I REGULATION of NEUROPEPTIDE RELEASE in the SCN

REGULATION OF NEUROPEPTIDE RELEASE IN THE SCN CIRCADIAN CLOCK: IN VIVO ASSESSMENTS OF NPY, VIP, AND GRP

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

by Jessica M. Francl December 2010

Dissertation written by Jessica M. Francl

B.S., Kent State University, 2003

M.S., The University of Akron, 2005

Ph.D., Kent State University, 2010

Approved by

Dr. J.David Glass , Chair, Doctoral Dissertation Committee Dr. Eric M. Mintz , Member, Doctoral Dissertation Committee Dr. Robert V. Dorman , Member, Doctoral Dissertation Committee Dr. Brian P. Bagatto , Member, Doctoral Dissertation Committee Dr. William Lynch , Member, Doctoral Dissertation Committee

Accepted by

Dr. James L. Blank , Chair, Department of Biology Dr. John R. D. Stalvey , Dean, College of Arts and Sciences

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TABLE OF CONTENTS

TITLE PAGE...... i

APPROVAL PAGE...... ii

TABLE OF CONTENTS...... iii

LIST OF ABBREVIATIONS...... iv

LIST OF FIGURES...... vii

ACKNOWLEDGEMENTS...... ix

DEDICATION...... xi

INTRODUCTION...... 1

SPECIFIC AIMS...... 22

MATERIALS AND METHODS...... 27

RESULTS...... 39

DISCUSSION...... 61

REFERENCES...... 82

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LIST OF ABBREVIATIONS

5-HT...... 5-hydroxytryptamine

8-OH-DPAT...... (±)8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide

ACSF...... artificial cerebral spinal fluid

AP5...... (2R)-amino-5-phosphonovaleric acid

AVP...... arginine vasopressin

BB2...... bombesin receptor 2

BSA...... bovine serum albumin

CalB...... calbindin cAMP...... cyclic AMP

CNS...... central nervous sytem

CSF...... cerebral spinal fluid

CT...... circadian time

DD...... constant darkness

DMH...... dorsomedial hypothalamus

DR...... dorsal raphe nucleus

GABA...... γ-aminobutyric acid

GHT...... geniculohypothalamic tract

GRP...... gastrin-releasing peptide

IGL...... intergeniculate leaflet

LD...... light:dark

MAPK...... mitogen-activated protein kinase

MR...... median raphe nucleus mRNA...... messenger ribonucleic acid

NMDA...... N-methyl-D-aspartate

NPY...... neuropeptide Y p-ERK...... extracellular signal-regulated kinase

PAC1...... pituitary adenylate cyclase-activating peptide receptor 1

PACAP...... pituitary adenylate cyclase-activating peptide

PCPA...... para-chlorophenylalanine

Per1...... period gene 1

Per2...... period gene 2

PHI...... peptide histodine isoleucine

PKA...... protein kinase A

PRC...... phase response curve

PVN...... paraventricular nucleus of the hypothalamus

PVT...... paraventricular nucleus of the thalamus

RGC...... retinal ganglion cell

RHT...... retinohypothalamic tract

RIA...... radioimmunoassay

SCN...... suprachiasmatic nucleus

SP...... substance P

SS...... somatostatin

TBS...... Tris-buffered saline

VIP...... vasoactive intestinal polypeptide

VPAC2...... vasoactive intestinal polypeptide receptor 2

Y1...... NPY receptor subtype 1

Y2...... NPY receptor subtype 2

Y5...... NYP receptor subtype 5

ZT...... zeitgeber time

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LIST OF FIGURES

Figure 1 ...... 6

Figure 2 ...... 7

Figure 3 ...... 12

Figure 4 ...... 29

Figure 5 ...... 32

Figure 6 ...... 36

Figure 7 ...... 37

Figure 8 ...... 40

Figure 9 ...... 41

Figure 10 ...... 42

Figure 11 ...... 44

Figure 12 ...... 45

Figure 13 ...... 46

Figure 14 ...... 49

Figure 15 ...... 50

Figure 16 ...... 51

Figure 17 ...... 52

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Figure 18 ...... 53

Figure 19 ...... 55

Figure 20 ...... 56

Figure 21 ...... 57

Figure 22 ...... 59

Figure 23 ...... 60

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ACKNOWLEDGEMENTS

First, I would like to acknowledge and thank my doctoral adviser, Dr. J. David

Glass, for his encouragement, generosity, and support. From improving my writing skills to reminding me to be patient and stay positive when experiments fail, Dr. Glass’ mentorship has provided me a strong foundation with which to begin my career. It has been a privilege to work in his lab for the past five years and his superior expertise as a researcher has left me with the skills necessary to become an independent scientist. I am extremely grateful for the opportunities that his guidance has afforded to me.

I would also like to thank the members of my dissertation committee, Drs. Eric

Mintz, Robert Dorman, and Brian Bagatto. Their valuable time, guidance, assistance, and instruction – with my research as well as in the classroom – have helped make this work possible. Their feedback from my prospectus defense is greatly appreciated and has made this dissertation stronger.

Next, I would like to recognize the present and past members of the Glass lab.

Thank you to Jessie Guinn, Jr., Allison Brager, Adam Stowie, Gagandeep Kaur, Raja

Thind, Steve Hammer, Randy Roberts, and Christina Ruby for their friendship and advice. I value all of the fun times we have spent together in the lab, at conferences, and at our random picnics. My microdialysis experiments lasting six hours in the middle of the night would have been (more) torturous without the company of Boy Jessie, Gagan, and a couple of good movies. I would also like to thank our technicians Amelie Cornil

for her assistance during my early time in the Glass lab, and Marc DePaul for his expert lab organization and computer skills. Additional thanks to my Kent State University professors as well as the administrative, stockroom, and vivarium staff members.

Lastly, special thanks to my wonderful boyfriend, Jim Ferrell, for his love and support. He has never failed to provide me with encouragement, especially when it was most needed. His understanding nature has made my transition from graduate student to postdoctoral researcher a little bit easier, and I only hope I can provide as much support for him in his graduate career as he has for me.

DEDICATION

I dedicate this dissertation to my parents, Chris Braun and Jim Francl, and to my stepfather, Matt Braun. Their love, sacrifices, and endless support have given me the personal strength and encouragement to fulfill my goals, and for that I will always be grateful to them.

CHAPTER I

INTRODUCTION

The suprachiasmatic nucleus (SCN) of the hypothalamus is responsible for generating and maintaining circadian rhythms in mammals. Interneuronal communication in the SCN occurs via neurotransmitter and neuropeptide release, with glutamate, γ-aminobutyric acid (GABA), serotonin (5-HT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and arginine vasopressin (AVP) being the neurochemicals most commonly associated with SCN regulation (reviewed by Reghunandanan and Reghunandanan, 2006). Photic and nonphotic stimuli are received by the SCN and this information is integrated by the SCN, ultimately resulting in a cohesive rhythm output that is entrained to stimulus input. Much data is currently available regarding in vitro actions and rhythmic profiles of release of these SCN neurochemicals through bath application procedures, electrophysiological recordings, immunocytochemistry, and in situ hybridization. However, to date there are few reports on the in vivo release of these agents, and notably neuropeptides from the

SCN. The studies performed here used brain microdialysis techniques coupled with sensitive radioimmunoassay to determine the in vivo release over a 24-hr period of three

SCN neuropeptides (NPY, VIP, and GRP) in a freely-behaving animal model;

additionally, these studies examined the effects of pharmacological agents on the release of these peptides, including both photic and nonphotic stimuli. The results of these experiments demonstrate that these neuropeptides are released rhythmically in the SCN, and that they are of synaptic origin. Such information sheds light on the possible functions of these neuropeptides with respect to photic and nonphotic regulation of the mammalian SCN circadian clock.

The experiments presented here have been published as the following: Glass et al., 2010; Francl et al., 2010a, Francl et al., 2010b. The methods used couple in vivo microdialysis with radioimmunoassay to detect and quantify neuropeptide release with greater specificity, with respect to timing, compared to previously employed methods such as in situ hybridization or immunohistochemistry. This system also allows for the examination of the intact SCN circadian clock without the loss of important neuronal inputs, such as what occurs in in vitro hypothalamic slice preparations. Microdialysis also permits experimental manipulation of the circadian clock via reverse drug perfusions, while concurrently monitoring and quantifying changes in neuropeptide release due to those experimental manipulations. Taken together, these details lend to important advantages of using microdialysis compared to other methods, and the experiments presented here represent the first use of microdialysis and radioimmunoassay to detect and measure NPY, VIP, and GRP release from the hamster circadian clock.

The circadian clock

A circadian rhythm refers to any behavior or physiological function that occurs with a period of approximately 24 hr. Proper control and regulation of circadian biological rhythms, such as sleep-wake cycles, body temperature fluctuation, and appetite/foraging is critical for homeostasis and survival (reviewed by Panda and

Hogenesch, 2004; Refinetti and Menaker, 1992). In mammals, circadian rhythms are generated and regulated by the biological clock, the suprachiasmatic nucleus (SCN), located within the hypothalamus (Inouye and Kawamura, 1979). The SCN is influenced by many external factors, the most important being the cycle of environmental light and dark due to the rotation of the Earth, to ensure that the organism retains a suitably-timed relationship with its external environment (Pittendrigh, 1981).

The SCN consists of paired nuclei of approximately 10,000 neurons each, positioned dorsal to the optic chiasm (Kulhman and McMahon, 2006; van den Pol, 1980).

Numerous studies have provided evidence for the role of the SCN as the biological clock.

Isolated neurons of the SCN exhibit a circadian rhythmicity in electrical firing rate in vitro, with peak firing rate occurring during the subjective daytime (Green and Gillette,

1982; Groos and Hendriks, 1982; Shibata et al., 1982). Unilateral or bilateral lesions to the SCN abolished nocturnal activity, drinking, sexual behavior, and the rhythmic fluctuation of CSF vasopressin levels in rats (Jolkkonen et al., 1988; Södersten et al.,

1981; Stephan and Zucker, 1972). Lastly, the shortened circadian activity rhythms of the tau mutant hamster are restored to a normal period by transplantation of wild-type SCN tissue (Ralph et al., 1990), while activity of SCN-lesioned animals are restored by

transplantation of fetal SCN (Lehman et al., 1987) but not with non-SCN tissue

(DeCoursey and Buggy, 1989). These experiments demonstrate that the SCN is an independent oscillating brain nucleus capable of producing and maintaining internal physiological rhythms as well as behavioral rhythms.

The SCN is entrainable to external environmental “zeitgebers” or temporal cues

(ZTs) (Aschoff and Pohl, 1978), including photic (light) and nonphotic (wheel-running, social interaction) inputs. In a normal light:dark cycle (LD) ZT 12 refers to the time of lights off, while in constant darkness (DD), CT 12 refers to “circadian time 12”, or the time an animal begins its daily onset of activity. The entrainment of the SCN to these

ZTs occurs in a phase-dependent manner in that these stimuli, when applied at certain times of an animal’s circadian period, have the ability to reset or shift the timing of the clock. In a study by Pittendrigh and Daan (1976), photic stimuli in the form of a brief light flash phase-dependently induced shifts in timing of behavioral activity in nocturnal animals housed in constant darkness (DD) and were graphically represented as a phase response curve (PRC). Light pulses that were given during the subjective day did not affect the timing of circadian behavioral activity, while photic stimuli perceived during the early subjective night postponed activity rhythms (resulting in a phase delay) or advanced the activity rhythm of the organism (resulting in a phase advance) when applied in the late subjective night. Similar results were seen in other animals that were given brief light pulses, including hamsters and squirrels (DeCoursey, 1960; reviewed by

Mistlberger et al., 2000; Takahashi et al., 1984; Figure 1).

