Phase Regulation of the Scn Circadian Clock: Serotonergic and Neuropeptidergic Mechanisms

PHASE REGULATION OF THE SCN CIRCADIAN CLOCK: SEROTONERGIC AND NEUROPEPTIDERGIC MECHANISMS

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

Gagandeep Kaur

December 2009

Dissertation written by Gagandeep Kaur

B.V.Sc.&A.H., Punjab Agricultural University, 2005

Ph.D., Kent State University, 2009

Approved by

Dr. J.David Glass , Chair, Doctoral Dissertation Committee Dr. Eric M. Mintz , Member, Doctoral Dissertation Committee Dr. Sean L. Veney , Member, Doctoral Dissertation Committee Dr. Mary Ann Raghanti , Member, Doctoral Dissertation Committee Dr, Stephen B. Fountain , Member, Doctoral Dissertation Committee

Accepted by

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

TABLE OF CONTENTS

TITLE PAGE……….……………………………………………………………………...i

APPROVAL PAGE…….………………………………………………………………... ii

TABLE OF CONTENTS……………………………….………………………………. iii

LIST OF ABBREVIATIONS……….……………………………………………………iv

LIST OF FIGURES……………………………………………………………………...vii

ACKNOWLEDGEMENTS……………………………………………………………...ix

DEDICATION……….…………………………………………………………………...xi

INTRODUCTION………………………………………………………………………...1

SIGNIFICANCE AND SPECIFIC AIMS……………………………………………….32

MATERIALS AND METHODS………………………………………………………...38

RESULTS…...... 50

DISCUSSION……………………………………………………………………………67

REFERENCES……………………………...... 91

iii

LIST OF ABBREVIATIONS

5-HIAA…………………………………………………...... ….5-hydroxyindoleacetic acid

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

8-OH-DPAT………………………………………8-hydroxy-2-di-m-propylamino tetralin

ACSF………………………………………………………...artificial cerebral spinal fluid

ACTH………………………………………………….…….adrenocorticotropic hormone

AVP…………………………………………………………………...arginine vasopressin

BMAL1 …brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 cAMP………………………………………………..3,5-cyclic adenosine monophosphate

CRE ……………………………………………..cAMP response element binding protein

CRH………………………………………………………corticotropin releasing hormone

CSF……………………………………………………………………..cerebrospinal fluid

CT……………………………………………………………………………circadian time

DD……………………………………………………………………….constant darkness

DRN…………………………………………………………………..dorsal raphe nucleus

GABA…………………………………………………………………γ-aminobutyric acid

GHT……………………………………………………………geniculohypothalamic tract

GRP…………………………………………………………….…gastrin releasing peptide

IGL…………………………………………………………………..intergeniculate leaflet i.p……………………………………………………………………………intraperitoneal

LD………………………………………………………………………………...light:dark

LL…………………………………………………………………………….constant light

LLb…………………………………………………………………...…brief constant light

MRN………………………………………………………………....median raphe nucleus

NMDA……………………………………………………………….N-methy-D-aspartate

NPY………………………………………………………………………..neuropeptide Y

PACAP……………………………………..pituitary adenylate cyclase activating peptide

PC ……………………………………....polycarbonate-polyether copolymeric membrane

PCPA……………………………………………………………..parachlorophenylalanine

PK2 …………………………………………………………………...... prokineticin 2

PRC…………………………………………………………………..phase response curve

PVN…………………………………………………………….….paraventricular nucleus

RIA...…………………………………………………………………..radioimmunoassay

RHT………………………………………………………………retinohypothalamic tract

SCN…………………………………………………………….…suprachiasmatic nucleus

SERT……………………………...... serotonin transporter protein

SON………………………………………………………………….….supraoptic nucleus

SSRI…………………………………………………selective serotonin reuptake inhibitor

VIP……………………………………………………….vasoactive intestinal polypeptide

V1a ………………………………………………………………..vasopressin receptor 1a

V1b ………………………………………………………………..vasopressin receptor 1b

V2 …………………………………………………………………..vasopressin receptor 2

ZT……………………………………………………………………………zeitgeber

LIST OF FIGURES

Figure 1 ...... 4

Figure 2 ...... 5

Figure 3 ...... 6

Figure 4 ...... 8

Figure 5 ...... 11

Figure 6 ...... 22

Figure 7 ...... 41

Figure 8 ...... 43

Figure 9 ...... 48

Figure 10 ...... 51

Figure 11 ...... 52

Figure 12 ...... 54

Figure 13 ...... 56

Figure 14 ...... 57

Figure 15 ...... 58

Figure 16 ...... 59

Figure 17 ...... 61

Figure 18 ...... 62

Figure 19 ...... 63

vii

Figure 20 ...... 65

Figure 21 ...... 66

viii

ACKNOWLEDGEMENTS

First of all, I would like to thank my doctoral advisor Dr. J. David Glass for his

guidance and support. I feel honored to have been mentored by a great scientist like him.

Science has ups and downs, but his determination, consistently positive attitude and excellent writing skills are lessons to be followed. Not to forget, his time devoted towards improving my writing skills will serve as a strong foundation in my future career. My sincere gratitude to Dr. Glass for believing in me as a graduate student.

Next, I would like to thank the members of my advisory committee, Dr. Eric Mintz, Dr.

Sean Veney and Dr. Maryann Raghanti, for their valuable time and guidance. Special thanks to Dr. Mintz for answering all my important as well as trivial queries instantly and making complicated things simpler.

I would like to thank all the Glass lab members, Jessie Francl, Jessie Guinn, Marc

Depaul, Steve Hammer, Allison Brager and Christina Ruby for being wonderful friends and for all the fun and support. I treasure the time we spent together in lab and outside.

Sharing a project with Jessie Francl was a great experience. I am thankful to Amelie

Cornill for her help with animal handling during my early days in the lab and to Marc

Depaul, for his great help with computer programming and softwares.

A special thanks to Dr. Douglas Kline for his encouragement and direction to bring out the best in me as a teacher. Additionally thanks to Pat Williams and Donna Warner for their help with registering classes and getting official paperwork done.

Finally, a special thanks to my soul mate and husband Raja Thind, for his love, support

and confidence in me. For always putting me before himself in career and comfort.

Throughout these four years, there were ups and downs, but I have had so much love and

support from him, that this journey always seemed smooth. And thanks to my elder sister

Sukhdeep and brother Japjit for their unconditional love and support.

DEDICATION

I dedicate this dissertation to my wonderful, loving and caring parents, Mr. and Mrs.

Iqbal Singh. To their hard work, sacrifices, blessings and never ending support. They

have and will always be my strength and inspiration. Their encouragement and belief in me had made all this possible, it is their hard work that will shine as a gem in my career.

INTRODUCTION

The Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the master

circadian clock in mammals (Inouye and Kawamura, 1979; Klein et al., 1991; Stephan

and Zucker, 1972). It acts as body’s endogenous pacemaker and generates nearly 24 hour

(i.e. circadian) rhythms of physiologic, metabolic and electrical activities. Several studies

provide credible evidence for the SCN being body’s internal clock. The pacemaker

property of the SCN is demonstrated by persistence of circadian oscillations with a period

of approximately 24 hours in hypothalamic slice cultures containing the SCN (Groos and

Hendriks, 1982; Inouye and Kawamura, 1979; Shibata et al., 1982). This spontaneous

firing rate of SCN neurons is higher during the subjective light phase (Gillette and

Reppert, 1987; Groos and Hendriks, 1982; Mihai et al., 1994; Pennartz et al., 2002;

Shibata et al., 1982). The SCN is capable of spontaneously changing its rate of

metabolism since the rhythmic uptake of 2-deoxy glucose increases during subjective

daytime in the SCN slices (Newman and Hospod, 1986).

Following lesions of SCN efferents, several brain regions which previously exhibit

neural rhythmicity lose such characteristics while the SCN itself displays persistent

circadian neuronal rhythms for prolonged periods (Inouye and Kawamura, 1979).

Isolation of the SCN region of the hypothalamus results in loss of locomotor and sleep

wake rhythms in rats (Inouye and Kawamura, 1979). In mice, circadian rhythms of wheel

running activity are abolished by bilateral ablation of the SCN but not by unilateral

ablation (Schwartz and Zimmerman, 1991). Bilateral lesions of the SCN also disrupt

circadian changes in the plasma levels of hormones such as corticosterone (Abe et al.,

1979; Moore and Eichler, 1972), thyrotropin (Abe et al., 1979) and adrenal corticotropin

hormone (ACTH) rhythms in rat (Cascio et al., 1987). Complete destruction of the SCN

also eliminates the body temperature rhythm (Abe et al., 1979). These studies implicate

the SCN as an autonomous oscillator which directs expression of body’s physiological,

metabolical and behavioral rhythms.

The tau mutation at a single autosomal locus in Syrian hamsters causes shortening of

the free running period of circadian rhythms with the homozygous mutants exhibiting a

free running period of about 20 hours and heterozygotes of about 22 hours compared to

24 hours in wild types (Ralph and Menaker, 1988). Transplantation of the SCN from

either tau mutant or wild type hamsters into lesioned hosts of opposite genotype, results

in restoration of circadian rhythms in the lesioned animals with the period characteristic

of the donor genotype (Ralph et al., 1990). This shows that the tau mutation, which

affects the free running period of circadian rhythms, acts at the level of the SCN.

Timing of the master clock is regulated by a variety of endogenous and exogenous signals, including photic and nonphotic inputs. To entrain the SCN to these external

zeitgebers (entraining agent), environmental information is relayed through different

pathways. The SCN responds to external zeitgebers in a phase dependant manner. If a

stimulus causes activity to begin before ZT 12, the shift in the phase of the SCN is called

an advance (Figure 1). On the other hand, activity onset later than ZT 12 following a stimulus is referred to as phase delay shift (Figure 2).

Photic phase shifting:

Although the SCN can be entrained by many environmental factors, the most important entraining stimulus is the daily environmental light dark cycle (Morin, 1994; Morin and

Allen, 2006; Pittendrigh, 1975). The entrainment of the SCN by daily photoperiod guarantees that the organism maintains an appropriate time-bound relationship to its environment (Pittendrigh, 1975). Photic stimuli reach the SCN through direct and indirect

neural pathways. The SCN is entrained to the environmental light dark cycle by photic

inputs received directly from the retina via the retinohypothalamic tract (RHT; Figure 3;

Moore and Eichler, 1972; Johnson et al., 1988; Pickard, 1982), with glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as major neurotransmitters of this pathway (Hannibal, 2002). The RHT is essential for photic entrainment; its

destruction leads to desynchrony with the environmental light dark cycle (Johnson et al.,

1988). Photic inputs also reach the SCN indirectly via the geniculohypothalamic tract

(GHT) of the intergeniculate leaflet (IGL; Johnson et al., 1989).

Daan and Pittendrigh (1976) showed that short duration light pulses induce phase

dependant shifts in the wheel running rhythms of nocturnal rodents. In their study,

animals housed in constant darkness (DD) were exposed to brief light pulses. Phase

delays were observed in response to light pulses during the early subjective night, phase

advances during the late subjective night and no effect was seen during the subjective

day. Similarly in a study by Takahashi et al (1984), hamsters kept in constant dark were

DAYS *

00 02 04 06 08 10 12 14 16 18 20 22 24 HOURS

Figure 1: Schematic demonstration of phase advance in the behavioral activity of a

Syrian hamster housed under constant conditions in response to a nonphotic stimulus.

Each horizontal row represents one circadian day. The dark blocks represent behavioral activity. Asterisk indicates the time of presentation of the stimulus.

* DAYS

00 02 04 06 08 10 12 14 16 18 20 22 24

HOURS

Figure 2: Schematic demonstration of phase delay in the behavioral activity of a Syrian hamster housed under constant conditions in response to a nonphotic stimulus. Each horizontal row represents one circadian day. The dark blocks represent behavioral activity. Asterisk indicates the time of presentation of the stimulus.

Figure 3: Diagrammatic representation of photic and nonphotic inputs to the SCN. Photic information is relayed to the SCN from the retinal ganglion cells via the retinohypothalamic tract (RHT) utilizing glutamate and PACAP as neurotransmitters.

Nonphotic information reaches the SCN directly from the raphe nuclei using serotonin

(5-HT) as neurotransmitter and indirectly via the geniculohypothalamic tract (GHT) of the intergeniculate leaflet (IGL) with neuropeptide Y (NPY) as transmitter. MRN,

Median raphe nucleus; DRN, dorsal raphe nucleus.

exposed to 1 hour duration of light pulses. The phase shift responses of the free running

rhythm were similar to that obtained by Daan and Pittendrigh (1976). The phase response

curve (PRC; plot of phase shift vs. the time of stimulus) to photic stimuli based on the

study of Takahashi et al (1984) is depicted in Figure 4. In the tau mutant hamsters, PRC

to light pulse is more variable compared to the wild types. Standard light pulses can

induce large phase shifts of nearly 12 hours in tau mutant hamsters exposed to DD for

prolonged periods (Scarbrough and Turek, 1996; Shimomura and Menaker, 1994). After

49 cycles in DD wild type hamsters showed phase delays to light pulse at CT15, where as

tau mutant hamsters showed extremely large shifts which were difficult to categorize as

advances or delays (Scarbrough and Turek, 1996).

Expression of the proto-oncogene c-fos is a significant indicator of cellular activation in

SCN neurons (Rusak et al., 1990). Light exposure at specific times, which alters the circadian phase of activity rhythms, causes c-fos expression in the retinirecipient SCN cells (Rea, 1989; Rusak et al., 1990; Selim et al., 1993). Apart from c-fos, light pulses also induce expression of a large array of immediate early genes, most of which have unknown function in the SCN ((Aronin and Schwartz, 1991, Potterfield and Mintz,

2007). Light exposure has no phase shifting effect during the day but during night it leads to phase shifts along with the induction of clock genes, per1 and per2 (Yan and Silver,

2002).

Nonphotic phase shifting:

Nonphotic inputs can affect and entrain the SCN like photic stimuli. Non-photic influences in the form of behavioral manipulations like cage changes, social interactions

1 ADVANCE

PHASE SHIFT (HOURS) SHIFT PHASE -1 DELAY

-2 06121824

CIRCADIAN TIME (HOURS)

Figure 4: Schematic representation of photic phase response curve to 1 hour saturating

light pulses in hamsters housed under constant dark (DD). The plot represents the

magnitude of phase shifts in response to light pulses at different times. Modified from

Takahashi et al, (1984).

(Mrosovsky, 1988), novel wheel exposure (Reebs and Mrosovsky, 1989), sleep

deprivation or pharmacological interventions (like serotonergic agonists,

benzodiazepenes) can reset the SCN clock with same effectiveness as the photic cues

(Mistlberger et al., 2000). Nonphotic cues phase shift the SCN during the day or light

period and are associated with a decrease in per1 and per2 expression and they also

attenuate the photic resetting during the night or dark period (Mrosovsky, 1996).

Nonphotic stimuli such as injections of saline (Mead et al., 1992), triazolam (Cutrera et

al., 1993) or forced running (Janik and Mrosovsky, 1992) phase advance the circadian

clock without the expression of immediate early gene c-fos in the SCN which is

expressed during photic phase shifts.

