, Carlsbad, CA, USA) for 1 h at room temperature. After rinsing with PBS, sections were mounted on gel coated slides, set out to dry, and coverslipped with Krystalon. Slides were stored at 4 C. Fluorescence images were captured using an Olympus BX-51 microscope. Surgical Procedure Hamsters were anesthetized (110 mg/kg ketamine, 22 mg/kg xylazine and 1.83 mg/kg acepromazine) and stereotaxically implanted with a 4 mm, 26-gauge guide cannula (Plastics One, Roanoke, VA, USA) aimed at the SCN. Stereotaxic coordinated were 1.1 m anterior and 1.7 mm lateral to bregma with the depth such that a microinjection needle inserted through the guide cannula would end at 7.4 mm below dura. The cannulas were implanted at a 10º angle toward the midline. Prior to cannula insertion, the skull was leveled between bregma and lambda. After surgeries, hamsters were individually housed in Plexiglass cages (24 cm x 45.5 cm x 21 cm) and given 24 h to recover in their original colony room. Hamsters were then placed in a continuously dark environment (DD), and were allowed to establish free-running activity rhythms. Cages were equipped with a running wheel (diameter 18 cm) that was monitored by a computer using ClockLab software (Actimetrics).

87 72 Microinjection After 10 days, hamsters received the following treatments in randomized order: microinjection of 125 pmol gastrin-releasing peptide (GRP; Phoenix Pharmaceuticals, Belmont, CA, USA) and 29.3 pmol neuropeptide Y (NPY; Phoenix Pharmaceuticals), each dissolved in phosphate buffered saline (PBS), both GRP and NPY, or PBS alone. Microinjections were given with a 32-gauge needle that extended beyond the guide cannula by 5.2 mm and was attached by polyethylene tubing to a 1 µl Hamilton syringe. The polyethylene tubing was filled with distilled water and an air bubble was introduced at the tip to avoid dilution of injected substances. Injections were given with the aid of night vision goggles while the hamsters were gently restrained by hand. The final volume of each microinjection was 250 nl, which were administered over a period of 10 s. After each microinjection, the needle was left in place for s. Microinjections were given at circadian time (CT) 6 ± 15 or 13 ± 15 min. Following microinjection, hamsters were returned to their home cages. Histology for behavioral experiments After the completion of each behavioral experiment, animals were deeply anesthetized with sodium pentobarbital (200 mg/kg) and killed by decapitation. Hamster brains were extracted and post-fixed in 4% paraformaldehyde. Coronal sections (100 µm thick) were sliced on a vibratome and counterstained with hemotoxylin. Injection sites were verified using light microscopy. Only animals with visible needle tracts that were within 300 µm

88 73 of the SCN were used in this study. Six animals (16% of total) were excluded from this study because the microinjection sites were farther then 300 µm from the SCN. Data Analysis Phase shifts in the circadian activity rhythm were quantified using the linear regression method (Daan and Pittendrigh, 1976). A line was fitted to activity onsets that occurred on the 10 days preceding the microinjection. The onsets of activity for each day were initially determined by ClockLab data analysis software (Actimetrics), and then visually inspected for artifacts. Days when the software could not calculate an onset were omitted from further calculations. A second line was fitted to activity onsets that occurred 4-10 days after microinjection. Days 1-3 post-microinjection were not used in the data analysis to avoid including transient effects. Phase shifts were determined by the difference between the two regression lines on the day following treatment. All of the work involved in phase shift calculations was performed by individuals blind to experimental treatments. Phase shifts are shown as mean ± standard error. Differences between groups were evaluated using a one-way ANOVA and the Tukey-Kramer test. Results Experiment 1 tested the hypothesis that NPY would inhibit phase delays induced by microinjection of GRP into the SCN region. There was a significant difference amount experimental groups injected at CT 13 (one-way ANOVA, P = ).

89 74 Microinjection of GRP induced delays of the circadian activity rhythm (-0.67 ± 0.12 h, n = 8) that were significantly different from animals that received NPY (0.16 ± 0.10 h, n = 9), a cocktail of GRP and NPY (-0.05 ± 0.08 h, n = 9), or vehicle (0.07 ± 0.07 h, n = 9) (Tukey-Kramer test, P < 0.05) (Figs. 3A and 3B). Experiment 2 tested the hypothesis that GRP would attenuate NPY-induced phase advances. There was a significant different among experimental groups at CT 6 (oneway ANOVA, P = ). Microinjection of NPY into the SCN region induced advances of the circadian activity rhythm (0.53 ± 0.12, n = 11) that were significantly different from animals that received GRP (-0.16 ± 0.03 h, n = 7), a cocktail of NPY and GRP (-0.07 ± 0.10 h, n = 7), or vehicle (0.05 ± 0.06 h, n = 8) (Tukey-Kramer test, P < 0.05) (Figs. 3B and 3C). Discussion The results of this study suggest that GRP and NPY have mutually inhibitory effects on the other s influence on circadian rhythmicity. During the early subjective night, microinjection of NPY into the SCN region blocked the phase shifting effects of GRP. Previous studies have shown that NPY (Lall and Biello, 2003a) or an NPY receptor agonist (Gamble et al., 2005) is able to attenuate light-induced phase advances in the late subjective night, even when NPY is administered up to 60 min after the light pulse (Lall and Biello, 2002;Lall and Biello, 2003b). However, the data on the ability of

90 75 GRP ΔФ = NPY ΔФ = A B GRP/ NPY ΔФ = SAL ΔФ = C D Fig. 3A. Representative actograms of circadian activity rhythms before and after microinjection of (A) GRP, (B) NPY, (C) GRP/NPY cocktail, and (D) SAL. Each line represents one 24 h period. Dark bars depict wheel-running activity. All injections were given with the aid of night vision goggles at CT 13 (indicated by a white circle). The regression lines used for calculating the phase shifts are shown along the onsets of activity, and the calculated phase shifts are shown.