The clock is also entrainable to nonphotic stimuli, such as novel wheel running or social interaction. These stimuli have a phase-advancing effect on the clock during the day, and minimal phase-delaying effects during subjective night in animals housed in DD

(reviewed by Mistlberger et al., 2000; Mrosovsky, 1988; Reebs and Mrosovsky, 1989;

Figure 2). The SCN neuronal phase-dependent response to photic and non-photic stimuli is possibly mediated through neuropeptide and neurotransmitter release and ultimately results in the overall projected activity and biological rhythms of an organism.

Therefore, it is important to quantify the release of SCN neuropeptides, as well as establish the timing of release, in order to better ascertain their functions in circadian timekeeping.

Figure 1. Graphical representation of photic phase response curve in hamsters. The graph represents magnitude and direction of phase shifts in response to a light pulse. Adapted from Mistlberger et al., 2000.

Figure 2. Graphical representation of nonphotic (wheel-running) phase response curve in hamsters housed in constant darkness. The graph represents magnitude and direction of phase shifts in response to a novel wheel. Adapted from Mistlberger et al., 2000.

Organization of the SCN

The neurons of the SCN are heterogeneous in content and are loosely categorized anatomically into “shell” and “core”, though some researchers suggest these groups may be too simplistic, as various portions of the SCN can be delineated by several neuropeptide and neurotransmitter types that do not fit the “core/shell” organization

(Antle and Silver, 2005; Morin and Allen, 2006; Morin, 2007). The core neurons are located in the ventral SCN in close contact with the optic chiasm, while neurons of the shell are located dorsally and surround the core (Abrahamson and Moore, 2001; Moore and Speh, 2002). These two neuronal areas receive different cellular inputs and are thought to have different functions. The core SCN neurons are responsible for receiving photic input from the retina and projecting this information, likely in the form of peptide release, to the dorsal shell neurons. The shell neurons then integrate these photic signals with other photic and nonphotic signals to produce a rhythm that is then projected to other brain areas (Albers et al., 1991; Kalsbeek and Buijs, 2002).

Evidence for this mechanism of intra-SCN communication stems from the organization of afferent and efferent neurons, as well as the content of those neurons. A series of SCN retrograde analyses demonstrated that specialized retinal ganglion cells

(RGCs) innervate the SCN via the retinohypothalamic tract (RHT), are intrinsically photosensitive, and can entrain the circadian clock to external light in the absence of a functioning traditional phototransduction pathway consisting of rods and cones (Berson et al., 2002; Freedman et al., 1999; Moore et al., 1995; Pickard, 1985). These RGCs depolarize following exposure to light (Warren et al., 2003) and circadian entrainment to

a light schedule was absent in math5 mutant mice, which lack RGCs (Brzezinski et al.,

2005; Wee et al., 2002). Also, lesioning of the hamster RHT results in a loss of circadian entrainment (Johnson et al., 1988). This proposed retina-RGC-SCN pathway (via the

RHT) provides a direct photic input to the SCN and appears to be involved in the mediation of photic entrainment of biological rhythms.

Individual neurons of the SCN, both in intact animals and in in vitro cell cultures and hypothalamic slices, exhibit circadian rhythmicity in electrical firing and in gene expression (Green and Gillette, 1982; Moga and Moore, 1997; Sakamoto et al., 1998;

Yamaguchi et al., 2003; Yamazaki et al., 1998), and are responsive to light (Meijer et al.,

1998). Single SCN cells may function as separate clocks but also must be synchronized with other SCN neurons and with the exterior environment of the organism (Colwell et al., 2003). SCN activity, detected as spontaneous action potentials using electrophysiological recordings in hypothalamic slices, is circadian in nature and peaks during mid-day while being relatively low at night (Inouye and Kawamura, 1981;

Kuhlman and McMahon, 2006). Within neurons of the SCN core, transcription of “clock genes” (clock and bmal) produces proteins that dimerize to stimulate the transcription of other clock genes (period, cryptochrome). The protein products, Per and Cry, interact within the cytoplasm of SCN cells to inhibit further transcription of clock and bmal, thus resulting in a feedback loop (Albrecht, 2002; Antle and Silver, 2005; Silver and

Schwartz, 2005). These clock genes are regularly transcribed and translated, and display precise patterns of expression over the course of 24 hr. Furthermore, the per1 gene is light-sensitive and its transcription is upregulated in response to light pulses administered

during the night (Albrecht et al., 1997). It is this rhythmic transcription and translation of clock genes, occurring approximately every 24 hr, which is thought to be the basis of circadian rhythmicity (reviewed by Mendoza and Challet, 2009). It is possible that SCN communication and rhythm projection depends on neuropeptide release, both among

SCN neurons themselves and from the SCN to other brain areas (Harmar, 2003).

Therefore, it will be critical to detect and quantify peptide release from the SCN using in vivo techniques in living, undisturbed animals to ascertain their possible functions within the SCN.

Neuronal communication at the SCN

The neuropeptides and neurotransmitters of the mammalian rodent circadian clock include neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), gastrin- releasing peptide (GRP), arginine vasopressin (AVP), calbindin (CalB), substance P (SP), pituitary adenylate cyclase-activating peptide (PACAP), glutamate, γ-aminobutyric acid

(GABA), and serotonin (5-HT) (Abrahamson and Moore, 2001; Castel et al., 1993; De

Vries et al., 1993; Reghunandanan et al., 1998; Romijn et al., 1997; Romijn et al., 1999;

Smale et al., 1991; van den Pol and Tsujimoto, 1985). The neurons of the SCN receive information input from three main areas – the retinohypothalamic tract (RHT), the intergeniculate leaflet (IGL) via the geniculohypothalamic tract (GHT), and the midbrain

Raphe nuclei (Deurveilher and Semba, 2008; Moga and Moore, 1997; Moore and Lenn,

1972; Reppert and Weaver, 2001). The core of the SCN is innervated by neurons of the

RHT, which utilize primarily glutamate and PACAP as neurotransmitters. These

transmitters are implicated in communicating incoming photic information to the clock and are co-stored in axons projecting to retinoreceipient cells of the SCN (Hannibal et al.,

2000; Michel et al., 2006). Additionally, the IGL of the thalamus indirectly transmits photic information to the SCN, received from retinogeniculate neurons and utilizes NPY and inhibitory GABA, by way of the GHT (Lund and Cunningham, 1972; Moore and

Card, 2004; Moore and Speh, 1993). Neuronal projections from the median raphe nuclei, containing 5-HT mainly innervate the SCN core, though some projections reach the shell, and mediate nonphotic interactions (Hay-Schmidt et al., 2003; Kawano et al., 1996;

Meyer-Bernstein and Morin, 1996). These innervations serve to modulate function of the

SCN at different times during a normal 24-hr circadian cycle (Figure 3).

Transmission of photic information

The circadian clock is capable of adjusting to external environmental stimuli, the most significant being light (Herzog and Schwartz, 2000; Morin and Allen, 2006;

Pittendrigh and Minis, 1964). The entrainment of the SCN, and thus timing of an organism’s behavior, by light ensures that the organism maintains a time-appropriate relationship with its external environment. Photic information reaches the SCN directly via the retinohypothalamic tract (RHT) and indirectly via the geniculohypothalamic tract

(GHT). The RHT is responsible for directing photic input to the core of the SCN and utilizes the excitatory neurotransmitter glutamate (Hannibal, 2003; Liou et al., 1986;

Reghunandanan and Reghunandanan, 2006) as well as pituitary adenylate cyclase- polypeptide (PACAP; Hannibal et al., 2000; Beaulé et al., 2009).

Figure 3. Schematic demonstrating neuronal input to the SCN and corresponding sites of neuropeptide and neurotransmitter release. Adapted from Piggins et al., 2002. 5-HT, serotonin; AVP, arginine vasopressin; DR, dorsal raphe nucleus; GABA, γ-aminobutyric acid; GHT, geniculohypothalamic tract; GLU, glutamate; GRP, gastrin-releasing peptide; IGL, intergeniculate leaflet; MR, median raphe nucleus; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase-activating peptide; RHT, retinohypothalamic tract; SCN, suprachiasmatic nucleus; VIP, vasoactive intestinal polypeptide.

Glutamate is released in response to light at the terminus of the RHT at the SCN and is also released due to in vitro stimulation of the optic nerve in hypothalamic slices

(Meijer and Schwartz, 2003). When applied to the SCN, both glutamate and PACAP shift the circadian clock in a manner similar to that of light as predicted by the photic

PRC (Harrington et al., 1999; Shirakawa and Moore, 1994). Glutamate also induces shifts in the timing of electrical activity of SCN tissue and cells cultured in vitro, similar to those shifts caused by light (Franken et al., 1999), while application of glutamate receptor agonists and antagonists resemble and block light-induced phase shifts, respectively (Colwell and Menaker, 1992; Mintz et al., 1997; Rea et al., 1993a; Shibata et al., 1994b). Glutamate is responsible for transmitting photic signals from the retina to the circadian clock, and several neuropeptides and transmitters are then released within the

SCN to relay information to the rest of the brain.

The SCN core receives direct retinal innervation, and evidence suggests that photic information is received and first processed here. Almost all SCN neurons contain

GABA, and most co-express peptides in these neurons (Tanaka et al., 1997b). The most commonly identified peptides in core neurons are vasoactive-intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP) (Silver and Schwartz 2005). VIP is widely expressed in the mammalian brain, including rodents, cats, pigs, and humans (Larsson et al., 1976; Lorén et al., 1979; Obata-Tsuto et al., 1983), and in the CNS is associated with functioning in embryonic development (Cazillis et al., 2004; Wu et al., 1997) and immune functions (reviewed by Pozo et al., 2000). VIP is produced from preproVIP along with peptide histodine isoleucine (PHI), is related to PACAP, and binds to two

receptor variants in the SCN – VPAC2, and PAC1 (Hannibal and Fahrenkrug, 2003a).

Neurons immunoreactive for VIP have been demonstrated in the core SCN and are sometimes colocalized with GRP or GABA (Romijn et al., 1997). In situ hybridization experiments show that VIP mRNA expression varies over the light/dark cycle in rat SCN but not in constant conditions (Piggins and Cutler, 2003). Electrophysiological recordings indicate that bath application of VIP to SCN slices resets the firing rhythm of the neurons and that the shift is dependent upon the time of peptide application, in a manner similar to the shifts caused by light in vivo (Reed et al., 2001). VIP preferentially binds to the VPAC2 receptor, which is localized in the SCN – both in VIP-expressing core neurons and in most AVP-expressing neurons of the shell (Kalamatianos et al.,

2004). Activity of mice lacking the VPAC2 receptor is disrupted in DD, and individual

SCN neurons cultured from these animals fire with reduced rhythmicity and electrical amplitude compared to wild type (Aton et al., 2005). Conversely, mice overexpressing

VPAC2 entrain to an 8 hr shift more quickly compared to wild-type controls (Shen et al.,

2000). Mice lacking VIP showed normal activity rhythms in LD (possibly due to other peptidergic mechanisms of signaling) but displayed disrupted patterns of activity when released into DD, as well as an inability to shift activity in response to a light pulse

(Colwell et al., 2003). This may imply a role for VIP in signaling photic information rather than in generating endogenous rhythms.

The VPAC2 receptor variant that binds VIP in the SCN is associated with increased adenylyl cyclase and cyclic AMP (cAMP) activity in several organ systems, including the CNS and brain (reviewed by Laburthe et al., 2002). Following cAMP

activation, protein kinase A (PKA) is activated and is capable of phosphorylating many downstream targets which then activate other targets, including mitogen-activated protein kinase (MAPK). Actively phosphorylated MAPK has been detected in the SCN following photic stimulation (Coogan and Piggins, 2003; Obrietan et al., 1998).