Nonphotic inputs reach the SCN either via the GHT of the IGL utilizing neuropeptide

Y (NPY; Albers and Ferris, 1984; Biello et al., 1994; Marchant et al., 1997) and γ-amino

butyric acid (GABA) as neurotransmitters or via the midbrain raphe nuclei utilizing

serotonin (5-HT) as neurotransmitter (Figure 3; Meyer-Bernstein and Morin, 1996;

Mistlberger et al., 2000). In hamsters, the SCN receives a dense 5-HT innervation from

the median raphe nucleus (MRN) and this projection helps to maintain circadian

rhythmicity under constant light (LL) and light-dark conditions (Meyer-Bernstein and

Morin, 1996).

Nonphotic stimuli also shift the circadian rhythms in a phase dependant manner,

although different from photic stimuli. Mrosovsky et al (1992) showed that nonphotic

stimuli in the form of wheel running phase dependently shift the circadian rhythms with

large phase advances seen during the mid subjective day, smaller phase delays seen

during the late subjective night and early subjective day, and minimal effects during the rest of the subjective night (Figure 5; Mrosovsky et al., 1992). In tau mutant hamsters, the amplitude of PRC is also increased for the nonphotic stimuli, with peak advances occurring at earlier regions of the circadian cycle compared to the wild type (Mrosovsky et al., 1992).

Serotonin (5-HT)

5-HT is an important neurotransmitter with diverse roles in the central nervous system. 5-

HT afferents from the midbrain raphe nuclei innervate various areas of forebrain such as thalamus, hypothalamus, amygdala, caudate putamen, parieto-temporal cortex, hippocampus and the SCN (Azmitia and Segal, 1978). The midbrain MRN sends major serotonergic projections to the SCN, which terminate primarily in its retinorecipient region, where-as the dorsal raphe nucleus (DRN) projections innervate the IGL (Meyer-

Bernstein and Morin, 1996). In Syrian hamster, 5-HT immunoreactivity is restricted to the ventromedial part of the SCN (Card and Moore, 1984) which also receives robust

RHT (Johnson et al., 1988) and GHT (Johnson et al., 1989) innervations. The MRN as well as the DRN contribute to regulation of 5-HT release in the SCN since electrical stimulation of either of these nuclei increases 5-HT output from the SCN (Dudley et al.,

1999). Systemic treatment with the 5-HT antagonist metergoline causes a decrease in

DRN induced 5-HT increase but has minimal effect on the MRN induced 5-HT release

(Dudley et al., 1999). Microinjections of the 5-HT1A agonist and antagonist into the MRN had a pronounced inhibitory and stimulatory effect on the SCN 5-HT release respectively. Both these treatments produced a much lesser effect in the DRN, signifying

1 ADVANCE

0 PHASE SHIFT (HOURS) SHIFT PHASE DELAY

-1 4 8 12 16 20 24

CIRCADIAN TIME (HOURS)

Figure 5: Schematic representation of the phase response curve to non-photic stimuli for

hamsters housed in constant dark (DD). The plot represents the mean phase shifts to 3

hour bouts of novelty induced wheel running. Modified from Mrosovsky et al (1992).

that the somatodendrtic 5-HT1A autoreceptors of the MRN play a larger role in regulating

the SCN 5-HT output (Dudley et al., 1999). Serotonergic projections from the MRN

regulate the timing of activity onset in the SCN, and only a few 5-HT fibers in the SCN

are sufficient to maintain a normal entrainment pattern (Meyer-Bernstein et al., 1997).

5-HT is synthesized from the dietary amino acid tryptophan. Tryptophan hydroxylase is

the rate limiting enzyme in 5-HT synthesis (Lovenberg et al., 1967) and is limited to

neurons specialized for 5-HT production (Weissmann et al., 1987). The amount and

activity of tryptophan hydroxylase determines the amount of 5-HT synthesized in the

tissue. The expression of tryptophan hydroxylase in the 5-HT rich MRN and DRN is

circadian in pattern and is under the control of circadian clock by both endocrine

(corticosterone from adrenals) and behavioral (locomotor activity) inputs (Malek et al.,

2007). The MRN 5-HT neuronal afferents to the SCN display a circadian variation in tryptophan hydroxylase content in their perikarya and axon terminals, with highest levels in perikarya achieved during the early daytime and highest levels in axon terminals achieved before the onset of dark period. Similarly in the SCN, tryptophan hydroxylase content exhibits higher values before the onset of the dark period (Barassin et al., 2002).

These tryptophan hydroxylase levels in the axon terminals coincide with peak levels of the SCN 5-HT release (Barassin et al., 2002).

In vivo studies have shown that the 5-HT neural release from the hamster SCN is circadian in pattern with peak levels obtained at the light to dark transition and levels falling to basal values during remainder of the dark phase (Dudley et al., 1998). This 5-

HT rhythm persists under DD conditions confirming its circadian nature (Dudley et al.,

1998). Microdialysis studies in rats have shown similar results, with peak SCN 5-HT

release obtained at the beginning of subjective night (Barassin et al., 2002). A similar pattern was seen in the release of 5-HT metabolite 5-hydroxyindole-acetic acid (5-

HIAA), but with a peak during the dark period 2-3 hours after the 5-HT peak (Barassin et

al., 2002; Glass et al., 1992). In the diurnal rodent Arvicanthis ansorgei, the SCN 5-HT content shows high levels during the daytime and low levels during night (Cuesta et al.,

2008). The SCN 5-HT release profiles in the nocturnal and diurnal rodents’ verify that the activity of 5-HT neurons is closely related to the arousal levels of the animal (Jacobs and Fornal, 1999). It is further supported by a study where wheel running during midday increased the SCN 5-HT levels (Dudley et al., 1998). There is a strong relationship between the behavioral state and activity of the 5-HT system (Mistlberger et al., 2000).

5-HT is closely associated with the SCN clock functioning, as shown by several studies. P-chlorophenylalanine (PCPA), a reversible inhibitor of 5-HT synthesis, suppresses circadian locomotor activity rhythms, plasma corticosterone and ACTH rhythms for several days following treatment (Honma et al., 1979). Phase shifting effects of the short acting benzodiazepene triazolam are diminished by neurochemical lesions of

5-HT afferents to the SCN by the specific neurotoxin 5,7-dihydroxytryptamine (Cutrera et al., 1994) and in the forebrain by p-chloroamphetamine (Penev et al., 1995). In rats, electrolytic raphe lesions disrupt circadian locomotor rhythms (Levine et al., 1986).

Similarly, in Syrian hamsters, lesions of the MRN that eliminate 5-HT fibers in the SCN, cause changes in phase of the activity cycle (Meyer-Bernstein and Morin, 1996).

Several studies point to a direct role of 5-HT in SCN function. Application of 5-HT to

SCN neurons is largely inhibitory to their neuronal activity (Nishino and Koizumi, 1977).

The 5-HT agonist quipazine induces phase advances in SCN slice culture along with a decrease in fos protein expression (Prosser et al., 1994). The same agent when applied during the subjective night inhibits light induced phase shifts of locomotor activity (Rea

et al., 1994) and c-fos expression (Glass et al., 1994; Glass et al., 1995; Selim et al.,

1993). In vitro studies have shown that 5-HT and its agonists phase advance the

spontaneous neuronal activity of the SCN neurons in slice preparations during mid

subjective day and phase delay during the subjective night (Prosser et al., 1990; Prosser et

al., 1993; Prosser, 2003; Shibata et al., 1992). These effects can be blocked by the 5-HT

antagonist metergoline (Prosser et al., 1993; Prosser, 2003). The phase advances

observed in these studies are similar in time and pattern to those observed in vivo in

response to behavioral stimuli.

In vitro studies have shown that the circadian clock (in mice and rats) is readily phase

shifted by application of 5-HT and its agonist (±)-2-diropyl-amino-8-hydroxyl-1,2,3,4-

tetrahydronapthalene (8-OH-DPAT). But in vivo studies (in hamster and mice) where 5-

HT agonists are applied directly to the SCN show inconsistent results. One possible

explanation for these inconsistencies is that the SCN isolated in vitro has reduced

endogenous 5-HT signaling, thus increasing its sensitivity to any later 5-HT stimulation

(Prosser et al., 2006). In support of this explanation, it was seen that pre-treatment of

mouse SCN brain slices with 5-HT, 8-OH-DPAT, L-tryptophan or fluoxetine, blocked

any phase shifts induced by subsequent 5-HT or 8-OH-DPAT treatment. Thus the amount

of 5-HT signaling in the SCN can alter 5-HT receptor expression and therefore, any phase shifting responses to the serotonergic stimuli. Also since the brain slice preparations are de-afferented and bathed in solutions different from the normal extracellular fluid, their physical properties may be affected (Prosser et al., 2006). The change in the SCN afferent signaling is important because injections of 8-OH-DPAT into the SCN of animals pretreated with PCPA, to block 5-HT input to the SCN induces significantly larger shifts (Ehlen et al., 2001). On the other hand, perfusing 5-HT into the hamster SCN diminishes the 2 day constant light potentiated phase shifts produced by 8-

OH-DPAT (Knoch et al., 2004).

Role in nonphotic phase resetting

Various lines of evidence suggest an important role for 5-HT in mediating nonphotic phase shifting of the SCN. The pattern of activity of 5-HT neurons is closely related to the behavioral state (Jacobs and Fornal, 1999). In vivo 5-HT release in the hamster

(Dudley et al., 1998) and rat SCN (Barassin et al., 2002) peaks at the beginning of subjective night, which is also the point of activity onset. Wheel running has a time dependant effect on 5-HT release from the hamster SCN (Dudley et al., 1998). Thus changing activity of the 5-HT neurons and its release in correlation with animal’s activity cycle could provide a direct behavioral feedback to the SCN (Dudley et al., 1999). As reviewed by Mistlberger et al (2000), if 5-HT release in the SCN is considered important for nonphotic phase shifting, then procedural manipulations that reduce 5-HT levels in the SCN should decrease the phase shift responses to nonphotic stimuli. This is complemented by a study in which 5-HT depletion in brain by p-chloramphetamine

abolishes all the phase shifts induced by short acting benzodiazepine triazolam (Penev et al., 1995). Another means to evaluate role of 5-HT in nonphotic phase shifting is by studying drugs that block or activate 5-HT receptors. 5-HT agonists (including 8-OH-

DPAT) phase shift spontaneous neuronal activity of the SCN neurons in slice preparations (Prosser et al., 1990; Prosser et al., 1993; Shibata et al., 1992). Systemic administration of 8-OH-DPAT in hamsters phase advances the SCN, with maximal affects during the mid-day (Bobrzynska et al., 1996; Tominaga et al., 1992). Phase advancing effects of the 8-OH-DPAT are also seen following its bilateral microinjections into the hamster SCN (Challet et al., 1998) or into the third ventricle (Edgar et al., 1993).

The phase advances observed in these studies are similar in time pattern to those observed in vivo in response to behavioral stimuli. Overall activation of the serotonergic system has a nonphotic effect on the rat SCN, with the receptors 5-HT1A and 5-HT7 playing major role (Cuesta et al., 2009).

Based on some studies, the role of 5-HT in nonphotic phase shifting is considered controversial. Studies have shown that raphe lesions (Meyer-Bernstein and Morin, 1998), treatment with 5-HT antagonists (Antle et al., 1998) and depletion of 5-HT in the SCN

(Bobrzynska et al., 1996) have no inhibitory effect on activity induced phase shifts.

Moreover direct injections of 8-OH-DPAT into the SCN have little or no phase shifting effects (Challet et al., 1998; Ehlen et al., 2001; Mintz et al., 1997). Further, electrical stimulation of the midbrain raphe nucleus that enhances 5-HT release in the SCN

(Dudley et al., 1999) causes phase shifts comparable to those caused by the behavioral activation (Glass et al., 2000; Meyer-Bernstein and Morin, 1999). But acute

administration of serotonergic reuptake inhibitors like clomipramine and fluoxetine

which also enhance the SCN 5-HT, have minimal phase shifting effect in vivo (Klemfuss

and Kripke, 1994; Yannielli et al., 1998) and in vitro (Sprouse et al., 2006). Despite the

fact that reuptake inhibitors increase extracellular 5-HT levels, they do not attenuate

photic phase resetting (Gannon and Millan, 2007). Based on these observations, it can be

said that 5-HT may play a modulatory role in the SCN functioning as opposed to an

essential role.

Role in photic phase resetting

Serotonin has been implicated in the modulation of photic signaling in the SCN as well.

Though 5-HT is known to play a role in the nonphotic phase shifting, some of its

receptors (5-HT3, 5-HT2C) are shown to have photic phase shifting like effects in the rat

SCN (Cuesta et al., 2009). 5-HT agonists inhibit light induced phase shifts of the

locomotor activity (Rea et al., 1994), inhibit light induced c-fos immunoreactivity (Glass

et al., 1994; Glass et al., 1995; Selim et al., 1993) and inhibit electrical activity of light

responsive SCN cells (Ying and Rusak, 1997). 5-HT agonists dose dependently inhibit

the SCN field potentials evoked by electrical stimulation of the optic nerve (Rea et al.,

1994). Treatment with the 5-HT precursor L-tryptophan increases extracellular levels of

5-HT in the SCN and in turn attenuates fos protein immunoreactivity and light induced

phase advances in the locomotor rhythms in hamsters (Glass et al., 1995). On the other

hand, 5-HT antagonist administration inhibits the L-tryptophan action on c-fos expression

(Glass et al., 1995) and also potentiates light induced behavioral phase shifts (Rea et al.,

1994). During the night time, serotonergic agonists and reuptake inhibitors decrease the

light induced phase shifts of the SCN (Gannon and Millan, 2007; Rea et al., 1994; Weber

et al., 1998). Thus overall, 5-HT has an inhibitory effect on photic phase shifting of the

SCN.

(±)-2-diropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT)

Non photic phase resetting of the mammalian circadian rhythms is mediated in part by

5-HT action on the SCN (Mistlberger et al., 2000). In vitro studies show that 5-HT

agonists produce phase-advances of the SCN at midday (Prosser et al., 1990). 8-OH-

DPAT, a 5-HT1A,7 agonist, produces phase-advances of the SCN neuronal firing in vitro

(Prosser et al., 1993; Shibata et al., 1992) and in vivo in rats (Edgar et al., 1993). These

phase advances are not abolished by activity restraint (Bobrzynska et al., 1996). Reverse

microdialysis perfusion of the SCN with 8-OH-DPAT or 5-HT at ZT 0 phase-advanced

the hamster circadian rhythms by acting through 5-HT7 receptor subtype (Ehlen et al.,

2001). In vitro studies in rat have also shown that the phase-advances induced by 8-OH-

DPAT are due to its action on 5-HT7 receptor using cAMP as second messenger

(Lovenberg et al., 1993; Sprouse et al., 2004b) which activates protein kinase A which in

turn opens calcium dependant potassium channels (Prosser, 2000).

Phase advances by systemic or local administration of 8-OH-DPAT are accompanied

with a decrease in the SCN per1 and per2 expressions (Ehlen et al., 2001; Tominaga et

al., 1992; Horikawa et al., 2000). Pre-exposure to 2 days of constant light significantly

enhances phase advances by the intra SCN 8-OH-DPAT at ZT 0 but not at ZT 6 or 18,

implicating that at the later times targets outside the SCN may be responsible for the

phase shifts (Figure 6; Knoch et al., 2004). Systemically administered 8-OH-DPAT can

also act indirectly on the SCN through the midbrain raphe nuclei with its effects mediated

by GABAergic transmission (Mintz et al., 1997). In a microdialysis study by Dudley et al

(1998), it was seen that systemic 8-OH-DPAT decreased the SCN 5-HT output. This

could be due to the direct action of 8-OH-DPAT on the somatodendritic 5-HT1A autoreceptors (Dudley et al., 1998). These studies show the importance of 5 8-OH-DPAT as an important tool to study the effect of 5-HT on different features of the master clock.