91 76 Phase Shift (hrs) * GRP NPY GRP/NPY SAL CT6 * CT13 Fig. 3B. Mean ± S.E.M. phase shift in hours induced by microinjection of GRP, NPY, GRP/NPY, and SAL near the SCN at CT 6 and CT 13. NPY is different from all other groups at CT 6 (*), and GRP is different from all other groups at CT 13 (*) (Tukey- Kramer, P < 0.05).

92 77 NPY ΔФ = GRP ΔФ = A B NPY/GRP ΔФ = SAL ΔФ = C D Fig. 3C. Representative actograms of circadian activity rhythms before and after microinjection of (A) NPY, (B) GRP, (C) NPY/GRP cocktail, and (D) SAL. Each line represents one 24 h period. Dark bars depict wheel-running activity. All injections were given with the aid of night vision goggles at CT 6 (indicated by a white circle). The regression lines used for calculating the phase shifts are shown along the onsets of activity, and the calculated phase shifts are shown.

93 78 NPY to inhibit light-induced delays in the early subjective night is mixed. Some reports indicate that NPY can attenuate light-induced delays (Lall and Biello, 2003a;Lall and Biello, 2003b). However, others show that NPY in the early subjective night does not attenuate light-induced delays, even when administered at high concentrations (Gamble et al., 2005). In addition to altering behavioral phase shifts during the late subjective night, NPY has been shown to inhibit the effects of light on the induction of clock genes (Brewer et al., 2002;Gamble et al., 2006). Taken together, these data support the idea that the transmission of photic information to the circadian clock mechanism does not follow a linear pathway. The fact that NPY is capable, at least in some circumstances, of blocking both light and GRP-induced phase delays would suggest that NPY is acting downstream from GRP signal reception in the SCN to inhibit light-induced phase shifts. However, GRP-induced phase delays are also dependent on the level of glutamatergic neurotransmission in the SCN (Kallingal and Mintz, 2006), and glutamatergic input from the retinas is thought to be upstream of GRP signaling. This suggests that there are parallel pathways for photic information within the SCN, including both GRP-dependent and GRP-independent routes. This idea is supported by data from GRP receptor knockout mice that show a moderately attenuated phase-shifting response to bright light (Aida et al., 2002), but not complete inhibition. There is a continually growing body of evidence that indicates that GRP plays a major role in photic resetting of the circadian clock by acting as a signal that communicates photic conditions to the oscillator cells of the SCN. GRP immunoreactivity is localized in the retinorecipient regions of the rodent SCN (Moore et

94 79 al., 2002;Morin et al., 2006), GRP receptors are localized in the dorsal SCN (Karatsoreos et al., 2006), microinjection of GRP near the SCN induces expression of c-fos in the dorsal SCN (Antle et al., 2005;Piggins et al., 2005), and microinjection of GRP into the third ventricle induces Per1, Per2, and the phosphorylated form of extracellular regulated kinase (p-erk) in the dorsal SCN (Antle et al., 2005). Additionally, the presentation of a light pulse to animals kept in constant conditions induces Per1 expression in GRPcontaining cells (Dardente et al., 2002). Together, these data strongly support the idea that GRP plays a significant role in transmitting photic information within the SCN, thereby affecting the circadian phase and cellular activity of the SCN. Non-photic input, mediated at least in part by NPY, would then inhibit these effects of GRP through the suppression of GRP-induced period gene expression. Although NPY does not alter circadian phase when injected during the early subjective night, it does induce phase advances when given during the subjective day, both in vivo (Albers and Ferris, 1984;Huhman and Albers, 1994) and in vitro (Biello et al., 1997;Shibata and Moore, 1993). Because the results of Experiment 1 indicated that NPY can inhibit GRP-induced delays in the early night, and because exposure to light during the subjective day can inhibit the phase shifting effects of NPY (Biello and Mrosovsky, 1995), we tested whether GRP inhibits NPY-induced advances. The results of Experiment 2 indicate that NPY-induced phase advances are attenuated by GRP, and suggest the possibility that NPY-induced phase advances are regulated by the level of stimulation being provided by GRP release within the SCN. The precise signaling mechanism for the interaction between GRP and NPY-induced signaling is unknown.