Additionally, inhibiting either PKA or MAPK kinase in rats blocked VIP- or VIP- agonist-induced phase advances in late night (Meyer-Spasche and Piggins, 2004), further supporting a role for VIP and the VPAC2 receptor in mediating incoming photic information to the SCN.

Gastrin-releasing peptide (GRP) is a 27-amino acid peptide widely present in the mammalian brain as well as within SCN neurons (McDonald et al., 1979; Mikkelsen et al., 1991; Roth et al., 1982). It is implicated in mediating a wide range of physiological functions, including memory processing (Flood and Morley, 1988; Roesler et al., 2004), satiety (Hampton et al., 1998; Stein and Woods, 1982), thermoregulation (Brown et al.,

1988), and circadian rhythms (Albers et al., 1991; McArthur et al., 2000). In the SCN, neurons of the RHT project directly onto GRP-expressing neurons (Tanaka et al., 1997a) and GRP is often localized in VIP-containing SCN neurons (Aïoun et al., 1998). GRP binds preferentially to the BB2 receptor subtype, which is widely expressed in the SCN

(Gonzalez et al., 2008). GRP, when applied via bath application to SCN slices, is capable of shifting peak SCN cellular firing in a manner similar to that of light (McArthur et al.,

2000). In vivo, GRP microinjection delayed hamster activity rhythms in early night and advanced activity in late night (Albers et al., 1991; Kallingal and Mintz, 2007; Piggins et al., 1995). Administration of GRP by intracerebroventricular injection at night caused

behavioral phase delays in mice, and these delays were absent in GRP receptor-deficient mice (Aida et al., 2002). A light pulse during the night increased c-fos expression in

GRP-containing SCN neurons (Earnest et al., 1993) and per2 gene expression (Dardente et al., 2002) in rats while injections of GRP into the third ventricle of hamsters induced an increase in c-fos in SCN neurons, similar to light (Antle et al., 2005; Piggins et al.,

2005).

Expression of GRP mRNA within the SCN varies over a 24-hr LD cycle, with peak levels occurring during the dark phase (Zoeller et al., 2006). Concurring with these results, GRP immunoreactivity measured in rat SCN neurons was highest during the day and low at night (Okamura and Ibata, 1994), suggesting a time delay between mRNA production and peptide expression. GRP content in the rat SCN, measured in cells immunoreactive for antiserum against GRP, exhibited a peak near the end of the light phase, and this content did not vary significantly under constant darkness conditions

(Shinohara et al., 1993). Similar to VIP, these data suggest a role for GRP and the BB2 receptor in mediating photic phase shifting.

Recently, information regarding a new SCN neuropeptide has emerged, named little SAAS. Little SAAS is derived from the proSAAS prohormone, is localized in the ventral subregion of the SCN (overlapping with VIP- and GRP-containing neurons), and little SAAS neurons in rat are responsive to photic stimulation as indicated by increased c-Fos detection following a light pulse (Atkins et al., 2010; Lee et al., 2010). In vitro stimulation of the optic nerve in the SCN brain punch preparation resulted in a significant increase in little SAAS release, detected by mass spectrometry (Hatcher et al., 2008).

Hatcher et al. (2008) also demonstrated that little SAAS is capable of producing shifts in

SCN electrical firing rates, such that application in early subjective night (CT 14) produced phase delays. Lastly, it was shown that little SAAS was colocalized with a small number of VIP-containing neurons, and a larger number of GRP-containing neurons, within the SCN (Atkins et al., 2010). The newly-discovered presence of little

SAAS could play a role as an additional modulator and/or mediator of photic stimulus input to the SCN, possibly working in conjunction with VIP, GRP, or both. Therefore, it is necessary to examine the endogenous release of VIP and GRP from the SCN clock to determine the effects on the release of these two neuropeptides of photic input from both a normal LD cycle and from light pulses at night, as well as in the absence of light.

Transmission of nonphotic information

Nonphotic stimuli presented during the subjective day are also capable of shifting the SCN clock. Neuropeptide Y (NPY) is extensive in the central nervous system of mammals, and is thought to be involved in the neuroendocrine control of several physiological functions including food intake and appetite control (Clarke et al., 1985;

Kalra et al., 1988), cardiovascular regulation (Edvinsson et al., 1987; Pedrazzini et al.,

1998), anxiety and pain management (Broqua et al., 1996; Heilig et al., 1989; Hua et al.,

1991; Nakajima et al., 1998), as well as circadian rhythms (Albers and Ferris, 1984;

Biello et al., 1996; Beillo et al., 1997).

With respect to circadian rhythms, NPY released from the IGL is thought to be involved in mediation of phase shifting due to nonphotic stimuli, possibly through several

receptor subtypes (Gribkoff et al., 1998). NPY applied in vitro or injected into the SCN in vivo during the subjective day causes phase shifts similar to those caused by nonphotic stimuli, such as novel wheel running (Gamble et al., 2005). Conversely, NPY administered during the early subjective night blocks light-induced phase delays, though

NPY during the late subjective night seems to have little or no effect on phase advaincing

(Weber and Rea, 1997). Additionally, administering NPY antiserum prior to a photic stimulus enhanced shifting capabilities in hamsters (Biello, 1994), suggesting a role for

NPY in the modulation of photic input. It is thought that NPY Y2 receptors mediate nonphotic phase shifting during the subjective day both in vitro and in vivo (Golembek et al., 1996; Huhman et al., 1996; Soscia and Harrington, 2005), while NPY Y5 receptors mediate the ability of NPY to block photic phase shifting in the late subjective night

(Gamble et al., 2006; Yannielli and Harrington, 2000; Yannielli and Harrington, 2001).

NPY, as well as Y1, Y2, and Y5 agonists, are also capable of reducing per1 and per2 mRNA when applied to SCN cells in vitro (Brewer et al., 2002; Fukuhara et al., 2001).

These results suggest a role for NPY in directly mediating nonphotic input at the SCN

(resulting in changes in gene expression and subsequent shifting), as well as indirectly modulating photic input via the IGL (by suppressing light-induced shifts). Thus, information regarding the in vivo release of NPY from the SCN is necessary to further elucidate its role in entrainment, and here it was evaluated in the hamster over 24 hr.

Serotonin (5-HT) release from the raphe nuclei is also implicated in the modulation of nonphotic signals to the SCN, both directly from the midbrain raphe, and indirectly from the dorsal raphe to the IGL (Meyer-Bernstein and Morin, 1996). Both

midbrain and dorsal raphe nuclear stimulation induced phase shifting in hamsters, and these shifts were blocked with administration of metergoline, a 5-HT1A,2,7 receptor antagonist (Glass et al., 2000; Glass et al., 2003). Serotonergic fibers are dense within the SCN, namely at the core (Bosler and Beaudet, 1985; François-Bellan and Bosler,

1992; Moore and Speh, 2004) and evidence suggests that 5-HT has a suppressive effect on photic SCN cells (Ying and Rusak, 1994). Pretreating cultured rat SCN cells with 5-

HT attenuated glutamate-induced increases in Ca2+ (Quintero and McMahon, 1999), while microiontophoresis of 5-HT into the hamster SCN blunted spontaneous neuronal firing rates, possibly acting through the 5-HT7 receptor (Ying and Rusak, 1997). The effect of the selective 5-HT1A,7 receptor agonist, 8-OH-DPAT, on the release of GRP from the hamster SCN has already been examined by our lab (Francl et al., 2010), and here 8-OH-DPAT was administered directly into the hamster SCN to elucidate the actions of 5-HT on VIP release in vivo.

Efferent SCN projections

To project rhythms, the SCN must extend its influence to other brain areas.

Direct neuronal projections from the SCN to outlying brain areas in rat, mouse, hamster, and human brain were identified by lesioning the SCN as well as by retrograde and anterograde tracing experiments. SCN output areas are mainly within the hypothalamus and include the paraventricular nucleus of the hypothalamus (PVN), the dorsomedial hypothalamus (DMH), the medial preoptic nucleus, and the intergeniculate leaflet and paraventricular nucleus the thalamus (IGL and PVT, respectively) (Abrahamson and

Moore, 2001; Berk and Finkelstein, 1981; Dai et al., 1997; Kalsbeek et al., 1993; Stephen et al., 1981; Watts et al., 1987). In hamster and rat, the contents of these outward- projecting neurons contain various combinations of SCN neurotransmitters and neuropeptides, including AVP, VIP, GRP, and GABA (Buijs et al., 1993; Card and

Moore, 1984; Kalsbeek et al., 1993). One of the best-characterized SCN output paths involves the ultimate control of the hypothalamo-pituitary-adrenal axis by AVP. It was shown that SCN AVP had an inhibitory effect on corticosterone (Kalsbeek et al., 1996) such that increasing SCN AVP release resulted in a suppression of corticosterone secretion, and it is suggested that the SCN is responsible for the daily rhythms in CSF corticosterone secretion seen in mammals.

Both VIP and GRP projections originating in the SCN have been detected in other hypothalamic areas. VIP projections have been located specifically in the anterior PVT, medial DMH, and universally in the PVN, while GRP-containing neurons were localized to the medial DMH, PVN, and the supraoptic nucleus (Kalsbeek et al., 1993a; Novak et al., 2000; Teclemeriam-Mesbah et al., 1996). In hamsters that received either an autograft (hamster-to-hamster) or heterograft (mouse- or rat-to-hamster) of fetal SCN tissue following lesion, VIP-containing neuron terminals originating from the SCN were integrated into host PVT tissue. The degree of integration was correlated to restoration of circadian behavioral activity rhythms, indicating extra-SCN VIP projections may be involved in recovery of circadian rhythms (Sollars and Pickard, 1995).

VIP neuronal projections emanating from the SCN have been implicated in the modulation of circadian melatonin production and increased in nighttime release from the

pineal gland. VIP efferents from the SCN that projected to the PVN synapsed on approximately 30% of all spinal cord projecting neurons, which provides anatomical and structural evidence for SCN impact on autonomic activity (Teclemeriam-Mesbah et al.,

1996). VPAC2 peptide and mRNA expression was localized in the rat PVN

(Kalamationos et al., 2004; Kalló et al., 2004b). Lastly, VIP, as well as AVP, infusions into the rat PVN produced increased levels of plasma melatonin content (Kalsbeek et al.,

1993b), and it is possible that VIP originating from the SCN could mediate this effect.

The PVN is an integrator of incoming information from the SCN to the pineal gland, which produces melatonin with a daily circadian rhythm, and PVN or SCN lesions abolish melatonin production rhythms in mammals (Klein et al., 1983; Perreau-Lenz et al., 2003; Reppert et al., 1981).

However, the extent of SCN VIP involvement in pineal melatonin production is unknown. In rats, using bicuculline to block GABA-ergic transmission from the SCN to the PVN resulted in increases in the rate-limiting enzyme involved in melatonin production during the subjective daytime (Kalsbeek et al., 2000), and bicuculline prevented the inhibitory effects of light on melatonin secretion (Kalsbeek et al., 1999), suggesting that the SCN may provides inhibitory GABA-ergic projections to suppress melatonin synthesis via the PVN during the day. As GABA is the most abundant SCN neurotransmitter and is localized in nearly all SCN neurons (Moore and Speh, 1993), it is possible that SCN efferent neuronal projections containing VIP colocalized with GABA co-modulate SCN mediation of circadian melatonin production and secretion.

SPECIFIC AIMS

SPECIFIC AIM 1: Assess the in vivo release of three major neuropeptides (NPY, VIP, and GRP) from the SCN over a period of 24 hr.