L-tryptophan and Selective Serotonin Reuptake Inhibitors

L-tryptophan is the 5-HT precursor molecule (Siegel, 2006). Systemic administration of

L-tryptophan significantly increases extracellular 5-HT release in the SCN and attenuates

light pulse induced phase advances in Syrian hamsters (Glass et al., 1995). L-tryptophan

produces concentration dependant phase advances of the SCN neuronal firing which are

enhanced when combined with fluoxetine (Sprouse et al., 2006). The L-tryptophan

induced phase shifts of the SCN clock in vitro can be inhibited by 5-HT inhibitor PCPA,

indicating that the phase shifts are a result of L-tryptophan being converted into 5-HT

(Sprouse et al., 2004a). The inhibitory effects of L-tryptophan pre-treatment on 8-OH-

DPAT induced phase shifts, are also prevented by PCPA, further indicating that its

conversion to 5-HT is required for its action (Prosser et al., 2006).

5-HT is removed from the synaptic cleft by reuptake action of the serotonin transporter

protein (SERT) which is found in the medial and ventral regions of SCN (Amir et al.,

1998; Legutko and Gannon, 2001), a pattern very similar to that of the 5-HT afferents to

the SCN. Selective serotonin reuptake inhibitors (SSRI’s) like citalopram, fluoxetine and

clomipramine act by blocking SERT and increasing the available 5-HT at synapses.

SSRI’s are widely used as antidepressants but their effects on circadian activity

parameters have been studied little till date (Gannon and Millan, 2007). Local SCN perfusion of the 5-HT reuptake blocker citalopram increases the SCN 5-HT release

(Dudley et al., 1998). Clomipramine phase dependently affects hamster free running activity rhythms with phase advances during the early and mid day. Citalopram and fluoxetine when administered late in night, inhibit light induced phase advances hamster wheel running rhythms (Gannon and Millan, 2007). However chronic oral administration of fluoxetine does not affect the free wheel-running rhythms in hamsters (Klemfuss and

Kripke, 1994) or rats (Wollnik, 1992). Pre-treatment with clomipramine also inhibits phase advances in response to light pulses (Yannielli et al., 1998).

In vitro application of fluoxetine to SCN slice culture does not produce phase shifts, but when combined with L-tryptophan, robust phase advances in the SCN neuronal firing are

seen (Sprouse et al., 2006). Keeping in mind the circadian pattern of 5-HT release, during

the light period when the levels of 5-HT are low in the SCN, fluoxetine injections are

shown to have some effect on the clock. But during the night time, when the levels of 5-

HT are high in the SCN, fluoxetine exerts no effect on the circadian clock (Cuesta et al.,

2009). Similarly in the diurnal rodent Arvicanthis ansorgei, fluoxetine injections cause

behavioral phase advances only during the night time, as 5-HT levels are low during the

night and high during the day in these animals (Cuesta et al., 2008). Thus fluoxetine

shows high effect only when 5-HT levels in the SCN are low. A difference in the effects

of fluoxetine following acute and systemic administration has also been observed. In

Syrian hamsters, acute fluoxetine treatment decreases rapid eye movement sleep and

hypothalamic temperature in a dose dependant manner, whereas chronic fluoxetine

treatment only affects the hypothalamic temperature rhythms (Gao et al., 1992). Acute

systemic administration of fluoxetine at CT 22 induces fos immunoreactivity in the

ventrolateral region of the SCN, where as chronic administration of the same resulted in

significant attenuation of the fos expression (Mullins et al., 1999).

Brief Constant Light

Earlier work from our lab has shown that exposure to brief (2 days) constant light (LLb)

can significantly potentiate the magnitude of phase shifts induced by nonphotic stimuli

and pharmacologic treatments specifically, 5-HT agonist 8-OH-DPAT (Knoch et al.,

2004). Exposure to LLb also changes the phase response curve to systemic 8-OH-DPAT,

promoting large phase shifts (~12 h) at early morning and at midday, when otherwise this

agonist normally has no effect (Figure 6; Knoch et al., 2004). LLb even enhances phase-

advances produced by the intra-SCN 8-OH-DPAT (Knoch et al., 2006). Studies in

transgenic mice have shown that exposure to LL desynchronizes the SCN neuronal firing

but does not affect their ability to generate rhythms (Ohta et al., 2005).

The mechanism underlying the potentiating effect of LLb is not known, but could be

related to the daily rhythm of release of 5-HT from the SCN that peaks at the beginning

of the dark phase (Dudley et al., 1998). Exposure to LLb eliminates this night time peak

of the SCN 5-HT release (Knoch et al., 2004), and therefore may hypersensitize the

system to 5-HT or its agonists. Since 5-HT release in the SCN increases with increased

activity such as wheel running (Dudley et al., 1998), periods of low activity should

decrease the amount of 5-HT in the SCN (Prosser et al., 2006). Thus the larger phase

6 ADVANCE 0

-6 DELAY PHASE SHIFT (HOURS) SHIFT PHASE

-12

-18 0 6 12 18 24 30 ZT (HOURS)

Figure 6: Phase response curve for systemic 8-OH-DPAT in hamsters housed under

14:10 LD cycle after 2 day brief constant light (LLb) exposure. Modified from Knoch et al, (2004).

shifts induced by the 8-OH-DPAT following LLb exposure may be due to decreased

activity and hence decreased 5-HT seen during the LLb exposure (Knoch et al., 2004),

subsequently increasing the likelihood of greater phase advances to any serotonergic

stimulation (Prosser et al., 2006). A detailed study was undertaken by Duncan et al

(2005), on possible mechanisms underlying the potentiating effects of LLb. It was observed that short term exposure to LLb did not result in upregulation of the pre- or

postsynaptic 5-HT receptors, since there was no alteration in 5-HT binding sites after LLb exposure (Knoch et al., 2006). It was also seen that LLb suppresses SCN arginine vasopressin (AVP) mRNA expression. As neuronal activity is essential for the transcription of AVP gene (Arima et al., 2002), LLb may diminish SCN neuronal firing

and alter its output signaling. Moreover as the AVP mRNA is localized in the SCN shell

region, this may be the site of action of LLb. Exposure to LLb also causes attenuation of

the circadian pacemaker amplitude (seen as decreased locomotor activity, deceased wheel

running, decreased night time SCN 5-HT release (Knoch et al., 2004), decreased per and

AVP mRNA expression (Duncan et al., 2005), which may be the reason behind the

potentiating effect of LLb because attenuation of the pacemaker amplitude will result in

the clock becoming hyper-responsive to any stimuli, resulting in an exaggerated response

(Winfree, 1970).

Arginine Vasopressin

Arginine vasopressin (AVP) is a nonapeptide primarily synthesized in the

magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) and is

transported via their axons to the posterior pituitary (Brownstein et al., 1980), where it is

released into the blood stream upon stimulation. AVP is also produced by the

parvocellular neurons of the PVN, bed nucleus of stria terminalis, medial amygdala and

the SCN (Buijs and Kalsbeek, 1993). AVP acts through three G-protein coupled

receptors, AVP receptor 1a (V1a), AVP receptor 1b (V1b) and AVP receptor 2 (V2). V1a

and V1b act through phospholipase C where as V2 acts through cAMP as second

messenger system (Jard et al., 1987). Receptors V1a and V1b are expressed throughout

the brain including the SCN(Johnson et al., 1993; Thibonnier et al., 2002; Vaccari et al.,

1998).

AVP is thought to be the major output signaling peptide of the SCN and was first

localized in the SCN of rat by (Vandesande et al., 1975). It is synthesized in the

dorsomedial SCN by a neuronal population comprising about 37% of the total SCN

neurons in rat (Moore et al., 2002). In Syrian hamsters AVP cells are found

predominantly in the dorsomedial parts of the SCN but AVP positive fibers and terminals

are also located in the ventrolateral regions of the SCN (Van der Zee et al., 2002).

Specifically AVP immunoreactive perikarya are present in the dorsomedial aspect of

each nucleus, with highest number in the intermediate two thirds of the rostrocaudal axis.

However the AVP axons form a dense plexus in the dorsomedial part as well as in a

vertical column on the lateral aspect of the nucleus (Card and Moore, 1984). Similarly in

voles AVP positive fibers are present throughout the SCN although AVP immunoreactive

cells are present mainly in the dorsomedial part (Gerkema et al., 1994). AVP

immunoreactive cells are present in the SCN of several other species like the ground

squirrel (Reuss et al., 1989), tree shrew (Sofroniew and Weindl, 1980), common

marmoset (Wang et al., 1997) and cynomolgus monkey (Ichimiya et al., 1988).

The SCN generates a rhythm of AVP content in the cerebrospinal fluid (CSF) in many

species (Jolkkonen et al., 1988; Schwartz and Reppert, 1985; Uhl and Reppert, 1986).

The endogenous pacemaker generated rhythm of AVP in the CSF can be maintained in

rats for at least 10 days in absence of environmental lighting signals (Schwartz et al.,

1983). But upon exposure to long term LL, the CSF AVP rhythm first dampens and then

is disrupted (Schwartz et al., 1983). AVP content (Tominaga et al., 1992) and release

(Kalsbeek et al., 1995) from the rat SCN exhibit distinct diurnal rhythms with peak levels

seen during the early light phase and nadir during the dark phase. AVP is one of the

major neuropeptides of the SCN. It is one of the efferent signals of the SCN to other brain

areas (Buijs, 1996) and plays an important role in amplifying the output of the SCN due

to its excitatory actions (Ingram et al., 1996; Liou and Albers, 1989; Mihai et al., 1994).

Although SCN AVP neurons are not solely responsible for maintaining the SCN neural

activity ( Liou and Albers, 1989; Mihai et al., 1994), as a neurotransmitter AVP may play

a role in the intra-SCN communication along with other neurotransmitters by

synchronizing the oscillations generated by individual SCN neurons (Reghunandanan and

Reghunandanan, 2006).

A variety of studies implicate AVP of the SCN origin in regulation of circadian

rhythmicity of locomotor behavior. V1a knockout mice show attenuated circadian

rhythmicity of the locomotor activities (Li et al., 2009). In Syrian hamsters, SCN lesions

decrease the number of AVP immunoreactive neurons in several forebrain areas and

disrupt circadian wheel running rhythms (Delville et al., 1998). AVP immunoreactivity in the SCN differs with the general locomotor and wheel running rhythmicity, with higher levels seen in non-rhythmic voles (Gerkema et al., 1994). The higher levels represent high protein levels, which means more synthesis or lower rate of release, which may be responsible for lack of rhythmicity, and therefore point towards a role of AVP in regulating the locomotor and or wheel running rhythms in voles (Gerkema et al., 1994).

The SCN cultures from the non-rhythmic voles lack a circadian pattern in AVP release, where as cultures from rhythmic voles exhibit a significant circadian pattern, further strengthening support for a role of AVP in the regulation of circadian locomotor behavior

(Jansen et al., 2000). Since AVP immunoreactivity in the SCN decreases with age in voles as does circadian patterns of wheel running activity (Van der Zee et al., 1999), AVP may play a role in maintaining circadian rhythmicity throughout the lifespan of this species (Caldwell et al., 2008).

Tau mutant hamsters show circadian rhythms in AVP mRNA expression with higher levels during the early subjective day and lower levels during subjective night, even under DD (Scarbrough and Turek, 1996). But the AVP mRNA expression in tau mutants is reduced compared to the wild type hamsters. The decreased AVP mRNA levels in the tau mutant hamsters may be responsible for their different locomotor activity patterns such as decreased total activity, variable activity onsets and fragmentation of their active period (Scarbrough and Turek, 1996). Tau mutants also show lower levels of AVP immunoreactivity, but higher rate of AVP release from the SCN compared to the wild types (Van der Zee et al., 2002). This higher level of release may be a compensatory

mechanism to increase synchronization within the SCN and to increase the strength of

rhythmicity (Van der Zee et al., 2002) which is otherwise weaker in tau mutants. Tau mutant hamsters demonstrate weak coupling amongst individual oscillators of the SCN

clock (Shimomura and Menaker, 1994). Since a change in coupling effects the free

running period as well as phase shift responses of the circadian clock (Pittendrigh and

Daan, 1976), the decreased AVP mRNA levels may be important in this. AVP can play a role in coupling individual SCN oscillators (Scarbrough and Turek, 1996) as AVP

neurons make synapses within the nucleus (Daikoku et al., 1992) as well as across the

midline, thus connecting two nuclei (van den Pol and Tsujimoto, 1985). Also, the

different response of the SCN to a light pulse after prolonged DD in tau mutant and wild type hamsters correlates with differences in the SCN AVP and VIP mRNA levels in the two genotypes (Scarbrough and Turek, 1996).

The naturally occurring AVP mutant Brattleboro rat has a single base deletion in Exon

B of the AVP gene, which results in an inability to process the translated gene to peptide

(Schmale et al., 1984; Schmale and Richter, 1984). AVP deficient Brattleboro rats have been used to study the diverse roles of AVP in circadian rhythms or otherwise. Although

Brattleboro rats show circadian organization of behavior, it is seen with lower amplitude

(Peterson et al., 1980). Brattleboro rats show decreased amplitude of slow wave and paradoxical sleep rhythms (Brown and Nunez, 1989). Thus the hypothalamic AVP may not be essential for generation of sleep-arousal rhythms but it may participate to modulate these rhythms. The presence of rhythmic neuronal activity in the SCN of Brattleboro rats, similar to the heterozygotes, negates role of AVP in generating rhythms of basal activity

(Ingram et al., 1996). Also, since Brattleboro rats have intact rhythms in neuronal activity

of the SCN (Ingram et al., 1996), wheel running (Groblewski et al., 1981; Stoynev and

Nagai, 1996) and drinking behavior (Groblewski et al., 1981), AVP neurons in the SCN

do not seem to play a role in generation of these rhythms; nevertheless a modulatory role

cannot be ruled out.

AVP immunoreactive neurons of the SCN have terminals in the PVN which

synthesizes and releases corticotropin releasing hormone (CRH). This then acts on the

adrenal gland to release ACTH. AVP released from the SCN exerts an inhibitory role on

secretion of CRH from the PVN (Buijs et al., 2003; Kalsbeek et al., 2006). A polysynaptic pathway from the SCN to the adrenal gland is present, which points to a direct affect of the SCN AVP on the adrenal ACTH release (Buijs et al., 2003). In the european ground squirrel, AVP immunoreactivity in the SCN is positively correlated with the body temperature rhythm; after hibernation, gradual re-appearance of spontaneous rhythmicity is accompanied by an increase in AVP immunoreactivity in the SCN (Hut et al., 2002). AVP cell numbers and its transport and release in the SCN differ in mouse lines different for thermoregulatory nest behavior (Van der Veen et al., 2005), indicating a role of AVP in circadian organization of behavior. Exposure to short photoperiod, suppresses AVP mRNA expression in the SCN of Siberian hamster (Duncan et al., 1995).