95 80 One possibility is that GRP and NPY are co-localized to the same cells (Fig. 3D) and that these inputs from each neuropeptide activate opposing intracellular signaling cascades. A second possibility is that each neuropeptide does not prevent the SCN cells from responding to the other, bur rather prevents the change in clock phase from being communicated to the rest of the SCN. GRP appears to activate a select subset of neurons in the dorsal SCN, which are then likely to transmit that information to other parts of the nucleus (Antle et al., 2005;Piggins et al., 2005). Blocking communication from these GRP-responsive neurons could inhibit the transfer of phase information to the rest of the SCN. A direct interaction between GRP and NPY is supported by neuroanatomical evidence: NPY fibers in the SCN show broad overlap with both GRP-containing cell bodies and GRP fibers (Fig. 3D) (Morin et al., 2006). It is important to note however that the effects of NPY on GRP-induced phase advances in the late subjective night were not examined in this study. There is evidence indicating that phase delays and advances are not always mediated by the same factors, or signaling events (Cheng et al., 2004). However, a consensus exists in the literature that NPY is able to block light-induced advances in the late night. Therefore, we hypothesize that NPY would also attenuate GRP-induced advances when administered near the SCN in the late subjective night.

96 Fig. 3D. Photomicrographs showing overlap of GRP (red) and NPY (green) immunoreactivity in the hamster SCN. Overlap is found to be greatest in the lateral portion of the nucleus, from the ventral border to about half way up the dorsal-ventral extent of the SCN. 81

97 82 Acknowledgements The authors would like to thank Veronica Porterfield and Erin Gilbert for their assistance. This research was supported by NIH Grant NS and the Kent State Department of Biological Sciences. Reference List Aida R, Moriya T, Araki M, Akiyama M, Wada K, Wada E, Shibata S (2002) Gastrinreleasing peptide mediates photic entrainable signals to dorsal subsets of suprachiasmatic nucleus via induction of Period gene in mice. Mol Pharmacol 61: Albers HE, Ferris CF (1984) Neuropeptide Y: role in light-dark cycle entrainment of hamster circadian rhythms. Neurosci Lett 50: Antle MC, Kriegsfeld LJ, Silver R (2005) Signaling within the master clock of the brain: localized activation of mitogen-activated protein kinase by gastrin-releasing peptide. J Neurosci 25: Antle MC, Silver R (2005) Orchestrating time: arrangements of the brain circadian clock. Trends Neurosci 28: Biello SM, Golombek DA, Harrington ME (1997) Neuropeptide Y and glutamate block each other's phase shifts in the suprachiasmatic nucleus in vitro. Neuroscience 77: Biello SM, Mrosovsky N (1995) Blocking the phase-shifting effect of neuropeptide Y with light. Proc Biol Sci 259: Brewer JM, Yannielli PC, Harrington ME (2002) Neuropeptide Y differentially suppresses per1 and per2 mrna induced by light in the suprachiasmatic nuclei of the golden hamster. J Biol Rhythms 17: Challet E, Pevet P (2003) Interactions between photic and nonphotic stimuli to synchronize the master circadian clock in mammals. Front Biosci 8: s246-s257.

98 83 Cheng HY, Obrietan K, Cain SW, Lee BY, Agostino PV, Joza NA, Harrington ME, Ralph MR, Penninger JM (2004) Dexras1 potentiates photic and suppresses nonphotic responses of the circadian clock. Neuron 43: Daan S, Pittendrigh CS (1976) A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J Comp Physiol 106: Dardente H, Poirel VJ, Klosen P, Pevet P, Masson-Pevet M (2002) Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light. Brain Res 958: Earnest DJ, DiGiorgio S, Olschowka JA (1993) Light induces expression of fos-related proteins within gastrin-releasing peptide neurons in the rat suprachiasmatic nucleus. Brain Res 627: Gamble KL, Ehlen JC, Albers HE (2005) Circadian control during the day and night: Role of neuropeptide Y Y5 receptors in the suprachiasmatic nucleus. Brain Res Bull 65: Gamble KL, Paul KN, Karom MC, Tosini G, Albers HE (2006) Paradoxical effects of NPY in the suprachiasmatic nucleus. Eur J Neurosci 23: Harrington ME, Nance DM, Rusak B (1985) Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract. Brain Res Bull 15: Huhman KL, Albers HE (1994) Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness. Peptides 15: Janik D, Mikkelsen JD, Mrosovsky N (1995) Cellular colocalization of Fos and neuropeptide Y in the intergeniculate leaflet after nonphotic phase-shifting events. Brain Res 698: Kallingal G, Mintz EM (2006) Glutamatergic Activity Modulates the Phase Shifting Effects of Gastrin Releasing Peptide and Light. Eur J Neurosci 24: Karatsoreos IN, Romeo RD, McEwen BS, Silver R (2006) Diurnal regulation of the gastrin-releasing peptide receptor in the mouse circadian clock. Eur J Neurosci 23: Karatsoreos IN, Yan L, LeSauter J, Silver R (2004) Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus. J Neurosci 24: Lall GS, Biello SM (2002) Attenuation of phase shifts to light by activity or Neuropeptide Y: a time course study. Neuroscience 119:

99 84 Lall GS, Biello SM (2003a) Attenuation of circadian light induced phase advances and delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist. Neuroscience 119: Lall GS, Biello SM (2003b) Neuropeptide Y, GABA and circadian phase shifts to photic stimuli. Neuroscience 120: LeSauter J, Kriegsfeld LJ, Hon J, Silver R (2002) Calbindin-D(28K) cells selectively contact intra-scn neurons. Neuroscience 111: Moore RY, Speh JC, Leak RK (2002) Suprachiasmatic nucleus organization. Cell Tissue Res 309: Morin LP, Meyer-Bernstein EL (1999) The ascending serotonergic system in the hamster: comparison with projections of the dorsal and median raphe nuclei. Neuroscience 91: Morin LP, Shivers KY, Blanchard JH, Muscat L (2006) Complex organization of mouse and rat suprachiasmatic nucleus. Neuroscience 137: Mrosovsky N (1991) Double-pulse experiments with non-photic and photic phaseshifting stimuli. J Biol Rhythms Pickard GE (1982) The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection. J Comp Neurol 211: Piggins HD, Antle MC, Rusak B (1995) Neuropeptides phase shift the mammalian circadian pacemaker. J Neurosci 15: Piggins HD, Goguen D, Rusak B (2005) Gastrin-releasing peptide induces c-fos in the hamster suprachiasmatic nucleus. Neurosci Lett 384: Romijn HJ, Sluiter AA, Pool CW, Wortel J, Buijs RM (1996) Differences in colocalization between Fos and PHI, GRP, VIP and VP in neurons of the rat suprachiasmatic nucleus after a light stimulus during the phase delay versus the phase advance period of the night. J Comp Neurol 372: 1-8. Shibata S, Moore RY (1993) Neuropeptide Y and optic chiasm stimulation affect suprachiasmatic nucleus circadian function in vitro. Brain Res 615: Tanaka M, Hayashi S, Tamada Y, Ikeda T, Hisa Y, Takamatsu T, Ibata Y (1997) Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat. Neuroreport 8: Weber ET, Rea MA (1997) Neuropeptide Y blocks light-induced phase advances but not delays of the circadian activity rhythm in hamsters. Neurosci Lett 231:

100 Yannielli P, Harrington ME (2004) Let there be "more" light: enhancement of light actions on the circadian system through non-photic pathways. Prog Neurobiol 74:

101 CHAPTER IV The Influence of Gastrin-releasing Peptide on the Suprachiasmatic Nucleus Is Regulated by Glutamate, Serotonin, and the Supraoptic Nucleus Abstract Microinjection of gastrin-releasing peptide (GRP) into the third ventricle induces c-fos and the phosphorylated form of extracellular regulated kinase (p-erk) in a region just dorsal to GRP cells in the suprachiasmatic nucleus (SCN). Previously, we demonstrated that activation of NMDA receptors is necessary for GRP-induced phase shifts of the circadian clock. This study was designed to evaluate the interaction between GRP, glutamate, and serotonin in the regulation of circadian phase in Syrian hamsters. Microinjection of GRP into the third ventricle induced c-fos and p-erk expression throughout the SCN. Coadministration of (±)-2-amino-5-phosphonopentanoic acid (AP5, NMDA antagonist) or 8-hydroxy-2-di-n-propylamino-tetralin (DPAT, 5-HT 1A,7 agonist) with GRP limited GRP-induced expression in the SCN to a subset of cells dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP-induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. C-fos and p-erk immunoreactivity in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei also increased following the ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Adult male Syrian hamsters were given microinjections 86

102 87 of GRP into the region just dorsal to the SON during the early night. GRP-induced c-fos activity in the SCN was similar to the pattern following ventricular administration of GRP. GRP or bicuculline (a GABA A antagonist) administered near the SON during the early night elicited phase delays of the circadian activity rhythm. These data suggest that GRP-induced phase-resetting is dependent on the levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate the SON as a potential regulator of photic signaling in the SCN. Introduction The mammalian suprachiasmatic nucleus (SCN) contains an endogenous clock that regulates physiological and behavioral circadian rhythms (Moore and Eichler, 1972;Moore and Card, 1985). The circadian clock responds to photic and nonphotic stimuli and expresses rhythms that occur in roughly 24 hour cycles. In Syrian hamsters (Mesocricetus auratus), light is an essential entraining cue. Photic information is transmitted to the SCN by the retinohypothalamic tract (RHT), which is a monosynaptic projection from the retina to the SCN (Hendrickson et al., 1972;Pickard, 1982). The RHT is essential for photic entrainment, for the surgical destruction of this projection leads to the inability to entrain to environmental light-dark cycles (Johnson et al., 1988). Glutamate (Mintz et al., 1999;Mintz and Albers, 1997;Ding et al., 1994) and pituitary adenylate cyclase activating polypeptide (Hannibal et al., 1997) are thought to be the critical neurotransmitters of this pathway.