Rationale and method: Much is known regarding the neuronal workings of the biological clock; however, these experiments fail to address the endogenous release of these neuropeptides from the SCN. Here, microdialysis experiments in conjunction with radioimmunoassay were undertaken to assess the release of NPY, VIP, and GRP from the

SCN in a freely-behaving animal model. Experiments were performed with a normal light schedule (14L:10D; LD) to assess effects of normal environmental light conditions on neuropeptide release, and with a constant darkness schedule (DD) to assess strictly circadian aspects of neuropeptide release in the absence of light for a period of 24 hr.

The methods used here have been previously employed in our lab to determine the in vivo release of 5-HT from the SCN (Dudley et al., 1998) and AVP (Francl et al., 2010), and the results from these experiments provided the basis of the timing for the remaining pharmacological experiments.

Hypothesis 1: It was hypothesized that NPY, VIP, and GRP would exhibit rhythmic diurnal patterns of release over the course of a 24 hr day under a normal 14L:10D LD

light schedule. Because NPY is not endogenous to the SCN but rather is present in afferent fibers from the IGL which receive photic stimulus input from the RHT and subsequently project to the SCN, it was hypothesized that NPY would peak during the day. VIP and GRP are implicated in photic information transmission; therefore, it was predicted that both VIP and GRP release would peak during the day in response to daily light. It was also hypothesized that housing animals in constant darkness would eliminate these release rhythms. NPY is released from the IGL which indirectly receives photic information. In the absence of photic stimulation it was predicted that rhythmic NPY release would be abolished. Likewise, much experimental data exists to suggest the functions of VIP and GRP within the SCN to be directly involved in signaling and/or regulation of incoming photic stimulus input, rather than in rhythm generation and maintenance. Therefore, it was expected that conditions of constant darkness would abolish any diurnal rhythmic release of VIP and GRP, similar to NPY.

SPECIFIC AIM 2: Validate the neuronal release of NPY, VIP, and GRP from the SCN using pharmacological and procedural methods.

Rationale and method: A sodium channel activator and a cocktail of calcium channel blockers were administered directly at the SCN via reverse-microdialysis to stimulate and inhibit, respectively, the neuronal release of NPY, VIP, and GRP. Use of these agents is necessary to validate the source of release of the neuropeptides being measured. A sodium channel activator was administered at the time of low endogenous peptide

release, based on the 24-hr LD data obtained in Specific Aim 1. A cocktail of calcium channel blockers (L-type) were administered to block the release of endogenous peptide prior to the time of its peak release, based on the 24-hr LD data. Lastly, data from animals that received a microdialysis probe implant >500 µm from the lateral portion of the SCN were analyzed. This was done to verify that the peptides collected in the microdialysate were of SCN-origin and were not diffusing from other local brain areas.

Hypothesis 2: It was hypothesized that a 1 hr perfusion via reverse-microdialysis of a depolarizing medium containing high [K+] and the sodium channel activator, veratridine, would cause an increase in the neuronal release of NPY, VIP, and GRP, while a similar perfusion of a cocktail of calcium channel blockers would cause a suppression in neuronal release of these SCN neuropeptides. It was also expected that data from animals that received microdialysis probes implanted >500 µm from the lateral margin would not resemble the data from the 24-hr LD profiles and would not exhibit any significant release pattern, thus confirming that the neuropeptides measured were of

SCN-origin.

SPECIFIC AIM 3: Determine the influence of a 5-HT1A,7 receptor agonist, 8-OH-DPAT, on the release of VIP from the SCN.

Rationale and method: 8-OH-DPAT, a 5HT1A,7 receptor agonist, was administered via reverse-microdialysis for 1 hr to determine the effects of serotonin on the release of VIP

from the SCN. Serotonin is partially responsible for mediating nonphotic signaling to the

SCN, while VIP is involved in photic signaling. Photic and nonphotic stimuli have the greatest effect on behavioral rhythms at opposing times of the circadian cycle (according to the photic and nonphotic PRC; Figures 1 and 2), and the SCN is heavily innervated by serotonergic fibers from the raphe nuclei (Bosler and Beaudet, 1985; Moore, 2004).

Additionally, our lab has shown an inhibitory effect of 8-OH-DPAT on GRP release in the SCN (Francl et al., 2010), which is also implicated in photic phase resetting. Thus it was important to ascertain a role, if any, for serotonin in the regulation and release of

SCN VIP.

Hypothesis 3: It was hypothesized that 5-HT plays a modulatory role in the release of the photic-related peptides of the SCN, and that administration via reverse-microdialysis of the 5-HT1A,7 receptor agonist, 8-OH-DPAT, for 1 hr would cause a subsequent suppression of VIP release from the SCN.

SPECIFIC AIM 4: Determine the influence of N-methyl-D-aspartatic acid (NMDA) and light on the release of VIP and GRP from the SCN.

Rationale and method: N-methyl-D-aspartic acid acts as a glutamate agonist by binding to NMDA receptors within the SCN. Glutamate is the neurotransmitter implicated in relaying photic signals from the retinohypothalamic tract to the SCN, where VIP and

GRP are the SCN neuropeptides likely involved in intra-SCN photic signal processing.

The effects of a 1 hr pulse of NMDA on VIP and GRP release from the SCN during the dark phase were assessed, as were the effects of a 1 hr light pulse, on release of these neuropeptides from the SCN. VIP and GRP are implicated in photic signaling at the

SCN, and VIP and GRP microinjected directly into the SCN in the early subjective night

(ZT 12-14) caused phase delays in hamsters (Piggins et al., 1995). This suggests possible roles for VIP and GRP to mediate photic information throughout the night; therefore, 1 hr

NMDA and light pulses were administered in early night (ZT 13-14).

Hypothesis 4: It was hypothesized that a 1 hr pulse of NMDA during the early night (ZT

13-14) would induce a peak in both VIP and GRP release from the SCN. Likewise, it was hypothesized that a 1 hr light pulse during the early night (ZT 13-14) would induce an increase in VIP and GRP release from the SCN.

CHAPTER II

MATERIALS AND METHODS

Animals

Adult male Syrian hamsters (Mesocricetus auratus), raised from breeder pairs obtained from Harlan Sprague-Dawley (Madison, IL) were maintained in a climate- controlled environmental chamber (20-22°C) under a 14L:10D photoperiod (LD; 200-

250 lux illuminance) and prior to experimentation were housed individually in circular polycarbonate cages (Raturn; Bioanalytical Systems Inc.; West Lafayette, IN). Rodent chow (Prolab 3000; PMI Feeds, Inc.; St. Louis, MO) and water were available ad libitum.

The experiments were approved by the Kent State Institutional Animal Care and Use

Committee and were conducted using the National Institutes of Health Guidelines for the

Care and Use of Laboratory Animals.

SCN Microdialysis

The microdialysis procedures used are similar to those described in previous studies on SCN monoamine neurotransmitter release (Glass et al., 2003), except that probes having larger molecular weight cutoff were used (CMA/12; 20kDa cutoff; CMA

Microdialysis, Inc.; North Chelmsford, MA). The in vitro efficiency of SCN

neuropeptide recovery in microdialysate, determined by incubating probes in an artificial cerebral spinal fluid solution (ACSF; NaCl, 126.5 mM; NaHCO3, 27.5 mM; KCl, 2.4 mM; KH2PO4, 0.5 mM; CaCl2, 1.1 mM; MgCl2, 0.85 mM; Na2SO4, 0.5 mM; D-(+)- glucose, 5.9 mM; bovine serum albumin, 0.1%; pH 7.5) of 125I-labeled peptide at 37°C and measuring radioactive label recovery in the microdialysate at a flow rate of 1µL/min, averaged 11%, 7%, and 8.6% for NPY, VIP, and GRP, respectively. For surgery, animals were anesthetized with sodium pentobarbital (Nembutal; 85 mg/kg) and received a probe implant with the tip aimed at the SCN (coordinates: AP: +0.03 from bregma, L:

+0.04 from midline, H: -0.80 from dura; head level). Surgical screws and dental acrylic were used to secure the probe to the skull. Probe location was verified histologically from frozen coronal sections stained with cresyl violet at the end of experimentation.

The animals were allowed to recover for 48-72 hr prior to microdialysis sampling. Over this period, the blood-brain barrier is reestablished (Benveniste, 1989) and animals exhibit normal circadian behavioral activity, as confirmed by actogram analysis. For microdialysis sampling, animals housed in a rotating bowl were connected via a tether attached to a harness that protected the inflow and outflow tubings (Figure 4). ACSF was perfused through the probe at a flowrate of 1.0 µL/min using a calibrated syringe pump

(CMA Microdialysis) and gas-tight syringe (Hamilton). The microdialysate flowed from the probe via PFTE tubing leading to 250 µL polyethylene non-stick tubes contained in an automated refrigerated fraction collector (BAS). Samples were frozen at -70°C until analysis.

Figure 4. Photograph demonstrating the microdialysis procedure. The animal is connected to the Raturn system by a belt and tether, which is attached to a sensor at the top of the system. The sensor detects the movement of the animal and adjusts the speed and direction of movement of the bowl cage to compensate, thus preventing twisting of the inflow and outflow tubing. This system allows for microdialysis to take place without interrupting the locomotor activity of the animal.

SCN microdialysate neuropeptide measurements

The content of NPY, VIP, and GRP in SCN microdialysate was measured by radioimmunoassay (RIA; Phoenix Pharmaceuticals, Inc.; Burlingame, CA). The assay is highly specific for these peptides, with low cross-reactivity of the primary antibody with other peptides (data supplied with the kit). Diluting the primary antibody shifted the standard curve to the left, increasing the sensitivity to ~0.2 pg/tube. The intra-assay coefficient of variability was 10.4%, 11.7%, and 10.8% for NPY, VIP, and GRP, respectively. The typical peptide yield was ~0.2-60.0 pg/sample. Briefly, standards and microdialysis samples were incubated in primary antibody for 48 hr at 4°C. Samples of

ACSF and drug cocktails equal in volume to microdialysis samples (60 µL) were run as controls, in addition to positive controls with known peptide content that were supplied with each kit. Next, samples were incubated in 125I-labeled peptide for 24 hr at 4°C.

Samples were then incubated in normal rabbit serum and goat-anti-rabbit secondary antibody for 90 min at room temperature, followed by centrifugation at 3600 rpm for 30 min at 4°C. The supernatant was carefully aspirated and the radioactivity of the pellets was measured by gamma counter (Cobra II; Packard Instruments (Perkin Elmer);

Waltham, MA).

Circadian locomotor activity measurements

In experiments where 24 hr profiles of peptide release under LD and DD were characterized, general locomotor activity rhythms were monitored from 4-5 days and 2 wks, respectively, prior to surgery and during the period of microdialysis sampling to

verify a normal pattern of circadian behavioral activity during sampling, and to establish subjective circadian time (CT) under DD. A representative actogram of an animal housed in LD conditions is shown in Figure 5. Locomotor activity was monitored using infrared motion detectors interfaced with a computerized data acquisition system

(Clocklab; Coulbourn Instruments, Whitehall, PA). In animals under DD, the onset of activity (defined as the first >10 min period of activity that was followed < 20 min later by a period of at least 1 hr of sustained activity) was designated as CT 12. In pharmacological drug perfusion experiments, circadian behavioral activity was monitored for 1 wk prior to surgery and during the period of microdialysis sampling and drug administration.

Figure 5. Representative double-plotted actogram of a hamster housed in LD prior to microdialysis probe implant surgery and experimentation. Each horizontal line indicates one 24 hr day. The day of surgery is noted, followed by two days of recovery. It should be noted that microdialysis experimentation does not disrupt the timing of general locomotor rhythmic behavior.

Experimental protocols

Daily profiles of SCN peptide release under LD and DD.