Since short photoperiods are associated with infertility in these animals (Duncan et al.,

1985), AVP thus may play a role in regulating reproduction in this species. Similar to the

Brattleboro rats, decreased AVP immunoreactivity in the SCN of ageing rats could be responsible for age related changes in the sleep wake rhythms (Roozendaal et al., 1987).

In grey mouse lemurs, ageing affects the diurnal rhythm of AVP immunoreactivity by delaying the peak to the beginning of subjective night compared to during midday in young animals (Cayetanot et al., 2005). This change may affect the efferent signals of the

SCN thus changing the expression of biological rhythms in this species with ageing.

SCN derived AVP has also been studied in relation to ageing, depression and other disease conditions in humans (as reviewed by Caldwell et al., 2008). The AVP content within the SCN of humans shows a circadian rhythm with a peak during the daytime

(Hofman and Swaab, 1993). This pattern is disrupted with age, as people above 50 years of age show a reverse pattern of AVP content with a peak at night compared to those less than 50 years of age (Hofman and Swaab, 1994). Decreased AVP mRNA expression is seen in the SCN of patients suffering Alzheimer’s disease (Liu et al., 2000). Moreover elderly patients suffering from depression, show an increased number of AVP neurons in the SCN (Zhou et al., 2001). In conclusion, AVP plays diverse modulatory roles in generation and maintenance of circadian rhythms.

Gastrin Releasing Peptide

Gastrin releasing peptide (GRP) is produced in the SCN ventral subnucleus by about

14% of the total SCN neuronal population in rats (Moore et al., 2002). In golden hamsters, GRP cell bodies are located in the central region of the SCN (Morin and Allen,

2006) and GRP immunoreactivity is highest in sleeping animals, lowest in awake morning hamsters, and intermediate levels are found in nocturnally active hamsters

(Schilling and Nürnberger, 1998). Similar results are seen in rats where GRP immunoreactivity is higher during the day than night (Okamura and Ibata, 1994). GRP

mRNA expression peaks during the middle of the subjective night in nocturnal mice and

at the end of the subjective day in diurnal mice (Dardente et al., 2004). The SCN GRP

levels do not exhibit a daily rhythm in DD but are increased in response to light exposure

(Shinohara et al., 1993). GRP acts through GRP receptors and the bombesin receptors.

The GRP receptor mRNA is localized heavily in the dorsal and medial aspects of the

SCN in mice. GRP receptor protein expression and binding in the SCN are modulated by

light with rhythmic expression under LD with a peak at ZT 12 but no rhythm under DD

(Karatsoreos et al., 2006).

GRP in the SCN is regulated by photic signals from the retina directly and plays an

important role in SCN entrainment, since the GRP-rich ventrolateral SCN region receives

direct input from the retinal terminals (Tanaka et al., 1997) and afferents from the RHT

(Hayashi et al., 2001). Following a light pulse at night, per1 gene is expressed in the GRP

neurons of rat SCN (Dardente et al., 2002). Microinjections of GRP cause phase-

dependant shifts of the hamster SCN, similar to light and microinjections of GRP into the

third ventricle (Antle et al., 2005) or the SCN (Piggins et al., 1995) during the early

subjective night cause phase delays. These results are supported by in vitro studies in the

hamster and rat where GRP application during the early subjective night phase delays and

during the late subjective night phase advances the SCN neuronal firing rhythms, with no

effect seen during the subjective day (McArthur et al., 2000). These phase shifting effects

of GRP can be blocked by pre-treatment with the GRP receptor antagonists (McArthur et

al., 2000). In the rat and hamster, a small proportion of the GRP immunoreactive cells

located in the retinorecipient region of the SCN are fos immunoreactive (Earnest et al.,

1993). Since light pulses induce expression of immediate early gene c-fos in the SCN

(Rea, 1989) this further strengthens the evidence for a role of GRP in photic phase

resetting. Exogenous GRP mimics action of photic stimuli as GRP microinjections

increase c-fos immunoreactivity in the SCN (Piggins et al., 2005). Presenting a light

pulse to animals otherwise under DD, increases c-fos expression in the retinorecipient

cells co-localized with GRP (Romijn et al., 1996).

Since GRP has a similar phase-shifting pattern as that of light pulses, it may be an

important messenger in the photic signaling cascade of the SCN. The rhythmic

expression of GRP receptor protein under LD may suggest that the photic-like phase

shifting effect of GRP could be mediated through these receptors (Karatsoreos et al.,

2006). GRP is thought to play a role in communication between the two phenotypically

distinct SCN regions or retinorecipient ventral SCN and autonomous dorsal SCN. It dose-

dependently phase delays the behavioral rhythms in wild type mice accompanied by

induction of per1, per2 and c-fos in the dorsal SCN. This phase shifting effect as well as

expression of per genes and c-fos in dorsal SCN is attenuated in mice lacking GRP

receptors (Aida et al., 2002). It is also seen that GRP microinjections increase c-fos

reactivity more in the dorsal SCN (Piggins et al., 2005). Thus GRP acting via

downstream mechanisms through its receptors may convey photic information from the

retinorecipient ventral SCN to the dorsal region.

SIGNIFICANCE AND SPECIFIC AIMS

Entrainment of the SCN to the daily light-dark (LD) cycle is critical for survival, health

and optimal performance. If the circadian clock becomes out of phase with the local time, as can happen when flying to new time zones or by working rotating shifts, it can disrupt sleep and impair cognitive performance. A strategy to find a solution for various chronopathies is to try to rapidly shift the circadian clock to the new day-night cycle.

Several factors are known to regulate the timing of the SCN. For example, 5-HT is thought to play an important role in non-photic phase resetting, since its agonists can phase shift the clock. The potential of 5-HT as a mediator of non-photic phase shifting could be exploited to find a therapy for conditions resulting from the jet lag and shift work. Under normal LD conditions, phase shifts to 5-HT or its agonist 8-OH-DPAT are small, in the range of 1-2 hours. Studies from our lab have shown that exposure to LLb markedly potentiates phase shifts induced by 8-OH-DPAT causing ~12 hour shifts. It is therefore important to study whether 5-HT can be used to re-entrain the SCN rapidly to large shifts of the LD cycle.

The present study was undertaken to exploit the potentiating effect of LLb on nonphotic phase resetting and to assess the potential actions of 5-HT on non-photic clock resetting and rhythm re-entrainment. Experiments were done to determine whether large

8-OH-DPAT induced phase shifts potentiated by LLb could accelerate the rhythm re-

entrainment to simulated jet-lag involving large (10 hour) advance shifts of the LD cycle.

Experiments were also undertaken to explore the in vivo circadian phases shifting effects

of increased endogenous 5-HT activity stimulated by precursor L-tryptophan and

reuptake inhibitor fluoxetine under LLb conditions. SSRI’s are widely prescribed for

depressive states related to disturbed circadian rhythms but their role in inducing phase

shifts in hamsters has not been studied yet. Their efficacy as antidepressants might

depend on alteration of circadian rhythm parameters via 5-HT. Their combined treatment

with LLb could result in improved therapeutic index and treatment of anxiodepressive

states as well as potentiated shifting of the circadian clock helping in jet lag. These

studies will also provide additional insight into regulation of the SCN by LLb and 5-HT.

The SCN is composed of thousands of neurons, each of which is capable of generating

own individual rhythm and act as a pacemaker. Synchronization of all these individual

outputs into a single signal requires regulated inter- and intra-cellular communication.

Along with other neurotransmitters including 5-HT, neuropeptides released from the

SCN are thought to play a role in synchronizing these different signals into a single

output. In vitro studies have shown that the ventrolateral SCN releases vasoactive

intestinal polypeptide (VIP) and GRP, where as the dorsomedial part mainly releases

AVP. The second aspect of this study therefore was to investigate the role of 5-HT in the

neuropeptidergic regulation. The SCN was exposed to 5-HT1A,7 agonist 8-OH-DPAT by

reverse microdialysis and its effect on release of the neuropeptides AVP and GRP were

examined. To determine that the action of 5-HT is specifically on neuropeptides released

from the SCN, daily release profile of AVP from the SCN was measured and its synaptic

release verified using in vivo microdialysis.

Aim 1:

A. To test the hypothesis that LLb potentiates rhythm re-entrainment response to the

5-HT1A,7 agonist 8-OH-DPAT during large phase-advances of the LD cycle.

Rationale: 8-OH-DPAT, a 5-HT1A,7 agonist, produces phase-advances of the SCN

neuronal firing in vitro (Prosser et al., 1993; Shibata et al., 1992) and in vivo

(Edgar et al., 1993). Exposure to LLb significantly enhances the phase advance

shifts produced by 8-OH-DPAT (Knoch et al., 2004). Based on this observation,

we hypothesize that LLb would dramatically accelerate 8-OH-DPAT induced re-

entrainment to large (10 hour) phase advances of the LD cycle, simulating jet lag.

Drug treatments were administered at ZT 0, the phase of 8-OH-DPAT PRC when

LLb has greatest potentiating effect on 8-OH-DPAT induced phase advances

(Knoch et al., 2004).

Approach: Syrian hamsters housed under 14:10 LD received intraperitoneal (i.p.)

injections of 8-OH-DPAT or vehicle, with or without exposure to ~2 day LLb.

Days to re-entrain to the new LD cycle were compared between the photoperiod

and the drug groups.

B. To test the hypothesis that LLb has potentiating effect on phase shifting responses

to stimulated endogenous 5-HT.

Rationale: Endogenous serotonergic activity can be boosted by the 5-HT

precursor L-tryptophan to increase its synthesis and release, or by use of reuptake

inhibitor fluoxetine to prolong its availability at the site of action. Higher amount

of 5-HT at the synapse should result in larger magnitude phase shifting response.

We hypothesize that LLb exposure should significantly amplify the phase-shifting

response to stimulated endogenous serotonergic activity. L-form of tryptophan

was used in these experiments since in vivo studies have shown that D-tryptophan

can neither phase shift the clock nor can block subsequent serotonergic phase

shifts (Prosser et al., 2006)

Approach: Syrian hamsters housed under 14:10 LD received i.p. injections of L-

tryptophan, fluoxetine, a combination of L-tryptophan and fluoxetine, or vehicle,

with or without exposure to ~2 day LLb. Magnitude of phase shifts was compared

between the photoperiod and drug groups.

Aim 2:

A. To confirm the circadian release of AVP from the SCN.

Rationale: In vivo microdialysis studies have shown circadian release of

neuropeptide AVP from the rat SCN, with a peak during the subjective day and an

all-time low at night (Kalsbeek et al., 1995). In Syrian hamsters, AVP

immunoreactivity (Schilling and Nürnberger, 1998) and AVP cell numbers (Van

der Zee et al., 2002) in the SCN show similar circadian rhythms. Rhythmicity of

the AVP content (Tominaga et al., 1992) and mRNA expression (Dardente et al.,

2004) in the SCN of Syrian hamsters is well maintained under DD. We

hypothesize that AVP is released from the hamster SCN in a circadian manner.

Approach:

a) Measure daily release profile of AVP from the SCN using in vivo microdialysis

and radioimmunoassay.

b) Confirmation that the measured peptide is released from the SCN only, using

hemiprobes for microdialysis.

c) Confirm the circadian release of AVP from the SCN by microdialysis in animals

free running under DD for at least 2 weeks.

d) Confirm the neuronal release of AVP from the SCN by reverse microdialysis with

high potassium perfusate.

e) Confirm the neuronal release of AVP by depleting extracellular calcium.

B. To test the hypothesis that 5-HT negatively regulates the release of neuropeptides

AVP and GRP from the SCN.

a) Rationale: In Syrian hamsters, 5-HT agonist 8-OH-DPAT and reuptake inhibitors

inhibit AVP induced offensive aggression (Delville et al., 1996; Ferris et al.,

1999) and flank marking (Albers et al., 2002; Ferris et al., 2001) behaviors. In line

with the opposing behavioral actions of 5-HT and AVP, we hypothesize that 5-

HT1A,7 agonist 8-OH-DPAT, should result in a decrease in the SCN AVP release.

5-HT neuronal release from the SCN is circadian in pattern with peak at the

beginning of the dark phase (Dudley et al., 1998). This release pattern is opposite

to that of the AVP peak release during the early subjective day. These opposing

rhythms may point towards a negative correlation between the SCN 5-HT and

AVP.

Approach: Measure the effects of 8-OH-DPAT on AVP release from the SCN

using reverse microdialysis and radioimmunoassay.

b) Rationale: In vitro studies have shown that 5-HT inhibits the calcium currents

produced by synaptic stimulation (Flett and Colwell, 1999). 5-HT depletion

enhances the GRP mRNA expression in the SCN during the light phase. This

inhibitory effect of 5-HT is mediated by the 5-HT1B receptor (Hayashi et al.,

2001). Here we want to study direct effect of 5-HT on GRP release from the

hamster SCN using 5-HT1A,7 agonist 8-OH-DPAT. We hypothesize that 5-HT

should result in a decrease in the SCN GRP output either by its direct action on

the GRP neurons or by decreasing the excitatory calcium currents.

Approach: Reverse microdialysis of 8-OH-DPAT in the SCN and its effect on

GRP release from the SCN will be studied using RIA.

METHODS AND MATERIALS

Animals

Adult male Syrian hamsters (Mesocricetus auratus), obtained from Harlan

(Indianapolis, IN, USA) or reared from breeders obtained from this source, were used for these studies. Hamsters were housed individually in polystyrene cages in light- and temperature-controlled (20±1ºC) environmental chambers. Animals were entrained to 14 hours light and 10 hours dark (14:10 LD) photocycle with light intensity of 250 lux. Food

(Prolab 3000, PMI Feeds, St. Louis, MO, USA) and water were provided ad libitum. The experiments were approved by the Kent State Institutional Animal Care and Use

Committee.

Circadian activity measurements and phase shift analysis

The circadian rhythm of general locomotor activity was recorded using overhead infrared motion detectors (BV-300DP, Digital Security Controls Limited, Toronto) positioned above each cage, as described previously (Ehlen et al., 2001). Sensor output was interfaced with a computerized data acquisition system (Clocklab; Coulbourn

Instruments, Allentown, PA, USA). Analyses of the rhythm characteristics and graphical output (actograms) were undertaken using the Clocklab/Matlab statistical package.

Locomotor activity was measured for a minimum of 7 days under LD before experimentation. To measure phase shifting response, a line based on the mean locomotor activity onsets for a 10 day pre-treatment period under LD was extrapolated to the day of 38

treatment under LLb for the test groups and to the treatment day under LD for the control

groups. A regression line based on activity onset for a 10 day post treatment period was

back extrapolated to the day of treatment. The difference between these two lines on the

day of treatment is considered the phase shift. Activity onset is defined as the first bout of

continuous activity sustained for a minimum 30 minutes. The rate of re-entrainment to 10

hour advance shifts of the LD cycle was determined by counting the number of days

required for stable entrainment of the locomotor activity rhythm to the new LD cycle.

Stable entrainment is defined as the first 10 days where activity onsets are aligned with

the beginning of the dark phase of the shifted LD cycle, with tau ~24.

LLb Protocol

Onset of the LLb is zeitgeber time 12 (ZT 12; time of lights-off under an LD cycle), and

is maintained over 2 days under the ambient room lighting (~250 lux) eliminating two

dark phases. This represents 50 hours of continuous lighting from the last dark phase to

the time of the drug treatments at ZT 0. To represent the circadian phase of the treatments

administered during the LLb exposure, ZT rather than circadian time (CT; convention for

free running conditions with no zeitgebers) was used. During these LLb exposures, there were no significant changes in the phase or period of free running circadian activity rhythm, hence making ZT equivalent to the CT as a phase marker.