103 88 Several reports have identified GRP as a primary candidate for the transduction of photic information within the SCN (Morin et al., 1992;Piggins and Rusak, 1993;McArthur et al., 2000;Antle et al., 2005;Piggins et al., 2005;Karatsoreos et al., 2004). GRP cells receive direct input from the RHT, suggesting that GRP plays a role in mediating the effects of light on the circadian clock (Abrahamson and Moore, 2001;Tanaka et al., 1997). GRP cell bodies are located in the ventral SCN in the rat (Dardente et al., 2002) and in the central region of the hamster SCN (Karatsoreos et al., 2004;Karatsoreos et al., 2006) and the receptors that GRP acts on are localized dorsally (Dardente et al., 2002;Karatsoreos et al., 2004;Karatsoreos et al., 2006). Animals in constant conditions presented with a light pulse express the immediate early gene c-fos in GRP cells (Earnest et al., 1993;Romijn et al., 1996). Exogenous application of GRP to the third ventricle (Antle et al., 2005), or near the SCN (Albers et al., 1991;Piggins et al., 1995), phase shifts the clock in a manner similar to light. The effects of GRP on the circadian clock are dependent on the level of excitation provided by activated NMDA receptors (Kallingal and Mintz, 2006) and can be opposed by neuropeptide Y (Kallingal and Mintz, 2007). Finally, previous reports indicate that the administration of GRP in the early subjective night increases c-fos (Piggins et al., 2005;Antle et al., 2005) and the phosphorylated form of extra-cellular regulated kinase (p-erk) (Antle et al., 2005) in a region just dorsal to GRP cell bodies (Antle et al., 2005). These data suggest that GRP plays an active role in mediating the effects of light on the circadian clock.

104 89 The current study was designed to determine the immunohistological and behavioral effects of GRP administered to the third ventricle in the presence or absence of glutamatergic or serotonergic signals within the SCN. Moreover, since earlier reports have identified the supraoptic nucleus (SON) as an output of the mammalian clock (Kalsbeek et al., 1995) and electrophysiological evidence indicates that the SON projects to the SCN (Saeb-Parsy et al., 2000), we investigated the activation of neuronal markers within the SCN and the behavioral response to GRP and a GABA antagonist following the administration of either substance to a region just dorsal to the SON. Materials and Methods Subjects 115 adult male Syrian hamsters (2 4 months) were bred from hamsters purchased from Harlan Sprague Dawley (Indianapolis, IN, USA). Animals were group-housed in a 14:10 h light:dark cycle with food and water available ad libitum. All experimental procedures were approved by the Kent State University Animal Care and Use Committee, and followed PHS guidelines for the care and use of laboratory animals. Surgical procedure Hamsters were anesthetized with an anesthesia cocktail (110 mg kg ketamine, 22 mg kg xylazine and 1.83 mg kg acepromazine) and stereotaxically implanted with a 4-mm, 26- gauge guide cannula (Plastics One, Roanoke, VA, USA) aimed at either the third

105 90 ventricle or a region just dorsal to the supraoptic nucleus (SON). Stereotaxic coordinates for the third ventricle were 1.2 mm anterior to bregma and 7.2 mm ventral to dura along the midline. Coordinates for the region just dorsal to the SON were 1.2 mm anterior and 1.7 mm lateral to bregma and 8.1 mm ventral to dura. Prior to cannula insertion, the skull was leveled between bregma and lambda. After surgery, hamsters were individually housed in Plexiglas cages (24 x 45.5 x 21 cm) and given 24 h to recover in their original colony room. Hamsters were then placed in a continuously dark environment (DD), and were allowed to establish free-running activity rhythms. These cages were equipped with a running wheel (diameter 18 cm) that was monitored by a computer using ClockLab software (Actimetrics). Microinjection After 10 days of free-running activity, microinjections were given with a 32-gauge needle that extended beyond the guide cannula by 5.2 mm, attached by polyethylene tubing to a 1 µl Hamilton syringe. The polyethylene tubing was filled with distilled water and an air bubble was introduced to avoid dilution of injected substances. Injections were given with the aid of night vision goggles while the hamsters were gently restrained by hand. The final volume of each microinjection was 1 µl for ventricular injections and 250 nl for SON injections, both of which were administered over a period of 10 seconds. After each microinjection, the needle was left in place for seconds. Microinjections were given at circadian time (CT) 13 ± 15 min. Following microinjection, hamsters were returned to their home cages.

106 91 Experiments 1 and 2 Hamsters implanted with guide cannulas aimed at the third ventricle received the following treatments in randomized order: microinjection of 0.5 nmol gastrin releasing peptide (GRP; Phoenix Pharmaceuticals, Belmont, CA, USA), 4.94 nmol (±)-2-amino-5- phosphonopentanoic acid (AP5; Research Biochemicals International, Natic, MA, USA), and 7.6 nmol 8-Hydroxy-2-di-n-propylamino-tetralin (DPAT; Sigma-Aldrich, St. Louis, MO, USA), each dissolved in phosphate buffer saline (PBS), a GRP/AP5 cocktail, a GRP/DPAT cocktail, or PBS. Following injection, hamsters were returned to their home cages for either one hour (c-fos) or thirty minutes (p-erk) before being euthanized and processed for immunohistochemistry. Experiment 3 Hamsters received microinjections aimed at the third ventricle of 0.5 nmol GRP, 7.6 nmol DPAT, and a cocktail of 0.5 nmol GRP and 7.6 nmol DPAT, each dissolved in PBS, or PBS. Following microinjections, hamsters were returned to their home cages and their behavioral rhythms were monitored. The order of injections was counterbalanced and a minimum of 10 days separated each injection. Experiment 4 Hamsters implanted with guide cannulas aimed at a region just dorsal to the SON received the following treatments in randomized order: microinjection of 0.5 nmol GRP, 4.94 nmol AP5, 7.6 nmol DPAT, a cocktail of GRP/AP5 or GRP/DPAT or PBS.