For these experiments, animals were kept in their home cage and in the same chamber to minimize any procedural behavioral stimulation. Also, samples were collected automatically and the system operation was monitored remotely in the light and dark phases by video to minimize contact with the animals. On the day of experimentation, the probe lining was connected to a syringe pump at ZT 4 under LD or

CT 4 under DD and perfusion with ACSF was initiated. After a 2 hr equilibration period, sample collection was initiated at ZT 6 or CT 6 with a sampling interval of 1 hr at a flow rate of 1.0 µL/min over the ensuing 24 hr period. The automated fraction collector was set with a time delay to account for the lag-time of effluent flow from the probe to reach the collection vial. Microdialysis probe implantation under DD was undertaken by anesthetizing the animals in darkness and masking the eyes with black tape to prevent retinal photic stimulation during the surgery. The microdialysis procedures, including probe connection, were undertaken under dim red light (0.5 lux).

Verification of neuronal SCN neuropeptide release.

Two separate pharmacological treatments delivered via intra-SCN reverse- microdialysis were used to confirm that peptides measured in the SCN microdialysates were from neuronal release (as opposed to procedural artifact). First, SCN peptide release was measured concomitantly with a pulse of depolarizing medium consisting of

ACSF with high [K+] (150 mM) and veratridine (100 µM; Sigma). Second, a calcium blocking mixture was used (diltiazem, 200µM; verapamil, 200µM; cinnarizine, 15µM; flunarizine, 12µM) in calcium-free ACSF containing EDTA (10 mM). Two days after microdialysis probe implantation, sampling for both treatments was initiated with a 2 hr equilibration period of normal ACSF. This was followed by 1 hr of baseline collection, then sample collection during a 1 hr pulse of depolarizing or Ca2+-blocking medium, followed by a 3 hr post-stimulation sample collection period with normal ACSF.

Controls sampled using normal ACSF throughout were run in parallel.

Nonphotic and photic treatment effects on SCN VIP and GRP release.

Photic (glutamatergic and light) and nonphotic (serotonergic) neurotransmitter mechanisms involved with regulating SCN VIP and GRP release were studied pharmacologically using reverse-microdialysis. To test the effects of nonphotic stimuli on VIP release, hamsters housed in a normal LD cycle were outfitted with an SCN microdialysis probe and underwent 2 hr of equilibration perfusion with ACSF. This was followed with 1 hr baseline collection preceding a 1 hr perfusion with 8-OH-DPAT (1.2 mM; ZT 6-7 for VIP). The experiment concluded with 2 hr of post-treatment sampling with normal ACSF. In this experiment, animals were subjected to an Aschoff Type II procedure, in that they were released into DD immediately following the initiation of the drug perfusion, and remained housed in DD until the end of the experiment. Lastly, to test the effects of photic stimuli on neuropeptide release, animals housed in a normal LD cycle received a 1 hr light pulse (approximately 250 lux; ZT 13-14 for both peptides) or a

1 hr reverse-microdialysis perfusion of NMDA (200 µM; ZT 13-14 for both peptides) following a 1 hr baseline collection. These experiments concluded with 2 hr of post- treatment sampling with normal ACSF.

Histological evaluations of intracranial implant sites

After completion of each experiment, microdialysis probe location was verified histologically. Hamsters were euthanized with sodium pentobarbital (Euthasol; 390 mg/kg), and brains were extracted and stored in 4% paraformaldehyde overnight. Brains were protected in 30% sucrose overnight and then cryosections of the SCN (20 µm) were stained with cresyl violet for light microscopic verification of probe implant location

(Figure 6).

Immunohistochemical evaluations of SCN neuropeptide expression

Immunohistochemistry was performed on representative SCN sections to demonstrate peptide-containing neuronal elements within the SCN using primary antibodies against NPY, VIP, and GRP (Figure 7, Bachem). Briefly, adult male hamsters

(n=1/peptide) were euthanized with sodium pentobarbital. Brains were removed and stored in 4% paraformaldehyde at 4°C. Brains were then transferred to a 30% sucrose solution for cryoprotection and 60 µm cryosections were collected in 24-count well plates

(Fisher Scientific). Sections were washed in Tris buffered saline with BSA (0.1%),

Triton-X (0.1%) and merthiolate (0.01%) (TBS) on a shaker at room temperature 2 times for 15 min each. Sections were then incubated in normal goat blocking serum for 60 min

Figure 6. Cresyl violet-stained SCN coronal section demonstrating microdialysis probe tract location. 3V, third ventricle; OC, optic chiasm; PT, probe tract; SCN, suprachiasmatic nucleus.

Figure 7. Coronal SCN section diagramming neurons immunoreactive for NPY, VIP and GRP. Scale bar is 200 µm; 3V, third ventricle; OC, optic chiasm; SCN, suprachiasmatic nucleus.

at room temperature on a shaker. The blocking solution was removed and sections were incubated in primary antibody made in rabbit (1:1000 for all peptides) for 48 hr at 4°C.

Sections were rinsed in TBS for 10 min on a shaker and incubated in FITC-conjugated donkey anti-rabbit IgG (Jackson Pharmaceuticals) for 90 min at room temperature on a shaker. Sections were then rinsed in TBS and water and were dried overnight before mounting on charged slides (Daigger) with Vectashield protection medium (Vector

Laboratories, Inc.).

Statistics

Drug effects were normalized as a percentage of the pretreatment baseline collections (1 hr) and were analyzed using a one-way randomized ANOVA. Treatment effects were determined using the Student Newman-Keuls test. Individual 24 hr profiles of peptide release were normalized by expressing values as a percentage of the daily mean. Daily variations in this release were analyzed as in previous experiments (Dudley et al., 1998) using a repeated measures ANOVA followed by Dunnet’s test procedure for comparing multiple group means (Zar, 1983). For all procedures the level of significance was set at p<0.05.

CHAPTER III

RESULTS

Daily profiles of SCN neuropeptide release under LD.

Raw neuropeptide amount collected in the microdialysate varied depending on peptide type. NPY recovery ranged from 0.2-4.0 pg/hr, while VIP and GRP collections ranged from 1.0-60.0 pg/hr. Daily patterns of release under LD conditions were seen for all neuropeptides measured. NPY release peaked at ZT 3 (204±29% of the daily mean; n=5; F4,23=4.200; p<0.001) and dropped sharply to its lowest level at ZT 8 (38±10% of the daily mean), then rose slowly as lights on approached (Figure 8). VIP release was greatest from mid-day to lights off, peaking at ZT 4 (211±19% of daily mean; n=7;

F6,23=2.291; p<0.005), while the nadir occurred during middle of the dark phase at ZT 16

(75±9% of the daily mean; Figure 9). GRP release was lowest during mid-day at ZT 7

(62±13% of the daily mean; n=4) and rose steadily throughout the night and early day to peak levels at ZT 4 (207±63% of the daily mean; F3,23=2.317; p<0.005; Fig. 10).

Additionally, data was collected from experimental animals where the microdialysis probe implant missed the SCN by more than 500 µm. In these animals, there was no detected pattern of rhythmic neuropeptide release (NPY: n=3; F2,23=0.437; p>0.9; VIP:

Figure 8. Double-plotted NPY release from the SCN measured under LD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 5 animals. Black bars indicate onset of the dark phase (ZT 12); * indicates p<0.05 compared to all other timepoints.

Figure 9. Double-plotted VIP release from the SCN measured under LD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 7 animals. Black bars indicate onset of the dark phase (ZT 12); * indicates p<0.05 compared to all other timepoints.

Figure 10. Double-plotted GRP release from the SCN measured under LD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 4 animals. Black bars indicate onset of the dark phase (ZT 12); * indicates p<0.05 compared to all other timepoints.

n=3; F2,23=1.262; p>0.20; GRP: n=3; F2,23=0.957; p>0.50), supporting the statement that the neuropeptides measured were of SCN-origin rather than from outlying brain areas.

Daily profiles of SCN neuropeptide release under DD.

In hamsters housed in DD for 2 weeks, NPY did not exhibit a rhythmic pattern of release under conditions of constant darkness, and no discernible peak release was detected, unlike what was seen under LD. Rather, NPY release fluctuated continuously over the course of the subjective day and night (n=3; F3,23=1.602; p>0.08; Fig. 11).

Under DD, VIP release was also arrhythmic and no evident pattern was detected (n=5;

F2,23=1.581; p>0.09; Fig. 12). Conversely, GRP was rhythmically released under DD and release was shifted and prolonged compared to the LD profile, and peak release was shifted from day to subjective night. GRP release was low from CT 7-14, and rose from its lowest level at CT 11 (68±14% of the daily mean; n=3) through the early subjective night to its highest point at CT 19 (129±14% of the baseline; F2,23=3.106; p<0.001) and continued to stay elevated for the remainder of the night (Figure 13).

Figure 11. Double-plotted NPY release from the SCN measured under DD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 3 animals. Dotted lines indicate the onset of activity (CT 12).

Figure 12. Double-plotted VIP release from the SCN measured under DD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 5 animals. Dotted lines indicate the onset of activity (CT 12).

Figure 13. Double-plotted GRP release from the SCN measured under DD conditions over 24 hr. Values plotted are normalized as a percentage of the daily mean±SEM for 3 animals. Dotted lines indicate the onset of activity (CT 12); * indicate p<0.05 compared to all other timepoints.

Verification of SCN neuronal neuropeptide release.

Administration of a 1 hr drug perfusion from ZT 12-13 of high [K+] ACSF + veratridine (100 µm) resulted in an increase in NPY release from the SCN (207±1.5% of the baseline; n=2; F4,9=7.507; p<0.05; Figure 14). NPY release was then reduced, but remained elevated (though not significantly) over baseline levels until the conclusion of the experiment. Drug perfusion from ZT 13-14 caused VIP levels to significantly increase to 199±28% of the baseline, which then decreased to baseline levels by ZT 14, at the conclusion of the drug pulse (n=4; F7,11=5.111; p<0.001; Figure 15). GRP release increased drastically (702±134% of the baseline; F6,9=5.659; p<0.02; n=2) before returning to baseline levels approximately 60 min following the conclusion of the perfusion from ZT 6-7 (Figure 16). Behaviorally, the 1 hr high [K+] + veratridine perfusion appeared to cause an increase in activity and grooming in the majority of animals compared to pre-drug baseline levels. This behavioral effect terminated approximately 10-15 minutes following the conclusion of the drug pulse and the replacement with normal ACSF. Behavior was then noted to be normal for the remainder of the experiment.

Conversely, a 1 hr perfusion of Ca2+-channel blockers in Ca2+-free ACSF delivered into the SCN from ZT 2-3 reduced VIP release to 55±8% of the baseline before returning to normal levels approximately 90 min after ending the perfusion (n=4;

F4,19=3.846; p<0.02; Figure 17). Likewise, administration of this drug cocktail from ZT

2-3 reduced GRP output to its lowest level (32±8% of the baseline; F5,15=6.500; p<0.005) occurring approximately 30 minutes after the conclusion of the drug perfusion (n=4;

Figure 18). NPY release was not assessed in conjunction with the calcium channel blocker cocktail for two reasons: 1) the level of detection of endogenous NPY was at the minimal level of the RIA kit, and administering a calcium blocker cocktail resulted in undetectable levels of NPY during the drug pulse and for 3-4 hr post-treatment until the end of the experiment and thus, results that could not be calculated; and 2) synaptic NPY release was previously assessed directly from the IGL, using both veratridine in high [K+]

ACSF and a calcium channel blocker cocktail (Glass et al., 2010). Behavioral effects were noticeable during the 1 hr Ca2+ reverse perfusion; namely, most animals exhibited reduced activity and a non-sleeping prone posture for most of the duration of the perfusion. These behaviors ceased in a manner similar to that of the high [K+] + veratridine experiments, ending approximately 15-20 min following the conclusion of the drug pulse. Animal behavior was noted as normal for the remainder of the experiment.