Experiment 1: Determining if LLb accelerates rhythm re-entrainment to large-

magnitude phase advance shifts of the LD cycle. 40

Male Syrian hamsters housed individually in a temperature controlled vivarium under

LD cycle were used. Animals were exposed to ~2 days of LLb followed by i.p. injection

of 8-OH-DPAT (Sigma, 5 mg/kg; 5 mg/ml DMSO) or vehicle (DMSO) at ZT 0 (Figure

7). Photic control groups received the same drug and vehicle treatments but were

maintained under LD. Immediately following the drug treatment animals were released

into the dark phase of the 10 hour advanced LD cycle. The extra 2 hours of darkness

following the drug treatments were necessary to avoid the blocking effect of light on

nonphotic phase shifting. The locomotor activity rhythms were monitored for up to 35

days post treatment to measure the rate of re-entrainment. The numbers of transition days

for stable adjustment (onset of activity at the new ZT 12 for several days) to the new LD

cycle were compared between the various treatment and photoperiod groups.

Statistics

The re-entrainment effects of 8-OH-DPAT under LLb versus LD were analyzed using two way ANOVA followed by the Student-Newman-Keuls post hoc test.

Experiment 2: Determining if LLb can amplify phase resetting responses to the

endogenous 5-HT.

Male Syrian hamsters housed individually in temperature controlled vivarium under LD

were exposed to ~2 days LLb followed by i.p. injections of L-tryptophan (Sigma, 50 mg/kg; 40 mg/ml DMSO), fluoxetine (Sigma, 10 mg/kg; 8 mg/ml DMSO), L-

tryptophan+fluoxetine or vehicle at ZT 0 (Figure 8). Control groups did not receive LLb exposure but the drug or vehicle treatments. Immediately following the drug treatments,

Old LD 14:10

LL Begin LLb

Treatment ZT0 *

New LD 14:10 00 02 0406 08 10 12 1416 18 20 22 24 Hours

Figure 7: Protocol for assessing the effect of 2 days of brief constant light (LLb) exposure

on 8-OH-DPAT mediation of re-entrainment to a 10 hour phase-advance of the LD cycle.

8-OH-DPAT or vehicle were delivered at ZT 0. Animals were released into darkness at

the beginning of drug or vehicle treatment followed by exposure to the dark phase of the

new LD cycle. LD controls did not receive LLb exposure. The top black bar represents

the dark phase of the initial LD.

animals were released into DD for at least 3 weeks. The magnitude of phase-shifting was

compared between the photoperiod and the drug groups.

Statistics

Data from the L-tryptophan/fluoxetine studies was analyzed using a two-way ANOVA

to study the effect of photoperiod. One way ANOVA followed by the Student-Newman-

Keuls post hoc test was used to compare the drug effects under different photoperiods.

Experiment 3: Determining the temporal release pattern of neuronal AVP from the

SCN.

Measuring AVP by In Vivo Microdialysis: Implantation of Dialysis Probe

Polycarbonate-polyether copolymeric membrane (PC) probes, with a molecular cut-off

20 KDa and an active membrane length of 2mm, (from CMA Microdialysis, Sweden)

were used for the microdialysis experiments. Under sodium pentobarbital anesthesia

(Nembutal; 50 mg/kg i.p.), animals were positioned with head immobilized and leveled

in a stereotaxic frame (David Kopf Instruments). Probes were stereotaxically aimed at the

lateral margin of the SCN to minimize physical damage to the clock. The stereotaxic

coordinates were +0.03 mm anterior to the bregma, +0.03 mm lateral to the midline

suture and -0.80 mm ventral to the dura, where 0.00 is the junction of bregma and the

midsagittal suture. The probes were firmly secured to the skull with three 1/8-inch

stainless steel anchor screws and cemented with dental acrylic. After 2-3 days of recovery

from the surgery, microdialysis was performed in freely behaving hamsters. To examine

the extent of contamination of microdialysates with AVP from outside the SCN,

hemiprobes prepared by occluding one half side of the dialysis membrane with epoxy and 43

LD 14:10

LL Begin LLb

* Treatment ZT0 *

00 02 0406 08 10 12 1416 18 20 22 24 Hours

Figure 8: Protocol for assessing the effect of 2 days of brief constant light (LLb) exposure

on serotonergic (L-tryptophan±fluoxetine) phase-resetting responses. Shown is a

treatment delivered at ZT 0 on the second day of constant light exposure. Animals were

releases from LLb to DD at the outset of the drug treatment. The top black bar represents the dark phase of the initial LD.

the remaining active membrane side aimed at the SCN medially were used. For

determining the circadian nature of the SCN AVP release, animals free running in DD for

at least 2 weeks were used. To avoid exposure to light during the probe implantation,

anesthesia was induced in dark and the eyes of the hamsters covered by sterile gauze pads held by surgical tape. The eyes were uncovered after surgery in the DD chamber with animals still under anesthesia. The in vitro efficiency of AVP recovery from the microdialysates was 11%; it was determined by incubating the probes in a solution of

125I-labeled peptide at 37°C and measuring the relative recovery of labeled peptide in the

microdialysate after perfusion with ACSF @ 1 µL/min.

Microdialysis Perfusion Studies

After 2-3 days of recovery from the surgery, microdialysis was performed in freely

behaving conscious hamsters. All the sampling experiments included connecting the inlet

part of the probe to the syringe pump (CMA/100, Bioanalytical Systems, West Lafayette,

IN) via a liquid swivel (Instech; Plymouth Meeting, PA) or raturn system (Bioanalytical

Systems, West Lafayette, IN) with polyethylene tubing. The outlet part of the probe was

connected via teflon tubing to either the automated refrigerated fraction collector

(Bioanalytical Systems, West Lafayette, IN) holding 250 µL polyethylene nonstick tubes

or 200 µL PCR tubes (Fisher) for manual collections. Probes were continuously perfused

with the filtered artificial cerebrospinal fluid (ACSF; 126.5 mM NaCl, 27.5 mM

NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 1.1 mM CaCl2, 0.9 mM MgCl2.6H2O, 0.5 mM

Na2SO4, 5.9 mM D-(+)-glucose, 0.1% bovine serum albumin; pH 7.3-7.5) at a flow rate

of 1μL/min. After 2-3 hours of equilibration, samples were collected at 1 h intervals by 45

the automated sampler or manually. Sampling continued for 24 hours for LD, DD release

profiles and the hemiprobe experiments. Both the raturn and swivel assemblies allow the

animals freedom of movement within the cage throughout the duration of experiments.

Samples were stored at -70ºC till analyzed for the peptide content using

radioimmunoassay.

Assessment of neuronal release: High [K+] perfusate

After 2-3 hours of probe equilibration, sampling was started at ZT 4. Following a 2 hour stable baseline collection with regular ACSF, a 1 hour perfusion of ACSF with 150 mM KCl and 100 µM veratridine (Biomol) was administered from ZT 6-7. The sampling was continued for 3 hours following the drug perfusion to collect a follow up baseline with regular ACSF.

Assessment of neuronal release: Ca2+-free perfusate

After 2-3 hours of probe equilibration, sampling was started at ZT 23. Following a 2 hour stable baseline collection with regular ACSF, a 1 hour perfusion of Ca2+-free ACSF

(regular ACSF with no CaCl2, 10.0 mM EDTA, 25.0 mM MgCl2.6H2O) with calcium

channel blockers diltiazem (Tocris, 200 µM), verapamil (Tocris, 200 µM), cinnarizine

(Sigma, 15 µM), and flunarizine (Tocris, 12 µM) was perfused from ZT 1-2. Sampling

was continued for 3 hours following the drug perfusion to collect a follow up baseline

with regular ACSF.

Radioimmunoassay 46

AVP was measured from the microdialysates by radioimmunoassay (RIA; assay kits

from Phoenix Pharmaceuticals, Burlingame CA, USA), as per the manufacturer’s

protocols. This assay is highly specific for rodent and human AVP. In brief, microdialysis

samples were incubated with 50% anti-AVP primary antibody for 48 hours at 4ºC.

Samples of ACSF and drug cocktails, equal to the microdialysate samples in volume (60

µL) were run as controls, in addition to the positive controls with known peptide content.

Next, samples were incubated with 125I-AVP for 24 hours at 4ºC. On the last day,

samples were incubated with normal rabbit serum and goat anti-rabbit secondary

antibody at room temperature for 90 minutes followed by centrifugation at 3600 rpm for

30 minutes to separate the antibody bound radioactivity from free radioactivity.

Supernatant was aspirated and radioactivity measured in the pellet by the gamma counter

(Packard Instruments Cobra II). Standard curves generated by using half primary antibody concentration showed a sensitivity of 0.1 pg. Intra- and inter-assay co-efficients of variation were 8.0% and 9.6% respectively.

Histology

Histological studies were performed following all the experiments to verify probe placement adjacent to the SCN. Animals were euthanized by i.p. pentobarbital sodium and phenytoin sodium (Euthasol, 0.1 mL). Brains were extracted and fixed overnight in

4% buffered paraformaldehyde at 4ºC, followed by cryoprotection in 30% sucrose

solution. Brains were frozen on dry ice and 20-30 μm thick sections obtained by

cryosectioning. Sections were mounted on positively charged microscope slides and

stained with cresyl violet and analyzed using light microscopy. Probe placement adjacent 47

to the SCN is marked by necrotic cells and presence of a probe tract (Figure 9). Only data

from animals with correct probe placements adjacent to the SCN was used for analysis.

Permissible limits for probe location were 250 microns lateral to the SCN; any

positioning anterior or posterior to the SCN was noted.

Statistics

Data for the 24 hour profiles of AVP release was expressed as a percentage of the daily

mean to minimize inter-individual variability in basal neuropeptide release in the SCN.

Repeated measures one way ANOVA was used to compare release across the different

time points. Post hoc Dunnets test was used for comparing multiple group means, so as to

define peak release from non peak release as done by Dudley et al (1998). Data from the

microdialysis pharmacology experiments was standardized by expressing the values as percentage of the baseline; where baseline is calculated as mean of the two pre-treatment samples. Treatments effects were analyzed using repeated measures one way ANOVA followed by the Student-Newman-Keuls post hoc test. The level of statistical significance was set at p<0.05 for all analyses.

Experiment 4: Determining serotonergic regulation of neuropeptides AVP and GRP release from the SCN.

5-HT regulation of SCN AVP release

The effect of 5-HT on AVP release from the SCN was assessed using reverse microdialysis with the 5-HT1A,7 receptor agonist 8-OH-DPAT during the subjective

daytime. After 2-3hours of probe equilibration, sampling was started at ZT 23. Following 48

Figure 9: Representative coronal section through the midposterior aspect of the SCN

showing a microdialysis probe tract (P) at the lateral margin of the nucleus. Section is

stained with cresyl violet. 3V, third ventricle; OC, optic chiasm.

2 hour baseline collection, animals were given 1 hour perfusion of the 8-OH-DPAT (1.2

mM in ACSF) from ZT 1-2. Sampling was continued for 3 hours following the drug

perfusion. Animals were exposed to darkness from the beginning of the 8-OH-DPAT

pulse till the end of follow up baseline collection, with drug perfusion and sample

collection performed under safe red light (~0.04 lux). Microdialysate AVP was measured

using RIA and probe placement was confirmed by histology.

5-HT regulation of SCN GRP release

The effect of 5-HT on GRP release from the SCN was assessed using reverse

microdialysis with 8-OH-DPAT during the subjective daytime. After 2-3 hours of probe

equilibration, sampling was started at ZT 4. Following 2 hour baseline collection, animals

were given 1 hour perfusion of 8-OH-DPAT (1.2 mM in ACSF) from ZT 6-7. Sampling

was continued for 3 hours following the drug perfusion to collect a follow up baseline.

Animals were exposed to darkness from the beginning of the 8-OH-DPAT pulse until the

end of sample collection. Drug perfusion and sample collection were performed under

safe red light (~0.04 lux). Microdialysate GRP was measured using RIA and probe

placement was confirmed by histology. RESULTS

Experiment 1: LLb accelerates 8-OH-DPAT induced rhythm re-entrainment to

large (10 hour) phase advance shifts of the LD cycle.

Re-entrainment responses to systemic administration of 5-HT1A,7 agonist 8-OH-DPAT

were tested with and without exposure to 2 days of LLb. Exposure to LLb significantly

accelerated 8-OH-DPAT induced rhythm re-entrainment to 10 h advance shifts (Kaur et al., 2009), with 8 out of 12 animals showing immediate re-entrainment (group mean

4.2±1.8 days; Figure 10, 11). Vehicle controls took significantly longer with a mean of

14.7±1.6 days (F1,21=19.2; p<0.001 versus 8-OH-DPAT). In animals not exposed to LLb,

8-OH-DPAT group re-entrained rapidly compared to the vehicles (means 11.2±1.7 versus

16.2±0.7 days, F1,9=7.8; p<0.02), but were slower compared to LLb exposed 8-OH-DPAT

treated animals (F1,15=5.6; p<0.03). There was no difference in the rate of re-entrainment

between vehicle groups under different photoperiods (F1,15=0.4; p>0.5). Suppression of

locomotor activity during the LLb exposure as seen in the previous studies by Knoch et al

(2004) was seen in all the actograms from animals exposed to LLb (Figure 11).

Experiment 2: LLb potentiates phase shifts responses to stimulated endogenous 5-

HT release.

Phase shifting responses to endogenous 5-HT activity enhanced by L-tryptophan,

fluoxetine or their combination was studied with or without exposure to LLb (Kaur et al.,

2009). LLb exposure has a significant potentiating effect on phase- resetting responses to 50

20 LLb

a* 5

20 12 11 0 LD LD b

15 a DAYS RE-ENTRAINMENT FOR

56 0 01238-OH-DPAT Veh

Figure 10: Potentiating effect of ~2 days brief constant light exposure (LLb, upper panel) on re-entrainment to 10 hour phase advance shift of the LD cycle following i.p. injection of 8-OH-DPAT or vehicle (Veh), compared to no LLb exposure (LD, lower panel).

Numbers in the bars represent number of animals in each treatment. Within a photoperiod group, bars with different letters are significantly different (p<0.05). Within drugs, bars with “*” are significantly different (p<0.03).

Figure 11: Representative double-plotted actograms of general locomotor activity showing re-entrainment responses to 8-OH-DPAT (A, C) or vehicle (B, D) injected i.p. at

ZT 0 for 10 hour phase advance shift in animals receiving 2 days of brief constant light exposure (left panels, shading represents LLb exposure) or no LLb exposure (right panels). Arrows denote time of injections. Modified from Kaur et al (2009).

the serotonergic treatments (F1,3=91.6; p<0.0001 versus LD; Figure 12, 13). In animals

exposed to LLb, groups with L-tryptophan and L-tryptophan+fluoxetine treatments, produced significantly greater phase advances compared to the vehicle (2.6±0.2 hours and 2.5±0.4 hours versus 1.5±0.3 hours respectively; F2,14=4.2; p<0.04). Fluoxetine

neither enhanced L-tryptophan shifting (p>0.20 versus L-tryptophan), nor had a phase

shifting effect of its own (p>0.29 versus vehicle controls). Animals under LD did not

show significant phase shifting effect of any drug treatment (F3,19=1.53; p>0.24).