107 92 Following injection, hamsters were returned to their home cages for one hour before being euthanized and processed for c-fos immunohistochemistry. Experiment 5 and 6 Hamsters prepared as in Experiment 4 received microinjections aimed at a region just dorsal to the SON of 125 pmol GRP or 0.9 nmol (-)-Bicuculline methobromide (BIC; Tocris, Ellisville, MO, USA), or PBS. Following microinjections, hamsters were returned to their home cages and their behavioral rhythms were monitored. The order of injections was counterbalanced and a minimum of 10 days separated each injection. Perfusion Hamsters used for immunohistochemistry were deeply anesthetized with sodium pentobarbital (200 mg kg) in the dark and their heads were covered in aluminum foil during trans-cardial perfusion. Each hamster was perfused with 150 ml of PBS followed by 250 ml of 4% paraformaldehyde. Hamster brains were extracted and post-fixed in 4% paraformaldehyde at 4ºC for 1-2 days prior to sectioning Immunohistochemistry Coronal sections (40 µm thickness) were sliced on a vibratome and free floating sections were saved in three vials. c-fos IHC (rabbit anti c-fos, 1:10,000; Santa Cruz Biotechnology) and p-erk IHC (rabbit anti-phospho-42/44 MAPK, 1:1,000; Cell Signaling Technology) were carried out using standard avidin-biotin labeling techniques.

108 93 Sections were washed with PBS and hydrogen peroxide. After additional rinses with PBS, sections were incubated in a solution containing 5% normal donkey serum in antisera diluent with 0.3% Triton-X for 1 hr. to reduce non specific labeling. Sections were then incubated overnight with primary antibodies in antisera diluent. Tissue was rinsed with PBS several times prior to incubation with biotinylated-sp-conjugated affinipure donkey anti-rabbit IgG (1:500; Jackson Immunoresearch). Sections were subsequently washed and then incubated in avidin-biotin complex (Vector Elite Kit, Vector Laboratories) for 1 hr. at room temperature. Following additional rinses with PBS, reaction product was visualized using DAB (Vector Laboratories). Sections were then mounted on gel coated slides, dehydrated through sequential series of alcohol washes and then cleared with Citrisolv and coverslipped with DPX. For GRP IHC (rabbit anti-grp, 1:1,000; Immunostar) and/or 5-HT IHC (goat anti-5-ht, 1:1,000; Immunostar), untreated hamsters were taken from the colony, perfused, and coronal sections (40 µm thickness) were sliced on a vibratome. Every third section was collected and stored in PBS. In all subsequent steps, sections were rinsed (3 x 5 min) in PBS between incubations. Sections were incubated in blocking solution containing 5% normal donkey serum in antisera diluent with 0.3% Triton-X for 1 hr., followed by an overnight room-temperature incubation of either one of the two or both primary antibodies. Sections were then incubated in Cy-3 conjugated donkey anti-rabbit (1:500, Jackson Immunoresearch) and/or Alexa 633 donkey anti-goat (1:500; Molecular Probes) for 1 hr. at room temperature. After rinsing with PBS, sections were mounted on gel coated slides, set out to dry, and coverslipped with Krystalon. Slides were stored at 4 C.

109 94 Histology for behavioral experiments After completion of each behavioral experiment, animals were deeply anesthetized with sodium pentobarbital (200 mg kg) and killed by decapitation. Hamster brains were extracted and post-fixed in 4% paraformaldehyde. Coronal sections (100 µm thickness) were sliced on a vibratome and counterstained with hemotoxylin. Injection sites were verified using light microscopy. Only animals with visible needle tracts that penetrated the third ventricle or were within 300 µm of the SON were used in this study. 23 animals (18% of total) were excluded from this study because the microinjection sites were either outside of the third ventricle or farther than 300 µm from the SON. Data analysis of phase shifts Phase shifts in the circadian activity rhythm were quantified using the linear regression method (Daan and Pittendrigh, 1976). A line was fitted to activity onsets that occurred on the 10 days preceding the microinjection. A second line was fitted to activity onsets that occurred 4 10 days after microinjection. Days 1 3 post-microinjection were not used in the data analysis to avoid including transient effects. Phase shifts were determined by the difference between the two regression lines. Differences between groups were evaluated using Student s t-test. Quantification of c-fos immunoreactivity To assess the distribution of labeled cells in the SCN following ventricular injections, images of the anterior, medial, and posterior SCN were captured using an Olympus BX-