Figure 14. Effect of a 1 hr reverse microdialysis of high potassium + veratridine on NPY release from the SCN. Values are normalized as a percentage of pre- drug baseline levels±SEM (n=2; closed circles) and are plotted with control ACSF values (n=5; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to baseline.

Figure 15. Effect of a 1 hr reverse microdialysis of high potassium + veratridine on VIP release from the SCN. Values are normalized as a percentage of pre- drug baseline levels±SEM (n=4; closed circles) and are plotted with control ACSF values (n=7; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

Figure 16. Effect of a 1 hr reverse microdialysis of high potassium + veratridine on GRP release from the SCN. Values are normalized as a percentage of pre- drug baseline levels±SEM (n=2; closed circles) and are plotted with control ACSF values (n=4; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

Figure 17. Effect of a 1 hr reverse microdialysis of a calcium channel blocker cocktail on VIP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=4; closed circles) and are plotted with control ACSF values (n=7; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

Figure 18. Effect of a 1 hr reverse microdialysis of a calcium channel blocker cocktail on GRP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=4; closed circles) and are plotted with control ACSF values (n=4; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to controls.

Nonphotic and photic treatment effects on VIP and GRP release.

8-OH-DPAT

Reverse microdialysis perfusion in mid-day (from ZT 6-7) with the 5-HT1A,7 agonist, 8-OH-DPAT, for 1 hr significantly reduced VIP release in the SCN compared to pretreatment baseline levels (24±12%; n=4; F7,14=3.317; p<0.005; Figure 19). This suppressive effect of 5-HT lasted throughout the duration of the drug perfusion and extended for at least 2 hr post-treatment until the end of the experiment. Minimal behavioral effects were noted during the 8-OH-DPAT perfusions, namely, several animals assumed a prone posture at the beginning of the drug administration. This effect was short-lived, and animals resumed normal behavioral approximately 15 min following the beginning of the drug pulse.

NMDA

Reverse microdialysis of the SCN with the glutamate agonist, NMDA, from ZT

13-14 caused a significant increase in both VIP (335±62%; n=3; F6,13=6.265; p<0.001;

Figure 20) and GRP (203±38%; n=2; F1,7=8.288; p<0.005; Figure 21). Behaviorally, animals appeared to exhibit increased activity based on visual observation, as well as increased grooming, for the duration of the NMDA pulse. Following the conclusion of the drug pulse, behavior appeared to return to normal, approximately 10-15 min following the end of the drug pulse and the replacement of normal ACSF.

Figure 19. Effect of a 1 hr reverse microdialysis pulse of 8-OH-DPAT on VIP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=4; closed circles) and are plotted with control ACSF values (n=7; open circles). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline, * indicates p<0.05 compared to control.

Figure 20. Effect of a 1 hr reverse microdialysis pulse of NMDA on VIP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=3). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline, * indicates p<0.05 compared to control.

Figure 21. Effect of a 1 hr reverse microdialysis pulse of NMDA on GRP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=2). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

Light

A 1 hr light pulse from ZT 13-14 significantly increased both VIP (342±117%; n=4; F6,14=3.539; p<0.001; Figure 22) and GRP release from the SCN (157%±17 of baseline; n=5; F9,6=4.910; p<0.05; Figure 23). VIP release was significantly elevated for

30 min, and then remained elevated (though not significantly) until the end of the experiment. For GRP, this stimulatory effect lasted approximately 30 min following the conclusion of the light pulse, and release then returned to near-baseline levels within 1 hr of the conclusion of the light pulse. During the light pulse experiments for both VIP and

GRP, all animals were awake upon the start of the experiment, and most slept during some portion of the light pulse. Following the conclusion of the light pulse and the return to normal dark conditions, the hamsters displayed normal nightly activities within 1 hr of the end of the light pulse.

Figure 22. Effect of a 1 hr light pulse on VIP release from the SCN. Values are normalized as a percentage of pre-drug baseline levels±SEM (n=4). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

Figure 23. Effect of a 1 hr light pulse on GRP release from the SCN. Values are normalized as a percentage of pre-treatment (light) baseline levels±SEM (n=5). Black bar indicates time of 1 hr drug administration; a indicates p<0.05 compared to baseline; * indicates p<0.05 compared to control.

CHAPTER IV

DISCUSSION

24 hr neuropeptide rhythms

Neuropeptides play critical roles in SCN regulation, including transmission of both photic and nonphotic entraining signals to the SCN as well as interneuronal communication within the SCN itself. For example, pituitary adenylate cyclase- activating peptide (PACAP) is localized in neurons of the RHT and is co-stored with glutamate (Hannibal et al., 1997; Hannibal et al., 2000; Hannibal and Fahrenkrug,

2003b). Mice that lack PACAP display reduced shifting response to both phase- advancing and phase-delaying light pulses as well as blunted c-Fos and phosphorylated

MAPK, both of which are indicators of regulation of gene expression by light (Colwell et al., 2004; Dragich et al., 2010). Many retinorecipient neurons of the ventral portion of the SCN contain VIP and sometimes GRP (Moore and Speh, 2002), likely indicating that

VIP- and GRP-ergic neuronal elements receive direct photic input from the retina via the release of glutamate and PACAP from the RHT (Morin et al., 2006).

Within the SCN, neuropeptides including NPY, VIP, GRP, and AVP are required for the production of normal behavioral rhythms and behavioral responses to photic or nonphotic stimuli. Electrical firing rates of SCN neurons maintained in a hypothalamic

slice varied with a period of 24 hr (Green and Gillette, 1982; Groos and Hendricks,

1982). When separated and cultured, these individual SCN cells also oscillated with a period of approximately 24 hr, though their phases were different (Honma et al., 1998;

Welsh et al., 1995). This indicates that while individual SCN neurons may act as circadian oscillators, a synchronizer must be present in the intact SCN which controls the timing of peak electrical firing, and neuropeptides (specifically VIP) have been implicated in performing this role.

Here, in vivo microdialysis and RIA were used to identify endogenous release patterns of NPY, VIP, and GRP from the hamster SCN circadian clock. Microdialysis provides a unique view into dynamic neurotransmitter and neuropeptide fluctuations in release at the synaptic level and also allows for examination of release due to experimental manipulations (Horn and Engelmann, 2001; McAdoo and Wu, 2008). This technique involves the sampling of extracellular materials by perfusion with ACSF in the area of interest. Brain microdialysis techniques have previously been used in our lab as well as others to measure glutamate, 5-HT, oxytocin, and AVP (Dudley et al., 1998;

Francl et al., 2010; Kalsbeek et al., 1995; Orlowska-Majdak, 2004; Portas et al., 2000;

Rea et al., 1993b). The efficiency of the microdialysis probe varies with the substance being measured, such that larger peptides like NPY have lower recovery than smaller peptides like AVP (Francl et al., 2010; present results). Efforts were made to mimic natural physiological conditions to ensure the highest molecular recovery possible, and included adding bovine serum albumin (BSA) to the ACSF, which was shown to increase

in vitro recovery (data not presented) as well as running microdialysis experiments using room temperature ACSF that included physiological ion concentrations and glucose.

In animals entrained to a 14:10 LD cycle, release of NPY, VIP, and GRP was rhythmic and peak release was in early to mid-day. NPY release peaked in early day and dropped quickly to nadir in mid- to late-day, then remained low for the majority of the dark phase. VIP exhibited a brief but significant peak in mid-day, then decreased rapidly and remained low for the entire dark phase. GRP increased immediately following lights on and remained high until mid-day. Release then dropped rapidly and slowly increased throughout the remaining day and into the dark phase. In constant conditions, the rhythmic pattern of release of NPY and VIP was lost, and no significant rhythm was detected. Only GRP release remained rhythmic in DD, though it did not closely resemble the LD rhythm. In DD peak release occurred over a broad portion of the mid-subjective night to early morning with low peptide levels in mid-day to early night. Additionally, the release of GRP was amplified in DD compared to LD, though this difference was not significant. The release of these neuropeptides from the SCN was confirmed by analyzing the data from those animals with a microdialysis probe implant >500 µm from the lateral portion of the SCN. In these animals, neuropeptide release did not resemble the 24 hr profiles in LD, the overall raw peptide amount (in pg) was significantly less than that collected from experimental animals, and no significant pattern or rhythm could be detected. Thus, it was confirmed that the neuropeptides measured were of SCN- origin, rather than diffusible peptides from neighboring brain regions.

Verification of synaptic peptide release

It was critical to verify that the neuropeptides measured from the SCN by microdialysis in the present experiments were synaptic in origin, rather than released through neuronal damage, leaked cellular content, or other procedural artifact. In these studies a sodium channel activator (veratridine) dissolved in high [K+] ACSF was administered via reverse-microdialysis to induce depolarization, and subsequently resulted in a significant increase in NPY, VIP, and GRP from the SCN. This method of verification has been utilized previously by our lab to assess SCN release of glutamate and 5-HT (Dudley et al., 1998; Rea et al., 1993) and by others to assess neuropeptide release from various brain areas (Lindefors et al., 1987; Orlowska-Majdak et al., 2003).

A second method of verification was performed by reverse-microdialysis of a calcium channel-blocking cocktail (L-type) dissolved in calcium-free ACSF. The result from these experiments was a significant suppression of neuropeptide release and this was similar to results seen measuring 5-HT (Dudley et al., 1998) and glutamate (Cousin et al., 1993). Within the SCN, the L-type calcium channel blocker nimodipine nearly eliminated electrical firing rhythms and blunted the amplitude of per1 expression in culture (Lundkvist et al., 2005; Pennartz et al., 2002), demonstrating a suppressive effect directly in the SCN. The alterations in neuropeptide release due to modification of neuronal sodium and calcium currents performed in the present study are therefore a robust indicator of the synaptic release of NPY, VIP, and GRP from the SCN.

Neuropeptide Y in the SCN

NPY is widely present in the CNS, and within the SCN specifically NPY is associated with nonphotic informational input but is not endogenously produced by SCN neurons. Rather, NPY-ergic neurons of the IGL in the thalamus project to the SCN via the GHT. Several lines of evidence support this relationship, including neuroanatomical tract studies showing a neuronal connection between the IGL and the SCN (Harrington et al, 1987; Morin and Blanchard, 1995; Pinato et al., 2009), IGL lesion studies demonstrating disrupted circadian rhythms and nonphotic entrainment following IGL destruction (Harrington and Rusak, 1986; Janik and Mrosovsky, 1994; Pickard, 1994;

Wickland and Turek, 1994), as well as evidence that microinjection of NPY into the SCN during the subjective day induced nonphotic-like behavioral phase shifts (Albers and

Ferris, 1984; Huhman and Albers, 1994; Kallingal and Mintz, 2007). NPY is capable of shifting the circadian clock during the subjective day while having little to no effect during the subjective night (Harrington and Schak, 2000), comparable to other nonphotic stimuli. Several studies demonstrate the suppressive effects of both NPY and nonphotic behavioral stimuli on the expression of per1 and per2 mRNA (Fukuhara et al., 2001;

Maywood et al., 1999; Maywood et al., 2002), and suppression of per1 by SCN microinjection of per1-antisense oligonucleotides induced nonphotic phase shifts in hamsters (Hamada et al., 2004). It has been suggested that suppression of period genes

(by nonphotic stimuli) at varying times of the subjective day could result in shifted peak timing of gene expression during the following circadian cycle, thus producing shifts in behavioral activity (Hamada et al., 2004).