Experiment 3: AVP is released from the hamster SCN with a distinct circadian

rhythm.

A. Daily profile of AVP release in the SCN

AVP release from the hamster SCN exhibits significant daily fluctuations (F5,23=2.53,

p<0.001; n=6) as determined by in vivo microdialysis with RIA. Peak AVP release

(149±22% of the daily mean) was seen during the early subjective day (ZT 2-4; Figure

14). The values decrease to lowest during the latter half of the subjective day with nadir

(65±14%) at the onset of the subjective night and gradually increase throughout the night.

The 24 hour release profiles measured with the hemiprobes were similar to those obtained by regular probes, with peak release seen during early subjective day and lower values observed during the latter part of the subjective day (Figure 16). The release showed a gradual increase during subjective night as seen before.

B. AVP release under free-running conditions

Hamsters housed under DD for ~14 days, with stable free running rhythms of locomotor

3.5 LLb

3.0 b* b*

2.5 ab*

2.02.03.5 a*

1.51.53.0

1.01.02.5

0.50.52.0 PHASE SHIFT (H) SHIFT PHASE

56 6 5 0.01.5 LD

1.0 a a a 0.5

a 65 6 0.0 6 Veh Trypt+Fluox Trypt Fluox

Figure 12: Potentiating effect of ~2 day brief constant light exposure (LLb, upper panel) on phase shifting responses to i.p. injection of vehicle (Veh), L-tryptophan and fluoxetine

(Trypt+Fluox), L-tryptophan (Trypt) or fluoxetine (Fluox) at ZT 0 compared to no LLb exposure (LD, lower panel). Numbers in the bars represent number of animals in each treatment. Within a photoperiod group, bars with different letters are significantly different (p<0.04). Within drug or vehicle treatments, bars with “*” are significantly different (p<0.01).

Figure 13: Representative double-plotted actograms of general locomotor activity showing the phase-resetting responses to L-tryptophan (A, E), L-tryptophan and fluoxetine (B, F), fluoxetine alone (D, H) and vehicle (C, G) injected i.p. at ZT 0 (arrows) in animals receiving 2 days of brief constant light exposure (LLb; left panels; shading represents the period of LLb exposure) or no LLb exposure (right panels). All the animals were released to DD at the time of injections. Note the suppression of activity under the

LLb exposure (Kaur et al., 2009).

200

* * * *

* * * * 150 * *

100 SCN AVP RELEASE SCN AVP (% DAILY MEAN) 50

0 6 12 18 0 6 12 18 0 6

ZT (HOURS)

Figure 14: Double-plotted daily release profile of AVP from the SCN of hamsters

maintained under a 14:10 LD cycle. Release of AVP from the SCN shows significant fluctuations across the 24 hours, p<0.001. Solid bars denote the dark phase of the LD cycle. Each point represents the mean ± SEM for 6 animals. *p<0.01 v.s. mean non-peak values. ZT refers to the zeitgeber time.

Figure 15: Representative double plotted actogram of hamsters used for microdialysis studies. The solid arrow represents the day of surgery followed by two days of recovery.

Shaded arrow represents the day of beginning microdialysis. It can be noted that microdialysis experimentation does not cause any change in the general locomotor activity rhythms.

250

200

150

100 SCN AVP RELEASE AVP SCN (% DAILY MEAN)

0 6 12 18 0 6 12 18 0 6 ZT (HOURS)

Figure 16: Double plotted daily release profile of AVP from the SCN of hamsters maintained under a 14:10 LD cycle using hemiprobes. Solid bars denote the dark phase of the LD cycle. Each point represents ± SEM for 2 animals.

activity prior to and during the microdialysis studies were used (Figure 15). As was seen

in hamsters housed under 14:10 LD cycle, a significant (F2,23=4.03, p<0.01; n=3) increase

in AVP release was seen during early subjective day time (CT 3), and lower values

during the latter part of the day and night (Figure 17). Maximal AVP release was

245±21% relative to the subjective daily mean.

C. Verification of neuronal release of AVP using high [K+]+veratridine

Synaptic release of AVP from the SCN was verified by local perfusion of ACSF with

high potassium (150 mM KCl) and veratridine (100 µM). Depolarization of the SCN by

high [K+] perfusate increased AVP release to 559±145% from a baseline of 100±15%.

This ~459% increase in release was seen within 60 minutes of treatment (F3,4=6.72, p<0.004, n=4; Figure 18). Following perfusion with the regular ACSF, the concentration of extracellular AVP in the SCN returned to basal levels.

D. Verification of neuronal release of AVP using Ca2+-free perfusate

Reverse microdialysis with calcium channel blocker cocktail (200 µM diltiazem, 200 µM

verapamil, 15 µM cinnarizine, 12 µM flunarizine) in calcium free ACSF significantly

decreased (F2,4=9.40, p<0.004; n=3) the SCN AVP release from a baseline of 100±6% to

42±9% confirming its synaptic release (Figure 19). AVP release recovered to baseline

levels on perfusion with regular ACSF.

300 * * * * 250

* * 200

150

100 SCN AVP RELEASE RELEASE AVP SCN (% DAILY MEAN)

0 6 12 18 0 6 12 18 0 6 CT (HOURS)

Figure 17: Double-plotted daily release profile of AVP from the SCN of hamsters maintained under constant dark (DD) for a minimum of 2 weeks. Release of AVP from the SCN shows significant fluctuations across 24 hours, p<0.001. Each point represents the mean ± SEM for 3 animals, *p<0.01 v.s. mean non-peak values. (CT; circadian time)

800 HIGH [K+] b* ACSF

600

a,b

400 a

SCN AVP RELEASE RELEASE AVP SCN BASELINE) (% a 200 a

a a a a a a a 0 5678 9 10 ZT (HOURS)

Figure 18: Effect of localized SCN perfusion with high potassium ASCF with veratridine

(HIGH [K+]) on AVP release. The shaded bar denotes 1 hour duration of perfusion.

Within a treatment group, points with different letters are significantly different from

baseline, p<0.004. Asterisks indicate significant differences between treatment groups,

p<0.003. Each point represents the mean ± SEM for 4 animals for HIGH [K+] and 6 animals for ACSF control.

150 CALCIUM BLOCK ACSF a a a a a 100 a a a

b* 50

SCN AVP RELEASESCN AVP (% BASELINE) b* b*

0 0 1 2345

ZT (HOURS)

Figure 19: Effect of localized SCN perfusion with calcium channel blockers in calcium free ACSF (CALCIUM BLOCK) on AVP release. The shaded bar denotes 1 hour duration of perfusion. Within a treatment group, points with different letters are significantly different from baseline, p<0.004. Asterisks indicate significant differences between treatment groups, p<0.01. Each point represents the mean ± SEM for 3 animals for calcium block and 6 animals for the ACSF control.

Experiment 4a: 5-HT has no effect on the SCN AVP release.

Reverse microdialysis with 8-OH-DPAT (1.2 mM) did not have a significant effect on the SCN AVP release (Figure 20). b. 5-HT negatively regulates the SCN GRP release.

Reverse microdialysis with 8-OH-DPAT (1.2 mM) significantly decreased (F3,8=4.358, p<0.003; n=4) the SCN GRP release from a baseline of 100±7.8% to 30.5±14.9% (Figure

21). The SCN GRP release recovered to baseline levels on perfusion with regular ACSF.

200

150

100 SCN AVP RELEASE SCN AVP (% BASELINE) 50

0 23 0 1 2 3 4 ZT (HOURS)

Figure 20: Lack of effect of localized perfusion with 8-OH-DPAT on AVP release in the

SCN. The shaded bar denotes 1 hour duration of perfusion. Each point represents the mean ± SEM for 3 animals.

200

150

100 SCN GRP RELEASE GRP SCN BASELINE) (% 50 * *

0 67 8 9 10 ZT (HOURS)

Figure 21: Effect of localized SCN perfusion with 8-OH-DPAT on GRP release. The shaded bar denotes 1 hour duration of perfusion. Points with asterisks are significantly different from the baseline p<0.003. Each point represents the mean ± SEM for 4 animals.

DISCUSSION

Brief Constant Light Potentiation of Serotonergic Phase Resetting

Exposure to LLb alters the shape of 8-OH-DPAT PRC and facilitates large magnitude

(type 0) phase shifts of the LD cycle; with large phase advances seen during early

morning (ZT 0) and large phase delays during the late night (ZT 18; Duncan et al., 2005;

Knoch et al., 2004; Knoch et al., 2006). LLb also potentiates behavioral phase resetting

related to wheel running and sleep deprivation (Knoch et al., 2004). Since the behavioral

activation stimulates the SCN 5-HT release (Dudley et al., 1998), it is hypothesized that

the large magnitude behavioral shifts potentiated by LLb could be in part mediated by the

5-HT release induced by the behavioral stimulations (Kaur et al., 2009). However there is

no direct evidence that links the potentiating effect of LLb to 5-HT. In the present studies,

animals receiving LLb exposure demonstrated significant phase advances to L-tryptophan,

thus confirming the existence of an in vivo clock resetting mechanism responsive to

endogenous 5-HT (Kaur et al., 2009). The present results also show that LLb exposure potentiates 8-OH-DPAT-induced phase resetting, hence promoting rapid re-entrainment to large magnitude phase advance shifts simulating jet lag (Kaur et al., 2009).

Previously we have shown that in Syrian hamsters LLb strongly potentiates phase-

resetting responses to nonphotic stimuli such as sleep deprivation, wheel exposure, intra-

SCN NPY and i.p. or intra-SCN 8-OH-DPAT (Duncan et al., 2005; Knoch et al., 2004;

Knoch et al., 2006). Although the mechanism for the potentiating effect of LLb is not

clear, several hypotheses have been put forth. The effect of LLb seems to be selective for

the nonphotic phase shifting responses as it does not enhance NMDA induced shifts in

the SCN, that mimic photic phase shifting (Landry and Mistlberger, 2005). In the present

study it was seen that LLb exposure significantly accelerates rhythm re-entrainment to

new 10 hour advanced LD cycle with majority (8/12) of animals showing immediate re-

entrainment to the new LD cycle. This could be a result of large phase shift induced by

combined treatment with 8-OH-DPAT and LLb at ZT 0, since previous study by Knoch et

al (2004), showed ~10 hour advance shifts in response to the same treatment. On the

other hand, response of the remaining animals (4/12) which took longer to entrain could

be a result of methodological errors (injections into the subcutaneous fat with slow

absorption of the drug). It should be noted that the LLb potentiated phase shifting

responses to L-tryptophan in this study are smaller than those seen with 8-OH-DPAT by

Knoch et al (2004). This is possibly due to greater binding affinity of 8-OH-DPAT to 5-

HT1A and 5-HT7 receptors, which are implicated in the phase advancing effects of

serotonergic stimuli. On the other hand, L-tryptophan increases 5-HT synthesis, possibly

affecting all 5-HT receptor subtypes, whether stimulatory or inhibitory. Also based on the

previous studies where 150 mg/kg of L-tryptophan increased the extracellular levels of 5-

HT by ~200% (Glass et al., 1995), the dose of 50 mg/kg used in the present study may

not have optimally stimulated 5-HT synthesis for phase-resetting (Kaur et al., 2009).

In the present study fluoxetine did not have a phase advancing response of its own or

any additive effect on phase advances to L-tryptophan. LLb exposure inhibits the

nighttime increase in 5-HT release and suppresses the locomotor activity (Knoch et al.,

2004) which could further decrease SCN 5-HT release. With decreased 5-HT release in

the SCN, inhibition of reuptake mechanism might not be as effective in increasing the

available 5-HT at the SCN synapses for phase advancing actions. On the other hand, L-

tryptophan, which increases the overall 5-HT synthesis and subsequently release, causes

significant phase advance responses due to increased production. The lack of an additive

effect of L-tryptophan with fluoxetine could have been due to different time frames of

action; the effect of fluoxetine might be terminated before the increased release of 5-HT

is achieved in response to L-tryptophan. The lack of phase shifting response to fluoxetine could also be related to findings that the therapeutic effects of fluoxetine do not act through or affect circadian rhythms in hamsters (Klemfuss and Kripke, 1994). Chronic treatment with fluoxetine is known to alleviate symptoms of obsessive compulsive disorder without any changes in the altered body temperature, plasma cortisol and melatonin rhythms (Millet et al., 1999; Monteleone et al., 1995). Further the therapeutic

effect of SSRI’s is due to their inhibitory action on the light induced phase advances

(Gannon and Millan, 2007) and not due to shifts induced by them. In a study by Gannon

and Millan (2007), it was seen that fluoxetine at a dose of 10 mg/kg did not inhibit light

induced phase advances in the hamster wheel running rhythms but at a dose of 20 mg/kg

did induce significant shifts. Hence the insignificant phase shifting responses to

fluoxetine might be because the dose of 10 mg/kg i.p. used in the present study was not

enough to obtain significant changes in the locomotor activity rhythms. However, i.p.

fluoxetine at dose rate of 5 and 10 mg/kg has been shown to increase extracellular 5-HT

in rat brain by 3- to 13-fold (Guan and McBride, 1988) although not specifically in the

SCN. Another reason could be the time of drug administration. In the present study the

injections were done at ZT 0 (the phase of the 8-OH-DPAT PRC when LLb has maximum potentiating effect [Knoch et al., 2004]). But this may not be the optimum time of injection and action for fluoxetine. The lack of effect of fluoxetine could also be due to species differences, since the hamster brain has more than 95% of monoamine oxidase A, which is more specific for 5-HT, compared to rat which has only 60-80% of MAO-A

(Edwards and Malsbury, 1978). This could result in a faster metabolism of the 5-HT at synapse once its reuptake is blocked, thus resulting in diminished effects or responses.

Our results are also supported by another study in rats where chronic rather than acute administration of fluoxetine had no effect on the circadian wheel running rhythm

(Wollnik, 1992).

In Syrian hamsters, a number of nonphotic and behavioral interventions other than LLb can induce similar potentiating effects. For example, exposure to a novel wheel in DD in hamsters previously housed under LD without wheel can induce phase advances of more than 12 hours when shifted to wheel during their light phase (Gannon and Rea, 1995).

These shifts were associated with continuous running for long periods. The re- entrainment rate to an 8 hour advance of the LD cycle in hamsters can be doubled by increasing activity in the form of running wheel confinement for 3 hours beginning shortly after release into DD from LD (Mrosovsky and Salmon, 1987). The short acting benzodiazepene triazolam can also significantly accelerate re-entrainment to 8 hour LD cycle shifts by ~50% (van Reeth and Turek, 1987). Vitamin B12 in drinking water when given to rats, doubles the wheel running activity during the re-entrainment period and

accelerates re-entrainment to the reversed LD cycle by ~30% (Tsujimaru et al., 1992).

Finally treatment with the 5-HT1A mixed agonist/antagonists (BMY7378 and S15535)

during the late night followed by a light pulse results in large (~5 hour) phase advance

shifts (Gannon, 2003; Gannon and Millan, 2006). The large phase shifts induced by these

interventions are similar to the ones seen in our LLb studies, suggesting a common pathway linked with behavioral activation (Kaur et al., 2009). It is interesting to note that our experiments are the only ones that show rhythm re-entrainment in the absence of running wheels (Kaur et al., 2009).