110 95 51 microscope. Additional images of extra-scn regions, including the SON, paraventricular nucleus (PVN), and cortex were also obtained. To assess the distribution of labeled cells following injections aimed at the SON, similar images were captured but the ipsilateral and contralateral components of the SON, SCN, and PVN were analyzed separately. Images were loaded into ImageJ software and the background was removed by setting a manual threshold to include labeled cells. The average area of a c-fos positive nucleus was determined and the total stained area was calculated. The average number of c-fos positive nuclei was estimated as the total stained area divided by the average size of positive nuclei. Differences between groups were analyzed using a 1-way ANOVA for third ventricle injections and a 2-way repeated measures ANOVA for SON injections. Dunnett s test was used to assess differences between each group and GRP injections. Quantification of p-erk immunoreactivity To assess the distribution of p-erk immunoreactivity in the SCN, SON, and PVN following ventricular injections, images of the anterior, medial, and posterior SCN were captured using an Olympus BX-51 microscope. Images were analyzed using ImageJ software. The optical density of each region was determined by subtracting the optical density of a nearby unstained brain region (background) from that of the selected region. Differences between groups were assessed by 1-way ANOVA. Dunnett s test was used to assess differences between each group and GRP injections.

111 96 Results Experiment 1: effects of ventricular administration of GRP on c-fos expression in the SCN, SON, and PVN This experiment was designed to test whether GRP-induced c-fos expression in the SCN is altered by coadministration of an NMDA antagonist or a 5-HT 1A,7 agonist. Animals received a microinjection of GRP (n=9), GRP and AP5 (n=8), GRP and DPAT (n=6), or vehicle (n=8). Significant differences between injection types were detected in the anterior (1-way ANOVA, p=0.02), medial (1-way ANOVA, p= ), and posterior (1-way ANOVA, p=0.004) SCN (Fig. 4A). At the level of the medial SCN, the number of c-fos expressing cells was significantly larger in GRP-injected groups than GRP/AP5, GRP/DPAT, or vehicle (Dunnett s test, p<0.05). At the level of the anterior and posterior portions of the SCN, the number of c-fos expressing cells was significantly larger in the GRP-injected groups than GRP/DPAT or vehicle (Dunnett s test, p<0.05). When further analysis was conducted to differentiate c-fos activation in the ventral and dorsal regions of the SCN, significant differences between treatment groups were detected in the ventral 1/3 (1-way ANOVA, p= ) and dorsal 2/3 (1-way ANOVA, p=0.0001) regions of the medial SCN (Fig. 4B). In both the ventral and dorsal regions, the number of c-fos positive cells was significantly larger in the GRP-injected groups than GRP/AP5, GRP/DPAT, or vehicle (Dunnett s test, p<0.05). Extra-SCN regions were also analyzed, including the SON, PVN, and a small part of the cortex from the same sections as the

112 97 Anterior Medial Posterior GRP GRP/ AP5 GRP/ DPAT SAL A c-fos Counts B # ^ * * GRP GRP-AP5 GRP-DPAT SAL ANT MED POST # # ^ Fig. 4A: (A) Representative photomicrographs of the hamster SCN at anterior, medial, and posterior levels following microinjection of GRP, GRP-AP5, GRP-DPAT, and SAL. (B) Mean + SEM c-fos counts induced by microinjection of GRP, GRP-AP5, GRP- DPAT, and SAL into the third ventricle at CT 13. There is a significant difference between groups (Anterior: 1-way ANOVA, p< 0.02, Dunnett s test, SAL and GRP- DPAT are different from GRP (*); Medial: 1-way ANOVA, p< , Dunnett s test, SAL, GRP-AP5, and GRP-DPAT are different from GRP (#); Posterior: 1-way ANOVA, p< 0.004, Dunnett s test, SAL and GRP-DPAT are different from GRP (^)).

113 # # * * * GRP GRP-AP5 GRP-DPAT SAL c-fos Counts # Ventral 1/3 Dorsal 2/3 Fig. 4B. Mean + SEM c-fos counts in the ventral 1/3 and dorsal 2/3 of the medial SCN induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13. There is a significant difference between groups (ventral 1/3: 1- way ANOVA, p< , Dunnett s test, GRP-DPAT, GRP-AP5, and SAL are different from GRP (*); dorsal 2/3: 1-way ANOVA, p< , Dunnett s test, GRP-DPAT, GRP- AP5, and SAL are different from GRP (#)).

114 99 SCN analysis. Significant differences between injection types were detected in the SON (1-way ANOVA, p= ; Fig. 4C) and the PVN (1-way ANOVA, p=0.001; Fig. 4C), but not the cortex (1-way ANOVA, p=0.08; Fig. 4D). In the SON and PVN, the number of c-fos expressing cells was significantly larger in GRP-injected groups than vehicle (Dunnett s test, p<0.05). Experiment 2: effects of ventricular administration of GRP on p-erk expression in the SCN, SON, and PVN This experiment was designed to test whether GRP-induced p-erk expression in the SCN is altered by coadministration of an NMDA antagonist or a 5-HT 1A,7 agonist. Animals received a microinjection of GRP (n=6), GRP and AP5 (n=7), GRP and DPAT (n=6), or vehicle (n=5). In general, the results were similar to those found for c-fos in experiment 1. Significant differences between injection types were detected in the anterior (1-way ANOVA, p=0.005), medial (1-way ANOVA, p=0.005), and posterior (1- way ANOVA, p=0.009) SCN (Fig. 4E). At the level of the anterior and medial SCN, the amount of p-erk immunoreactivity was significantly larger in the GRP-injected groups than GRP/AP5, GRP/DPAT, or vehicle (Dunnett s test, p<0.05). At the level of the posterior SCN, positive p-erk immunoreactivity was significantly larger in the GRPinjected groups than GRP/DPAT or vehicle (Dunnett s test, p<0.05). As in Experiment 1, when further analysis was conducted to differentiate p-erk activation in the ventral and dorsal regions of the SCN, significant differences between injection types were