In addition to mediating nonphotic phase resetting, NPY is implicated in the modulation of photic information received at the SCN. NPY phase-dependently attenuated photic phase shifts of SCN neuronal activity (Golombek et al., 1996; Yanielli and Harrington, 2001) as well as locomotor activity rhythms (Lall and Biello, 2002;

Weber and Rea, 1997). Additionally, hamsters receiving a microinjection of anti-serum to NPY prior to a phase-shifting light pulse displayed enhanced behavioral shifts (Biello,

1994) as did hamsters that received an NPY receptor antagonist microinjection prior to a light pulse (Yanielli et al., 2004).

The mechanisms by which NPY exerts its effects within the SCN are not yet fully understood. It is thought that the phase-shifting and phase shift-blocking actions of NPY are mediated by activation of different receptor subtypes that have opposing actions on cell firing and shifting (Gribkoff et al., 1998). The ability of NPY to shift neuronal firing rates and behavioral activity rhythms during the day is likely due to the Y2 receptor subtype (Golombek et al., 1996; Huhman et al., 1996), which is a distinctly separate receptor subtype than that which mediates blockade of photic phase shifts by NPY (the

Y5 receptor; Harrington and Hoque, 1997; Lall and Biello, 2003). Additional evidence comes from knockout studies which demonstrated that the spontaneous firing rhythm of hypothalamic tissue slice neurons maintained from mice lacking the NPY Y2 receptor

(Y2-/-) did not shift with midday application of NPY, while NPY did shift the rhythms of control slices with normal Y2 receptors (Soscia and Harrington, 2005). The presence of several different receptor subtypes therefore presents a mechanism by which NPY can exert dual (and opposing) effects in the SCN.

At the molecular level, NPY reduces the expression of both per1 and per2 genes

(Fukuhara et al., 2001; Maywood et al., 2002), although the method and timing of NPY administration may differentially affect per gene expression (Brewer et al., 2002). This coincides with evidence suggesting that regulators of nonphotic information to the clock

(including NPY and 8-OH-DPAT; Horikawa et al., 2000) as well as nonphotic behavioral stimuli (locomotor activity; Maywood and Mrosovsky, 2001) generally have a suppressive effect on per genes.

In the present study, in vivo NPY release was evaluated over 24 hr, with peak release occurring in the morning when nocturnal animals are relatively inactive. This peak was absent from animals housed in DD, indicating that NPY release is driven by the light-dark cycle and is independent of the animals’ nighttime period of activity. Rather, forced wheel running during the middle of the day induced NPY release, which suggests that the effects of behavior on NPY release are phase-dependent, in accordance with the timing of the nonphotic phase-response curve (Biello and Mrosovsky, 1996; Glass et al.,

2010). This is similar to the effect of behavioral activity on 5-HT release (also implicated in mediation of nonphotic phase shifting), such that wheel-running during midday stimulated 5-HT release but had little effect at night (Dudley et al., 1998). Additionally, the present experiments indicate that endogenous NPY release in hamsters was lowest in midday (ZT 6). Reduced NPY within the SCN at this time could potentially provide for large behavior-induced increases in NPY. This temporally agrees with the phase- advancing portion of the nonphotic phase-response curve, and our lab has shown that

forced midday (ZT 6-7) wheel running in hamsters significantly stimulated NPY release above baseline levels (Glass et al., 2010).

Rhythmic NPY immunoreactivity within the SCN has been measured in the rat, the Mongolian gerbil, and the diurnal 13-lined ground squirrel (Calzá et al., 1990; Fite et al., 2007; Jhanwar-Uniyal et al., 1990; Shinohara et al., 1993; Vidal and Lugo, 2006).

Diurnal release patterns of NPY were present in all cases (detected by immunohistochemistry or enzymatic assay of whole-SCN tissue punches), and these release patterns were absent or reduced in animals housed in DD, similar to the present results. Under LD, the timing of peak NPY release varied across species. The profile of

NPY release in rats is similar to the present results obtained in hamsters, in that peak

NPY release occurred at or near the transition period from dark to light. However, a second peak was detected at the transition from light to dark in rats (Calza et al., 1990;

Shinohara et al., 1993). In the Mongolian gerbil, NPY release was low in early day and subsequently peaked during both late day and late night, while in the thirteen-lined ground squirrel, NPY release was low during midday (similar to the present results) but peaked during mid-night. These proposed differences in SCN NPY release could be due to methodological differences (immunohistochemistry detects intraneuronal content, microdialysis detects extraneuronal release, while assay of whole SCN tissue detects both) or to species/housing differences (hamsters are housed in 14:10 LD, while rats are housed in 12:12 LD). Lastly, it is unclear how the detection of increased neuronal content (for example, by immunohistochemistry) correlates with increased neuronal release (detected by microdialysis). Increased neuronal content seemingly corresponds to

increased cellular activity, though this may also be interpreted as low neuronal activity, resulting in an increased intracellular pool of unreleased neuropeptide. The present results obtained by microdialysis demonstrate neuronal neuropeptide release in real time, thus offering a clear picture of neuronal activity and as well as the timing of possible downstream actions following NPY receptor binding in the SCN.

Vasoactive intestinal polypeptide in the SCN

VIP-containing neurons are localized mainly within the ventrolateral portion of the SCN (Abrahamson and Moore, 2001; Ibata et al., 1989) and receive direct neuronal projections from the retina (Ibata et al., 1989; Tanaka et al., 1993). VIP binds to three receptor variants, two of which are located in the SCN – PAC1 and VPAC2 – with

VPAC2 being the preferential receptor for VIP (Cagampang et al., 1998a, b; Usdin et al.,

1994). In vitro, VIP generally has a suppressive effect on cell firing rate, which is mimicked and blocked by selective VPAC2 receptor agonists and antagonists, respectively (Reed et al., 2002). Additionally, in vitro studies demonstrated that VIP application can delay peak cellular firing in the SCN when applied in early night, while application in late night caused a phase advance in neuronal firing (Reed et al., 2001).

Similar results were obtained when VIP was injected directly into the SCN, which caused phase-dependent delays and advances of behavioral activity in hamsters (Piggins et al.,

1995).

Per1 and per2 gene expression oscillates rhythmically in the SCN, with per1 expression peaking during the light portion of the day-night cycle (Tei et al., 1997) and

per2 peaking near lights-off (Takumi et al., 1998). It is thought that changes in the timing of peak period gene expression ultimately result in shifts of the circadian clock

(reviewed by Reppert and Weaver, 2002). VIP was shown to induce per1 and per2 levels in the SCN (Nielsen et al., 2002), similar to light, GRP, PACAP, and NMDA (Aida et al.,

2002; Albrecht et al., 1997; Moriya et al., 2000; Nielsen et al., 2001; Shearman et al.,

1997; Yan et al., 1999), indicating a role for VIP in conveying photic entraining information in SCN period gene induction and thus, photic phase shifts.

In these experiments, the 24 hr profile of VIP was measured from the hamster

SCN using microdialysis. VIP release exhibited a diurnal release pattern, with a significant but transient peak in neuropeptide release occurring from ZT 4-5. This is consistent with findings that reveal VIP mRNA in the rat SCN is highest in mid- to late- night, followed by peak VIP-like immunoreactivity approximately 2-6 hr later (Okamoto et al., 1991; Yang et al., 1993). Though it is unknown how increases in VIP immunoreactivity reflect increases in neuronal release, it seems that the present results temporally agree with existing data regarding changes in VIP production and content in the SCN over a normal 24-hr LD cycle. The duration of peak release of VIP was relatively short-lived (lasting 2 hr). The reason for this is unclear, though it has been shown that VIP-ergic neurons of the SCN receive VIP synapses (Daikoku et al., 1992; van den Pol and Gorcs, 1986), and that approximately one-third of all VIP-ergic neurons in the SCN contain VPAC2 receptors (Kalló et al., 2004b), which could indicate VIP release in the SCN is partially under control of a VIP-based negative-feedback

mechanism. Thus, the brief peak of VIP release in mid-day could have been terminated by VIP-VPAC2 interactions at VIP-releasing neurons.

In hamsters housed in DD, no clear pattern of VIP release could be detected. This is consistent with results from previous experiments showing that VIP-like immunoreactivity did not vary in rats housed in constant darkness (Shinohara et al., 1993;

Takahashi et al., 1989). Also, no circadian rhythm of VIP mRNA was detected in rats

(Takeuchi et al., 1992). However, there is conflicting evidence regarding the circadian profile of gene expression of the VIP VPAC2 receptor. Some reports indicate no circadian rhythmic expression of VPAC2 mRNA (Shinohara et al., 1999), while others demonstrate both a diurnal and circadian variation in expression in the SCN (Cagampang et al., 1998). It was furthermore shown that administering cysteamine to deplete SCN somatostatin (SS) levels induced a circadian rhythm of VIP content in rats housed in DD

(Fukuhara et al., 1994), suggesting that VIP may be regulated by both environmental light conditions as well as other SCN neuropeptides (SS) and neurotransmitters (5-HT, discussed below).

In addition to mediating environmental photic information, VIP may be associated with gating activity of SCN neurons. Gating refers to controlling the timing of spontaneous SCN neuronal firing, and/or to controlling the responsiveness of SCN neurons to external stimuli, such as what is seen in photic and nonphotic phase responses curves. In mice lacking the VPAC2 receptor, light pulses administered during the subjective night of the circadian cycle elicited increases in p-ERK and c-Fos (markers of increased cellular activity) as well as during the subjective daytime (Hughes et al., 2004)

when light normally has very little effect on SCN neurons. As stated above, VIP generally has a suppressive effect on firing rate. Increased VIP release during the early day, as shown by the present results, could subsequently inhibit SCN firing rates, thus making neurons unresponsive to light signals during the day. It was shown that in SCN slices prepared from mice lacking the VPAC2 receptor, incubation with VIP did not result in inhibition of sodium currents as the same treatment did in controls (Pakhotin et al.,

2006). Additionally, Pakhotin et al. (2006) observed that neurons lacking VPAC2 did not exhibit the typical circadian variation in spontaneous neuronal firing seen in wild-type

SCN neurons.

Despite this evidence, it remains unclear whether VIP is in fact directly involved in the maintenance of gating the responses of the SCN to incoming signals. According to the present experiments, VIP release is highest in early-day. If VIP does, in fact, suppress SCN firing, it would be expected that spontaneous SCN neuronal firing would not exhibit a daily peak during the day, shortly after peak VIP release. Rather, it could be that VIP signaling is indirectly involved by modulation of GABA-ergic neurons. Nearly all SCN neurons contain GABA (Moore and Speh, 1993), and a possible mechanism could involve release of VIP in early day, thus inducing suppressive currents in the SCN that inhibit GABA-ergic neurons, subsequently resulting in disinhibition and therefore daily increases in SCN firing rates.

Evidence also exists for a role of VIP in synchronization of SCN neurons, so that individually oscillating neurons cycle with a similar period and thus produce a single output. Dispersed SCN cells oscillate with differing periods (Welsh et al., 1995) while

neurons maintained in slice preparations or in culture oscillate with a unified rhythm

(Herzog et al., 1997; Quintero et al., 2003; Yamaguchi et al., 2003), indicating that neurotransmission or cell-cell contact may be required for some SCN neurons to remain synchronized over a circadian cycle. It was shown that application of VPAC2 receptor antagonists to normal SCN cells resulted in a loss of the typical mid-day peak in electrical firing, which also resembled the endogenous firing rates of neurons harvested from animals lacking VPAC2 (Cutler et al., 2003). In the SCN slice of mice lacking the

VPAC2 receptor, synchrony between SCN neurons was lost and this was correlated to a reduction in the daily peak in per gene expression (Maywood et al., 2006).

The present results may provide additional support to the argument that VIP plays a role in synchronization of SCN neurons. In the present experiments, it was shown that

VIP peak release occurred from ZT 4-5, just prior to the well-documented synchronized daily peak in SCN neuronal firing (Green and Gillette, 1982; Groos and Hendriks, 1982;

Shibata et al., 1982). The release of VIP at a specific subset of SCN neurons containing the VPAC2 receptor (possibly AVP-containing oscillators of the dorsal SCN) could provide a mechanism by which VIP synchronizes period gene expression rhythms in the

SCN.