The pathway through which LLb and these other potentiating interventions act is still

unclear but the midbrain raphe 5-HT system is a possibility. It is known that wheel running and sleep deprivation increase 5-HT released in the SCN and the IGL (Dudley et al., 1998; Grossman et al., 2004), both of which receive their 5-HT afferents from the

midbrain raphe nuclei (Meyer-Bernstein and Morin, 1996). Electrical stimulation of the midbrain raphe nuclei at midday induces phase advance shifts of the wheel running rhythm (Glass et al., 2000; Meyer-Bernstein and Morin, 1999) and LLb-potentiated phase

shifts to sleep deprivation are abolished by the 5-HT1A antagonist –(-)pindolol (Knoch et

al., 2006) implicating a serotonergic mechanism. In addition, the large behavior (sleep

deprivation, novel wheel exposure) induced shifts are mimicked by systemic serotonergic

treatments i.e. 8-OH-DPAT (Knoch et al., 2004) and L-tryptophan (Kaur et al., 2009).

A central question thus is what is the mechanism whereby LLb produces serotonergic

hypersensitivity? One hypothesis is that the potentiated response to serotonergic phase

advance influences depends upon the degree of post synaptic sensitivity to 5-HT

produced by experimental and or endogenous variations in the 5-HT tonus (Kaur et al.,

2009). Evidence supporting this view is that the deafferented mouse and rat SCN slices

with lesser 5-HT release exhibit larger (~4 hour) phase advances in response to 8-OH-

DPAT compared to in vivo (~1 hour) effects (Prosser, 2003; Prosser and Gillette, 1989;

Shibata et al., 1992; Sprouse et al., 2005). Conversely pre-treatment of the SCN slices

with 8-OH-DPAT desensitizes the phase resetting responses to following 8-OH-DPAT

treatment (Prosser et al., 2006). Also, phase advances to the intra-SCN 8-OH-DPAT are

significantly larger following pre-treatment with the 5-HT synthesis inhibitor PCPA

(Ehlen et al., 2001). Exposure to LLb decreases SCN 5-HT release at night (Knoch et al.,

2004), which may produce a depletion induced hypersensitization of the post synaptic

serotonergic response to subsequent serotonergic stimulation. This is supported by the

same study where reverse microdialysis of 5-HT into the SCN to restore the nocturnal

levels dampened the potentiating effect of LLb on 8-OH-DPAT induced phase advances

(Knoch et al., 2004).

An animal’s recent behavioral history (whether it is more or less active during the

night), could proportionately increase or decrease serotonergic tonus the next day. This

could result in down- or up-regulation of 5-HT receptor-mediated phase resetting (Kaur

et al., 2009), and is supported by the demonstration of a daily variation in the 5-HT1A receptor sensitivity (Lu and Nagayama, 1996). Nevertheless, a study by Duncan et al

(2005) showed that LLb does not upregulate 5-HT1A, 5-HT7 and 5-HT1B receptor binding in the SCN. This potentiation may occur at some signal transduction step downstream from the ligand binding. Since the LLb exposure causes significant suppressions of AVP

and per1 mRNA expression in the SCN, it may act through neuropeptide and/or clock

gene signaling pathways (Duncan et al., 2005).

Another proposed mechanism for the potentiating effect of LLb on nonphotic phase

resetting comes from limit cycle theory, where inhibition of circadian pacemaker

amplitude could render the clock more responsive to a phase shifting stimulus (Jewett et

al., 1991; Kronauer, 1990; Winfree, 1970). Such attenuation of the circadian pacemaker

by the LLb is evident from its suppression of locomotor activity, SCN 5-HT release

(Knoch et al., 2004), SCN AVP and per1 mRNA’s (Duncan et al., 2005; Sudo et al.,

2003) and SCN electrical activity (Shibata et al., 1984). For the SCN oscillator system, amplitude is considered to be an important determinant of the pacemaker response, which

if made sufficiently small could produce large (type 0) shifts, but under normal

conditions, produces small (type 1) shifts. Such effect on the pacemaker amplitude has

also been demonstrated in humans (Jewett et al., 1991). Thus in the present studies, the

attenuating effect of LLb on the circadian functions could produce a similar large phase

resetting response to 8-OH-DPAT and large phase shifts to the L-tryptophan and or fluoxetine treatments. However studies where LLb had no potentiating effect on photic

(NMDA) stimulation (Landry and Mistlberger, 2005), argue against the limit cycle

amplitude hypothesis, at least as it relates to light.

Circadian rhythm desynchronization is related to many chronopathological disorders,

like depressive syndromes associated with circadian clock phase advance and/or delay

disruptions (Duncan, 1996; Healy and Waterhouse, 1995; Lewy et al., 2006; Millan,

2006; Murray et al., 2005), or shift work and jet-lag related disorders. These disorders

usually result from the disrupted sleep wake cycles and out of phase light-dark cycles. To

treat such conditions, strategies for resynchronization of the clock phase with the LD

cycle are useful, however the phase shifts induced by most of these pharmacological and

natural (behavioral, photic) stimuli are relatively small (1-2 hours), thus limiting their

benefits especially in alleviating the jet lag malaise. Here the potential of LLb exposure that produces rapid and large shifts of the circadian clock has been explored. As seen in our previous studies, clock phase can be immediately advanced or delayed by as much as

12 hours (without transients), by appropriate co-application of LLb and 8-OH-DPAT

stimulation (Knoch et al., 2004). This kind of treatment can theoretically be used to

rapidly accelerate re-entrainment of the circadian clock to large advance (eastward) and

delay (westward) shifts of the LD cycle simulating jet-lag. Such was seen in hamsters,

where combined treatment with LLb and 8-OH-DPAT produced nearly immediate re-

entrainment in the majority (75%) of animals exposed to large (10 hour) phase advance

shifts of the LD cycle. Similar potentiating effects of LLb were also seen with L-

tryptophan and fluoxetine. From these results it is clear that the rapid phase-resetting

response to 5-HT agonists and larger phase shifting in responses to stimulated

endogenous 5-HT activity potentiated by LLb offers an important approach for

therapeutically adjusting circadian clock phase for treatment of chronopathological

disorders.

AVP Release Profile from the SCN

The present study utilized in vivo microdialysis and RIA to characterize endogenous

release of AVP from the SCN and to study role of 5-HT in regulation of this neuropeptide

in the SCN. Such measurements reveal that in hamsters entrained to 14:10 LD cycle, the release of AVP from the SCN is rhythmic with a peak during the early subjective day and

levels falling to the lowest during the later part of the day and early night. A similar

release profile measured with hemiprobes confirms that the AVP in microdialysate is of

the SCN origin. In hamsters free running in DD, the AVP release profile was similar to

that of LD entrained animals, with peak release during the early subjective day. The

persistence of AVP rhythm under constant environmental conditions indicates that this

rhythm is generated by endogenous pacemaker activity and is circadian in nature. In line

with the in vivo study by Kalsbeek et al (1995) in rats, where distinct diurnal rhythm in

AVP release from the SCN was observed with a peak during the midday and nadir during

the midnight, our study has shown a distinct rhythm in the AVP release from the hamster

SCN with peak release during the early subjective day and nadir during the later day or

subjective night. The difference in the time of peak release as seen with the microdialysis

studies by Kalsbeek et al (1995), could be due to species differences between the

experimental models. The release of AVP from the hamster SCN was confirmed by using

hemiprobes for microdialysis experiments to occlude AVP released from sources other

than SCN from entering the dialysate. This approach has been successfully used in our

lab for confirmation of 5-HT release from the SCN (Dudley et al., 1998).

A distinct circadian rhythmicity in CSF AVP content has been observed in cats

(Reppert et al., 1981), guinea pigs (Jones and Robinson, 1982), monkeys (Perlow et al.,

1982), rabbits (Gunther et al., 1984) and rats (Schwartz et al., 1983), with peak levels

after the beginning of the light period, lowest at the end of the light period and levels

rising throughout the dark period. The SCN is responsible for generating and maintaining

AVP rhythm in the CSF since rhythmicity is abolished in SCN lesioned animals

(Jolkkonen et al., 1988; Schwartz and Reppert, 1985; Uhl and Reppert, 1986).

Since neuropeptides like AVP become biologically active only when released into the

extracellular fluid, studies based on the cellular content and immunoreactivity of AVP

across the day night cycle should be interpreted with caution (Landgraf et al., 1998). The

present study measured peptide release directly from the extracellular fluid surrounding the SCN neurons using microdialysis, thus giving a clear picture of temporal changes in peptide release. This technique has been successfully used in our lab to measure the release of 5-HIAA (Glass et al., 1992), glutamate (Rea et al., 1993), 5-HT (Dudley et al.,

1998) and NPY (Glass et al, submitted) from the SCN of freely behaving hamsters.

Caution was taken to optimize the efficiency of the microdialysis procedure for peptide recovery. Considering the importance of temperature of the perfusion media in peptide recovery (Benveniste, 1989), ACSF at room temperature was used for continuous perfusion of the microdialysis probes. The ACSF used for these studies was isotonic to

CSF and had all the relevant ions and pH in physiologic range. As for most molecules,

equilibrium between the perfusion medium and the extracellular fluid is incomplete, it is

important to calibrate the microdialysis probes (Orlowska-Majdak, 2004). Efficiency

measured for the PC probes used in these microdialysis experiments was ~11%. This was

calculated based on the relative recovery, where concentration of the substance in the

perfusion medium to its concentration in the medium surrounding the probe is relative

recovery and is expressed in percentage (Orlowska-Majdak, 2004). The flow rate used for

the microdialysis studies should be as low as possible to avoid any rise of pressure inside

the probe, which impedes diffusion of molecules into the perfusate (de Lange et al.,

1997). In our studies a flow rate of 1 µL/min was used, which has been successfully used

in rabbits for AVP recovery from the hippocampus (Orlowska-Majdak et al., 2001).

Our results are consistent with both in vivo and vitro studies of AVP content and release

from the SCN. AVP release by the SCN is circadian in manner and is maintained in vitro

(Gillette and Reppert, 1987) over several cycles (Earnest and Sladek, 1986). In Syrian

hamsters, AVP immunoreactivity (Schilling and Nürnberger, 1998) and AVP cell

numbers (Van der Zee et al., 2002) in the SCN show similar circadian rhythms. A

circadian pattern is also seen in AVP mRNA expression in the SCN of Siberian hamsters

(Duncan et al., 1995). AVP mRNA expression in the SCN shows identical circadian

rhythm in nocturnal mice as well as diurnal rodent with highest levels during the late

subjective day (Dardente et al., 2004). AVP mRNA expression also shows matching

rhythms in the SCN of normal and Brattleboro rats, suggesting a circadian regulation and

no dependence on the peptide release (Uhl and Reppert, 1986). The AVP receptor V1a is

localized in the brain in rats (Johnson et al., 1993) and marmosets (Wang et al., 1997).

The V1a receptor mRNA expression follows a diurnal rhythm with a peak during the

subjective night (Kalamatianos et al., 2004a; Li et al., 2009; Young et al., 1993). This is

consistent with the finding that the SCN is more strongly activated by the application of

AVP during the night than during the day (Liou and Albers, 1989).This receptor rhythm

is present in Brattleboro rats, which rules out a role of AVP feedback regulating its

receptor expression (Young et al., 1993).

Neuronal origin of AVP release from the SCN

It is important to verify that the AVP sampled from the SCN by microdialysis in the

present experiments is a result of synaptic release and not possible leakage from damaged

neurons or other procedural influences. In the present study AVP release was significantly increased by high [K+]+veratridine-induced local depolarization of the SCN,

confirming its synaptic release. This response is consistent with our previous

microdialysis studies evaluating release of excitatory amino acid glutamate (Rea et al.,

1993) and neurotransmitter 5-HT (Dudley et al., 1998) from the SCN. These results are

also similar to previous studies in rat where reverse microdialysis with High [K+] in the perfusate increased AVP release from the SCN (Kubota et al., 1996) and the caudate nucleus (Orlowska-Majdak et al., 2003). As a second confirmation, depletion of Ca2+ in the perfusion media along with presence of calcium channel blockers markedly decreased

AVP release from the SCN. This response is similar to decrease seen in the release of 5-

HT from the SCN (Dudley et al., 1998). Blocking the L-type calcium channel is known to abolish the oscillations of the SCN neurons in vitro (Pennartz et al., 2002). In the present study, the addition of calcium channel blockers did not amplify the magnitude of inhibitory response when compared to other studies, since only L-type calcium channels

were blocked with the drugs, sparing the other type calcium channels. The response of

AVP release to alterations in the ionic currents of the ACSF provides strong evidence of its synaptic release from the SCN.

Circadian release of AVP from the SCN

In the present study AVP release measured from animals free running under constant conditions (DD) was similar to that seen under LD. This indicates that the rhythm of

AVP release from the hamster SCN is circadian in nature and is not dependant on the LD

cycle and is consistent with several in vivo and vitro studies. Circadian rhythmicity of the

AVP content (Tominaga et al., 1992) and the mRNA expression (Dardente et al., 2004) in

the SCN is maintained under DD. Rhythmicity of the AVP content in the SCN is

maintained under short term (Tominaga et al., 1992) as well as long term LL conditions

(Isobe and Nishino, 1998), suggesting that the SCN AVP levels are controlled and

generated by an endogenous pacemaker. AVP mRNA expression in the SCN shows

circadian variations which are maintained under DD (Cagampang et al., 1994) or LL

(Tominaga et al., 1992). No such significant variations are seen in the AVP release from

the SON under DD or LD (Cagampang et al., 1994). A recent study in hamsters has

shown that exposure to LLb suppresses SCN AVP mRNA expression (Duncan et al.,

2005), which is in agreement with the study by (Tominaga et al., 1992), where LL

decreased the mean AVP levels but did not affect its rhythmicity. Constant low light

conditions also decrease AVP staining intensity in the SCN of voles (Gerkema et al.,

1994).

Expression of AVP mRNA and peptide content are higher during the light period than

the dark (Duncan et al., 1995; Dardente et al., 2004; Cagampang et al., 1994) which corresponds to the SCN neuronal firing rate which is higher in light period (Gillette and

Reppert, 1987; Groos and Hendriks, 1982; Kalsbeek et al., 1995; Mihai et al., 1994;

Pennartz et al., 2002; Shibata et al., 1982; present study). Release of AVP from the rat

SCN has no autoregulatory influence, since direct application of V1/V2 antagonist had no

effect on AVP output (Kubota et al., 1996). As discussed earlier, AVP neurons are

present mainly in the dorsomedial SCN and express genes per1 and per2 abundantly

(Dardente et al., 2002). Rhythmic expression of the per mRNA is present in the AVP

neurons of the dorsomedial SCN even under constant conditions (Hamada et al., 2004).

The AVP gene transcription shows a circadian rhythm in the rat SCN culture with a peak

during the daytime (Carter and Murphy, 1989; Carter and Murphy, 1992; Reppert and

Uhl, 1987). Since circadian expression of the AVP gene is seen in immature fetal SCN

before the formation of afferent and intra SCN neural connections, AVP mRNA levels

are apparently under specific circadian control (Reppert and Uhl, 1987). Dynamic

changes in AVP gene transcription leads to diurnal variations in AVP mRNA levels in

the SCN. The AVP heteronuclear RNA (an indicator of gene transcription) and AVP

mRNA in the rat SCN show circadian variations with peak during the day time and nadir

during night. Similar changes in the heteronuclear RNA were also observed under

constant conditions (Yambe et al., 2002). It was suggested that the rapid turnover of the

AVP mRNA at night could also contribute to the diurnal changes. Thus the rhythmic

diurnal release pattern of AVP from the SCN seems to be generated at the gene level.