115 100 SON PVN GRP GRP/ AP5 GRP/ DPAT A SAL c-fos Counts B GRP GRP-AP5 GRP-DPAT SAL SON PVN * # Fig. 4C: (A) Representative photomicrographs of the hamster SON and PVN following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT13. (B) Mean + SEM c-fos counts induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13. There is a significant difference between groups (SON: 1-way ANOVA, p< , Dunnett s test, SAL is different from GRP (*); PVN: 1-way ANOVA, p< 0.002, Dunnett s test, SAL is different from GRP (#)).

116 c-fos Counts GRP GRP-AP5 GRP-DPAT SAL Fig. 4D: Mean + SEM c-fos counts in a small section of the cortex induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13. There is no significant difference between groups (1-way ANOVA, p< 0.08).

117 102 Anterior Medial Posterior GRP GRP/ AP5 GRP/ DPAT SAL A Optical Density * # # * ^ * # ^ 5 B 0 GRP GRP-AP5 GRP-DPAT SAL ANT MED POST Fig. 4E: (A) Representative photomicrographs of the hamster SCN at anterior, medial, and posterior levels following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT13. (B) Mean + SEM p-erk optical density induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13. There is a significant difference between groups (Anterior: 1- way ANOVA, p< 0.006, Dunnett s test, GRP-AP5, GRP-DPAT, and SAL are different from GRP (*); Medial: 1-way ANOVA, p< 0.006, Dunnett s test, GRP-AP5, GRP- DPAT, and SAL are different from GRP (#); Posterior: 1-way ANOVA, p< 0.009, Dunnett s test, GRP-DPAT and SAL are different from GRP (^)).

118 103 detected in the ventral 1/3 (1-way ANOVA, p=0.002) and dorsal 2/3 (1-way ANOVA, p=0.04) regions of the medial SCN (Fig. 4F). In the ventral region, the amount of p- ERK immunoreactivity was significantly larger in the GRP-injected groups than GRP/AP5, GRP/DPAT, or vehicle (Dunnett s test, p<0.05). In the dorsal region, however, p-erk immunoreactivity in the GRP-treated groups was significantly larger than vehicle but not the AP5 or DPAT-treated groups (Dunnett s test, p<0.05). Similar to Experiment 1, extra-scn regions of the hypothalamus were analyzed, including the SON and PVN. Significant differences between injection types were not detected in the SON (1-way ANOVA, p=0.1) or the PVN (1-way ANOVA, p=0.12) (Fig. 4G). Experiment 3: effects of a 5-HT 1A,7 agonist on GRP-induced phase delays Because GRP and serotonin immunoreactivity are co-localized to the same cells (Morin, 2006) (Fig. 4H), this experiment was designed to test whether microinjection of a 5- HT 1A,7 agonist would alter the phase shifting effects of GRP when administered into the third ventricle in the early subjective night. The close proximity of GRP and serotonin immunoreactivity in the hamster SCN suggests a potential interaction between these two systems to regulate photic entrainment. In order to investigate the behavioral effects of increased serotonergic neurotransmission on GRP-induced shifts, the effect of DPAT on GRP-induced phase delays was evaluated. Animals received a microinjection of GRP (n=9), GRP and DPAT (n=7), DPAT (n=11), or vehicle (n=12) and significant differences were detected between the injection types (1-way ANOVA, p=0.0001). GRP-

119 104 Optical Density (O.D.) * * * # 0 GRP GRP-AP5 GRP-DPAT SAL Ventral 1/3 Dorsal 1/3 Fig. 4F: Mean + SEM p-erk optical density in the ventral 1/3 and dorsal 2/3 of the medial SCN induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13. There is a significant difference between groups (ventral 1/3: 1-way ANOVA, p< 0.002, Dunnett s test, GRP-AP5, GRP-DPAT, and SAL are different from GRP (*); dorsal 2/3: 1-way ANOVA, p< 0.04, Dunnett s test, SAL is different from GRP (#)).

120 105 SON PVN GRP GRP/ AP5 GRP/ DPAT SAL A Optical Density B 0 GRP GRP-AP5 GRP-DPAT SAL SON PVN Fig. 4G: (A) Representative photomicrographs of the hamster SON and PVN following microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT13. (B) Mean + SEM p-erk optical density induced by microinjection of GRP, GRP and AP5, GRP and DPAT, and SAL into the third ventricle at CT 13 (SON: 1-way ANOVA, p< 0.1; PVN: 1-way ANOVA, p< 0.11).

121 Fig. 4H: Photomicrographs showing overlap of GRP (red) and serotonin (green) immunoreactivity in the hamster SCN. Overlap is found to be greatest in the lateral portion of the nucleus, from the ventral border to about half way up the dorsal-ventral extent of the SCN. 106