Gastrin-releasing peptide in the SCN

GRP-containing neurons are located mainly in the ventral portion of the SCN and are often in synaptic contact with VIP neuronal elements (Aïoun et al., 1998; Mikkelson et al., 1991; Romijn et al., 1997; Shinohara et al., 1993). Like VIP neurons, GRP-ergic

neurons receive a direct retinal input from the RHT (Tanaka et al., 1997), and GRP is implicated in the mediation of photic phase-shifting. Evidence for this role comes from both in vitro and in vivo studies that explored the role of GRP in the SCN. In vitro, GRP treatment to both rat and hamster SCN slices caused significant phase delays and advances of the neuronal firing rate rhythm, and this effect was blocked with co- administration of a selective BB2 antagonist (McArthur et al., 2000). Further support for the involvement of the BB2 receptor subtype comes from an experiment demonstrating attenuated per1 and per2 mRNA expression following a light pulse in mice lacking that

GRP receptor (Aida et al., 2002).

Microinjection of GRP alone, or in a cocktail with VIP and PHI, elicited phase shifts in behavior comparable to light (Kallingal and Mintz, 2006; Piggins et al., 1995), and induced c-fos, per1, and per2 gene expression in the SCN (Antle et al., 2005;

Dardente et al., 2002; Gamble et al., 2007). Rats that received a light pulse had increased immunoreactivity for c-Fos in neurons dually stained for GRP compared to controls

(Earnest et al., 1993). Like VIP, GRP is implicated in possibly mediating the SCN clock gene changes that result in a phase shift.

In a noteworthy experiment, it was shown that exogenous GRP application promoted cellular rhythmicity of SCN slice preparations obtained from behaviorally arrhythmic mice that lacked the VPAC2 receptor (Brown et al., 2005). In this study, a small portion of VPAC2 knockout mice were behaviorally rhythmic, while the majority exhibited arrhythmic behavior as well as decreased peak SCN firing rates, but not decreased overall firing rates. This indicates a reduced capability to gate SCN activity to

only occur at a specific time during a circadian cycle (Aton et al., 2005). Additionally, it was shown that continuous (>24 hr) application of GRP could “rescue” the arrhythmic

VPAC2 knockout slice and produce normal rhythms of neuronal firing rates. This promotion of rhythmic SCN activity was not reproduced by application of NMDA (an excitatory agent), indicating that the effect was not a generalized excitation event, but was mediated strictly through GRP-BB2 interactions. Lastly, Brown et al. (2005) demonstrated that application of GRP to wild-type neurons was detrimental to production of neuronal circadian rhythms, but promoted rhythmic firing in SCN neurons that were blocked pharmacologically by VPAC2 receptor antagonists. Evidently, the role of GRP in the functional circadian clock involves mediation of photic information, and possibly includes mediation of cellular rhythmicity with respect to gating the timing of SCN electrical activity in the absence of VIP.

The present results may provide additional support for the functional significance of GRP in the SCN. In this study, it was shown that GRP release from the SCN exhibited a significant increase in release immediately following lights-on, and remained elevated for several hours. GRP release for the remaining day and night was relatively low, only rising slightly during the middle and late night. These data provide support for the role of

GRP in induction of light-induced signals in the SCN. In contrast, hamsters housed in

DD exhibited a circadian pattern of GRP release, with maximal release occurring over much of the subjective night, followed by decreased release continuing over the subjective day. It appears that in the absence of light, GRP release is circadian in nature and may be clock controlled. While widely recognized as a neuropeptide involved in

mediating the transmission of environmental light information, it is possible that GRP is also involved in the mediation of cellular rhythmicity, either in the absence of light or as a biological redundancy to VIP. However, apart from the study by Brown et al. (2005), and a similar study that also showed GRP could acutely synchronize neurons lacking

VPAC2 (Maywood et al., 2006), little supplementary data exists supporting this type of biological purpose for GRP in the circadian clock. Further experiments investigating the effects of timed in vivo application of neuropeptide receptor blockers on behavioral rhythms are needed to elucidate the possible role of GRP in endogenous rhythm generation.

Serotonergic regulation of VIP

Serotoninergic innervation in the SCN is robust, with 5-HT implicated in the modulation of photic phase-resetting, as well as mediation of nonphotic phase-resetting. 5-HT innervation is well-described in mammals, including the rat, hamster, cat, monkey, and human (Moore and Speh, 2004; Ueda et al., 1983). Because VIP-containing neurons of the SCN are probable components of the photic RHT-to-SCN retinorecipient pathway

(Reuss and Decker, 1997; Tanaka et al., 1993) and these ventral SCN neurons receive serotonergic input (Bosler and Beaudet, 1985; Moore and Speh, 2004; Ueda et al., 1983), it is likely that VIP activity could be modulated by 5-HT. The present findings provide additional support to this argument, in that a 1 hr perfusion of the 5-HT1A,7 receptor agonist, 8-OH-DPAT, caused a significant and long-lasting suppression of VIP release from the SCN. This is in accordance with several other experimental lines of evidence

suggesting that 5-HT can negatively modulate photic shifting. First, increasing 5-HT by administering selective serotonin reuptake inhibitors attenuated photic phase shifting in the hamster (Gannon and Millan, 2007). Also, both 8-OH-DPAT and L-tryptophan (the precursor of 5-HT) caused a significant reduction of light-induced c-Fos in the hamster

SCN, while 5-HT or 8-OH-DPAT injection reduced light-induced phase shifts in behavioral activity rhythms (Glass et al., 1994; Rea et al., 1994). Lastly, our lab demonstrated the inhibitory effect of 8-OH-DPAT on GRP release from the SCN (Francl et al., 2010). On the contrary, inhibiting 5-HT by administering to hamsters a subcutaneous injection of a 5-HT1A receptor antagonist increased the magnitude of light- induced shifts (Smart and Biello, 2001). Additionally, injection of rats housed in DD with PCPA (an inhibitor of tryptophan hydroxylase and thus, 5-HT production) induced a significant peak in VIP mRNA that was not present in vehicle saline-injected controls

(Okamura et al., 1995). These data suggest that VIP release in the SCN, and ultimately behavioral activity rhythms, can be controlled by 5-HT.

Glutamatergic and photic regulation of VIP and GRP

In the SCN, photic stimulus inputs are mediated through the RHT by glutamate release, specifically through the NMDA receptor (Colwell et al., 1990). Support of this comes from experiments confirming the presence of the peptide precursor of glutamate,

N-acetylaspartylglutamate, in the RHT and retinorecipient neurons of the SCN (Moffett et al., 1990), the localization of glutamate in retinal neurons terminating in the SCN

(Castel et al., 1993) and that glutamate is released at the SCN upon optic nerve

stimulation (Liou et al., 1986). When microinjected into the SCN, NMDA induces a rapid induction of per1 gene expression (Asai et al., 2001) and light-like phase shifts in animal behavioral activity (Mintz et al, 1997; Novak et al., 2002), while light-induced per1 and per2 gene expression as well as Fos protein induction are reduced in animals pretreated with NMDA receptor antagonists (Abe et al., 1991; Moriya et al., 2000).

Lastly, NMDA receptor antagonists also block the phase-shifting effects of light (Colwell et al., 1991; Colwell et al., 1992), pointing to a functional role of glutamate-NMDA receptor interactions that modulate neuronal clock activity and behavioral rhythms.

The role of photic stimuli on the regulation of VIP and GRP release from the SCN was examined in the present study through the use of reverse microdialysis of the glutamate receptor agonist, NMDA, and light pulses. During and following 1 hr of

NMDA drug stimulation in early night, VIP release from the SCN significantly increased compared to both vehicle control levels and pre-treatment baseline levels. Release remained increased until near-mid night. This concurs with previous studies examining the effects of NMDA on the circadian clock, where 1 hr of NMDA applied to the SCN hypothalamic slice induced a decrease in VIP-like immunoreactivity (thus indicating increased VIP release) in the SCN but not other brain areas (Shibata et al., 1994a). It was shown that NMDA (Moriya et al., 2000) and VIP (Nielson et al., 2002) induce per1 and per2 gene expression during the night. If VIP is stimulated by NMDA, as suggested by the present results, then this provides additional evidence for the role of VIP in the photic gene induction pathway and activity shifts.

GRP release was transiently but significantly increased over baseline and vehicle control levels by NMDA reverse dialysis before rapidly dropping to vehicle control levels following the conclusion of the drug pulse. Data suggests that GRP-mediated phase delays by microinjection in early night are dependent on NMDA receptors, as these delays were blocked by co-administration with AP5, an NMDA receptor antagonist

(Kallingal and Mintz, 2006). This is in accord with the results of the present study in which a 1 hr reverse-microdialysis pulse of NMDA into the SCN region significantly increased GRP release. This, along with the differing 24 hr LD and DD release profiles, convincingly implies that GRP activity in the SCN is influenced by the environmental light cycle.

Summary

In total, these experiments represent the first to elucidate the timing of release of

NPY, VIP, and GRP in the hamster circadian clock by in vivo microdialysis. Previous experiments demonstrating patterns of neuropeptide release from the SCN over 24 hr often utilize in situ hybridization to examine mRNA expression or immunohistochemistry which evaluates intracellular peptide content. These experiments provide valuable information regarding the timing of activity of SCN neurons, though the temporal resolution may be somewhat inadequate, with evaluations often occurring every 4-6 hr.

Several studies have been performed using in vitro hypothalamic SCN slice preparations to collect and quantify neuropeptide release; however, this method involves isolation of the SCN and thus, the loss important neuronal inputs that regulate activity and timing of

the clock under normal conditions. The experiments performed here examine the real- time in vivo release of neuropeptides in a living animal model, with hourly collections that represent a temporal resolution much greater than those obtained in in situ hybridization or immunohistochemistry experiments. Microdialysis also allows for the detection of neuropeptide release in the undisturbed and intact SCN, under both normal lighting conditions and constant darkness conditions. Lastly, reverse microdialysis perfusions permit the administration of various drugs directly into the SCN while simultaneously quantifying changes in neuropeptide release due to those experimental manipulations. In conclusion, microdialysis represents a powerful tool to detect neuropeptide release under both normal and experimental conditions in a living animal model, thus allowing for improved overall understanding of the functional SCN circadian clock.

Future directions

The present microdialysis experiments reveal the neurophysiological timing by which the SCN functions through neuropeptide release. These results provide the first quantified measurement of in vivo NPY, VIP, and GRP release from the SCN. These experiments also confirmed the synaptic release of these neuropeptides and examined the effects of photic and nonphotic stimuli input on neuropeptide release. Many new questions arise regarding how the specific timing of release of these neuropeptides contributes to the overall behavior of an organism. It will be important to expand upon these results to evaluate what role, if any, these precise rhythmic patterns of neuropeptide

release play in generating and maintaining normal circadian rhythms in mammals.

Administering to hamsters housed in a normal LD cycle a specific neuropeptide receptor blocker immediately prior to the daily peaks in neuropeptide release, while simultaneously monitoring behavior, could potentially isolate the important contributions each neuropeptide makes in producing rhythmic behavior. Conversely, administering to hamsters housed in DD a daily microinjected bolus of a neuropeptide into the SCN at the time of peak release under LD could possibly induce behavioral patterns indicative of an

LD cycle, rather than the free-running patterns seen with a DD cycle. Experiments such as these would further our understanding of how each neuropeptide, with maximal release occurring at specific times of the day, contributes to the formation of rhythmic behavior and the maintenance of daily circadian rhythms in activity.

CHAPTER V

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