AVP gene transcription is maintained in a circadian fashion in vitro and this rhythmicity

is blocked by voltage dependant sodium channel blocker tetrodotoxin (Arima et al., 2002)

showing that synaptic activity is required for rhythmic AVP gene transcription. Since

tetrodotoxin application decreased the AVP heteronuclear RNA levels and rhythmicity,

ongoing neural activity is essential for circadian AVP gene transcription and peptide

release. It was also seen that forskolin, an adenylate cylase stimulator, increases AVP

gene transcription and a functional MAPK pathway is required for gene transcription

because MAPK inhibitor decreased AVP heteronuclear mRNA levels in the SCN (Arima

et al., 2002).

In a detailed study, Jin et al (1999) showed that the AVP gene promoter contains E box,

an element which is responsive to clock proteins, CLOCK and brain and muscle aryl

hydrocarbon receptor nuclear translocator like protein 1 (BMAL1). It was shown that

AVP gene regulation is positively regulated by the CLOCK-BMAL1 heterodimers by

binding necessarily to E box in the AVP gene, similar to the per genes. This E box in the

AVP gene is essential for its circadian expression since CLOCK gene knockout mice did

not show any circadian pattern in the AVP mRNA expression as well as in per gene

expression. It was also seen that the AVP content in the SCN and in its vasopressigenic

efferent projection areas was diminished in clock mutant mice. This study was further

confirmed by Silver et al (1999), where they showed that the circadian rhythm in day

night mRNA expression in CLOCK gene knockout mice was only abolished for the AVP

gene and not for the vasoactive intestinal peptide (VIP), substance P or cholecystokinin

expressions although these genes also have E box sequences.

In the mouse, the VPAC2 receptor for VIP is considered necessary for generation of

circadian rhythms, since the circadian rhythms of electrical activity of the SCN are lost in

VPAC2 deficient mice in vitro. These results were replicated in vivo by chronic

administration of VPAC2 receptor antagonists to the wild type mice (Cutler et al., 2003).

VIP signaling seems to be necessary to maintain rhythmicity of the AVP gene in the SCN

(Harmar et al., 2002). The VIP neurons of the SCN establish synapses amongst

themselves as well as with the AVP producing neurons of the SCN (Daikoku et al.,

1992). VIP receptor VPAC2 mRNA is present in almost all the AVP neurons in the

dorsomedial SCN in rat (Kalamatianos et al., 2004b). VIP application causes an increase

in AVP release from the SCN slices (Isobe and Nishino, 1996) and dispersed SCN cells

(Watanabe et al., 1998). VIP also causes phase dependant shifts of rhythmic AVP release from the SCN with phase advances seen during the mid or late subjective night and phase

delays during the late subjective day or early subjective night (Watanabe et al., 2000).

The VIP receptor VPAC2 is G protein coupled receptor and primarily acts through cAMP

as second messenger (Lutz et al., 1993) and the AVP gene has cAMP response element

(CRE; Mohr and Richter, 1990). As we know that adenylate cyclase stimulator forskolin increases AVP gene transcription in the SCN (Arima et al., 2002), thus through its action on the VPAC2 receptors and subsequently increasing cAMP, VIP may play a role in generation of the SCN AVP rhythms. Such a role is also indicated by the ability of VIP to cause phase dependant shifts of rhythmic AVP release from the SCN (Watanabe et al.,

2000). Further, the VPAC2 deficient mice, do not express circadian pattern of the core clock genes per1, per2 and cry 1 as well as of clock controlled AVP gene (Harmar et al.,

2002). Thus VIP may act as paracrine synchronizing agent of the SCN that mediates

information from the retinorecipient ventral SCN to the AVP rich dorsomedial SCN and helps to maintain the autonomous oscillations of the SCN (Kalamatianos et al., 2004b;

Harmar, 2003).

The SCN communicates with its target areas not only by neural synapses but also by

humoral signals (Bittman, 2009), since grafting of SCN encapsulated in semipermeable

capsule (to prevent neural growth) in previously lesioned animals, restores locomotor

activity rhythms (Silver et al., 1996). AVP is one of the principle efferent signals of the

SCN to other brain areas (Buijs, 1996) and plays an important role in amplifying the

output of the SCN due to its excitatory actions (Ingram et al., 1996; Liou and Albers,

1989; Mihai et al., 1994). Although SCN AVP neurons are not responsible for

maintaining SCN neural activity (Liou and Albers, 1989; Mihai et al., 1994), they may

play a role in intra-SCN communication by synchronizing the oscillations generated by

individual SCN neurons (Reghunandanan and Reghunandanan, 2006). There is also

evidence showing role of the SCN AVP in regulation of hormonal rhythms, like

corticosterone, leutinizing hormone and even clock control genes (Hamada et al., 2004;

Kalsbeek et al., 2008; Palm et al., 1999). AVP neurons of the SCN project to the

dorsomedial hypothalamus, where AVP inhibits the HPA axis activity by inhibiting

corticosterone release (Kalsbeek et al., 1992; Kalsbeek et al., 1996).

Prokineticin 2 (PK2) functions as an output molecule of the SCN. Its mRNA shows

circadian rhythm of expression with a peak during the light period and the peptide itself

has a negative effect on the locomotor activity (Cheng et al., 2002). In V1a knockout

mice, the circadian amplitude of the PK2 mRNA is dampened as is circadian locomotor

behavior (Li et al., 2009). This negative effect on the PK2 mRNA amplitude could be due

to decreased amplitude of the SCN firing or may involve another complex mechanism by

which AVP regulates other clock controlled genes (Li et al., 2009). AVP may even act

through the V1a receptor to boost the transcription of PK2.

AVP is the only neurotransmitter of the SCN that is shown to be released in a circadian pattern under constant conditions. AVP-deficient Brattleboro rats show attenuated

rhythms in a number of circadian parameters. Brattleboro rats show circadian rhythms of

the SCN neuronal firing but the amplitude is significantly lower than the wild types

(Ingram et al., 1996). These rats also show decreased amplitude of slow wave and

paradoxical sleep rhythms (Brown and Nunez, 1989). Further, under constant conditions,

Brattleboro rats do not exhibit free running rhythm or synchronization of the body

temperature, which otherwise is present in wild types (Murphy et al., 1999; Wideman et

al., 2000). Brattleboro rats also exhibit dampened rhythms of pineal melatonin synthesis

(Schroder et al., 1988). Recent studies from voles also implicate the SCN AVP in

regulation of locomotor rhythms (Gerkema et al., 1994; Jansen et al., 2000). All these

studies along with the roles listed before point to a regulatory role of the SCN AVP in

modulation of several circadian parameters.

Regulation of SCN AVP release by 5-HT

In Syrian hamsters, AVP positive fibers and terminals are present in the ventrolateral

region of the SCN (Van der Zee et al., 2002), which is densely innervated by the 5-HT

afferents (Meyer-Bernstein and Morin, 1996). Quipazine, a 5-HT antagonist, decreases c-

fos mRNA levels in the dorsomedial SCN (Prosser et al., 1994), which is an AVP rich

region. This may signify a direct interaction between 5-HT and AVP neurons in the SCN,

which is supported by the presence of 5-HT7 receptors on AVP neurons in the mouse

SCN (Belenky and Pickard, 2001). Further it is seen that (although not related to the

SCN), AVP and 5-HT together play a role in regulation of behavior. In Syrian hamsters,

5-HT has an inhibitory effect on the AVP induced behaviors, and AVP-facilitated

offensive aggression is inhibited by the 5-HT reuptake inhibitor fluoxetine in the

ventrolateral hypothalamus (Delville et al., 1996) and by 8-OH-DPAT in the anterior

hypothalamus (Ferris et al., 1999). Also AVP facilitated flank marking behavior is

inhibited by 8-OH-DPAT (Albers et al., 2002) and 5-HT reuptake inhibitors fluoxetine

and clomipramine (Ferris et al., 2001) in the anterior hypothalamus. Based upon these

behavioral studies, and the fact that 5-HT inhibits calcium currents produced by synaptic

stimulation in the SCN (Flett and Colwell, 1999), it was hypothesized that 5-HT might

negatively regulate the SCN AVP release. Contrary to this hypothesis however, are the

present results showing that 8-OH-DPAT did not have a significant effect on AVP

release from the hamster SCN. These results are consistent with an in vitro study, where

5-HT did not have any significant effect on AVP release from the SCN slice (Isobe and

Nishihara, 2002). Since 5-HT increases AVP release from the hypothalamic slice culture

(Kostoglou-Athanassiou and Forsling, 1998), this indicates differential serotonergic

regulation of the SCN parvocellular AVP neurons and hypothalamic magnocellular AVP

neurons (Isobe and Nishihara, 2002). In fact destruction of 5-HT afferents to the SCN has

no effect on AVP neuronal immunoreactivity in the rat SCN (Kawakami et al., 1985).

Also, in a study by Mirochnik et al (2005), it was seen that 5-HT depletion in fetal brain did not have any effect on AVP mRNA expression or neuronal size in the adult SCN.

However 5-HT depletion could have caused attenuated release of AVP, since an increase in number of AVP neurons and peptide concentration in the call bodies was observed.

The absence of any effect of 5-HT on the AVP release could be due to the specific nature of the agonist, as 5-HT action on the SCN AVP release might be mediated by

receptors other than 5-HT1A and 5-HT7 or by sites outside the SCN. Regarding the

nonphotic PRC, the maximum effect of 8-OH-DPAT would be expected around ZT 6,

but in the present study the drug perfusions were done from ZT 1-2, which might not be a

relevant time for significant drug effect. Further AVP release from the SCN peaks during

the early subjective day (Kalsbeek et al., 1995; present study) and has an excitatory

action on the SCN (Liou and Albers, 1989; Mihai et al., 1994; Ingram et al., 1996),

whereas 5-HT release from the SCN peaks at the beginning of the dark phase (Dudley et

al., 1998) and it has an overall inhibitory effect on the SCN neural activity (Nishino and

Koizumi, 1977). Based on these release profiles and effects on the SCN neuronal activity,

5-HT and AVP seem to have opposite effects and may tend to balance each other’s

actions. Further studies need to be done to clarify their interaction. Use of 5-HT itself, its

different agonists to specify the receptor subtype involved and higher dose of 8-OH-

DPAT, can shed some light on this.

Regulation of SCN GRP release by 5-HT

In Syrian hamsters, GRP expressing cells are present in the central region of the SCN

(LeSauter et al., 2002; Miller et al., 1996; Morin and Allen, 2006). These neurons play an

important role in photic phase resetting of the SCN, as they receive direct input from the

retinal terminals (Tanaka et al., 1997) and express c-fos in response to light exposure

during subjective night (Earnest et al., 1993; Romijn et al., 1996). Microinjections of

GRP into the third ventricle (Antle et al., 2005) or the SCN (Piggins et al., 1995) cause

phase-dependant shifts of the hamster locomotor activity rhythms similar to light.

Neuronal GRP release in the SCN of freely behaving hamsters was studied in our lab

using in vivo microdialysis and RIA. In hamsters maintained under LD, GRP release

exhibits a diurnal rhythm with peak release during the early subjective day (Glass lab,

unpublished data). Similar release profiles from hemiprobe microdialysis experiments

confirmed the SCN origin of the measured peptide. Synaptic release of the measured

GRP was confirmed by significant increase in peptide release in response to high

[K+]+veratridine in the perfusate. As a second confirmation, depletion of Ca2+ in the perfusion media caused a significant decrease in GRP release from the SCN (Glass lab, unpublished data). Our results are consistent with in vitro studies were GRP immunoreactivity in the SCN of hamsters (Schilling and Nürnberger, 1998) and rats

(Okamura and Ibata, 1994) was higher during the day than night.

The ventrolateral part of the SCN where GRP is produced is densely innervated by 5-

HT afferents (Hayashi et al., 2001). This close proximity if GRP cell bodies and 5-HT afferents may point towards a neuroanatomical interaction in modulating SCN functions.

Our studies show that 5-HT negatively regulates the SCN GRP release since reverse

microdialysis with 8-OH-DPAT significantly decreased the SCN GRP release. Our

studies are supported by a study in rats where 5-HT depletion enhanced GRP mRNA

expression in the SCN during the light phase. This inhibitory effect of 5-HT is mediated

by 5-HT1B receptor (Hayashi et al., 2001).

Several studies point to an important role of GRP in photic signaling. Microinjections

of GRP into the third ventricle (Antle et al., 2005) or the SCN (Piggins et al., 1995) cause

phase-dependant shifts of the hamster SCN, similar to light. These results are supported

by in vitro studies in hamster and rat where GRP application phase delays the SCN neuronal firing during the early subjective night, phase advances during the late subjective night but has no effect during the subjective day (McArthur et al., 2000). GRP microinjections also increase c-fos reactivity, especially in the dorsal SCN (Piggins et al.,

1995; Piggins et al., 2005). On the other hand 5-HT is known to have an inhibitory effect

on light induced phase shifts. 5-HT agonists inhibit light induced phase shifts of the

locomotor activity (Rea et al., 1994), inhibit light induced c-fos immunoreactivity (Glass

et al., 1995; Glass et al., 1994; Selim et al., 1993) and inhibit electrical activity of light

responsive SCN cells (Ying and Rusak, 1997). During the night, serotonergic agonists

and reuptake inhibitors decrease the light induced phase shifts of the SCN (Gannon and

Millan, 2007; Rea et al., 1994; Weber et al., 1998). It is also seen that GRP dose dependently phase delays the behavioral rhythms in wild type mice accompanied by induction of per1, per2 and c-fos in the dorsal SCN (Aida et al., 2002). On the other hand, phase advances by systemic or local administration of 8-OH-DPAT are

accompanied by a decrease in the SCN per1 and per2 (Ehlen et al., 2001; Tominaga et

al., 1992; Horikawa et al., 2000) expressions. If we consider the release profiles, 5-HT

release from the SCN peaks at the beginning of the dark phase (Dudley et al., 1998),

whereas GRP release from the SCN peaks during the early subjective day (Glass lab, unpublished data). These opposing actions of 5-HT and GRP on the photic phase shifting as well as their different release profiles point to a negative correlation between them, which is strengthened by our results.

Conclusion

Circadian pacemaker activity is controlled by complex interplay of different neurotransmitters and stimuli, of which 5-HT is an integral part. LLb exposure, mediates

a switch from type 1 to type 0 phase resetting, and offers an opportunity for strong

serotonergic and behavioral phase resetting of the mammalian circadian clock. Although

the mechanism underlying the hypersensitization effect of LLb is not clear, it has

potential importance in understanding the basis of nonphotic clock resetting and for

exploring the role of 5-HT in circadian nonphotic phase regulation. The present findings

also confirm an endogenous circadian rhythm in neuronal AVP release from the SCN that

occurs even in absence of photic cues. Since the SCN is heterogeneous in structure and

function, interaction between its different neurochemicals and cell types is important to

the synchronization of individual pacemaker neurons and their unified output. The

negative association between SCN 5-HT and GRP in the present study can be important

in determining the pacemaker response to different exogenous and endogenous stimuli.

On the other hand no direct effect of 5-HT on the SCN AVP release points to a different mode of interaction, where these two can interact at their target sites outside the SCN or show opposite action within the SCN.

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