Analysis of Light-Induced Immediate-Early Gene Expression in the Suprachiasmatic Nucleus

ANALYSIS OF LIGHT-INDUCED IMMEDIATE-EARLY GENE EXPRESSION IN THE SUPRACHIASMATIC NUCLEUS

A thesis submitted to Kent State University in partial fulfillment of the requirements for the degree of Master of Science

By,

Amanda S. Ohnmeiss

August 2009

Thesis written by Amanda S. Ohnmeiss B.S. Allegheny College, 2007 M.S., Kent State University, 2009

Approved by

_____ Dr. E. Mintz ______, Advisor

____ Dr. R. Dorman ______, Director, School of Biomedical Sciences

___Dr. T. Moerland______, Dean, College of Arts and Sciences

TABLE OF CONTENTS

PAGE

LIST OF FIGURES ………………………………………………………………………v

LIST OF TABLES………………………………………………………….………….…vi

ACKNOWLEDGEMENTS………………………………………………………..…….vii

Introduction………………………………………………………...... 1

Suprachiasmatic Nucleus Background…………………...... 1

Entrainment………………………………………………………………………..2

Common Light-Induced Genes of the SCN……………………………………….3

Glutamate………………………………………...………………………..8

Goals………………………………………………………………………9

Methods…………………………………………………………………….…….13

Animals...... 13

Experiment 1……………………………………………………………..13

Experiment 2……………………………………………………………..14

Experiment 3………………………………………………………….….14

Experiment 4………………………………………………………….….15

Immunohistochemistry……………………………………………..……15

Data Analysis…………………………………………………………….16 Results…………………………………………………………………………....18

iii

Discussion………………………………………………………………………..38

References……………………………………………………………………….47

LIST OF FIGURES

PAGE Introduction

Fig 1: MAPK sequence of events…………………………………………………6

Fig 2: Summary of light induced effects in the SCN………………………….....12 Methods

Fig 3: Representative diagrams of SCN division for cell counting……………...17

Results

Fig 4: Experiment 1 c-fos expression……………………………………………19

Fig 5: Experiment 2 c-fos expression……………………………………………21

Fig 6: Experment 3 actograms of vehicle/MK-801 injected animals……………23

Fig 7: Experiment 4 c-fos expression……………………………………………26

Fig 8: Experiment 4 egr1 expression………………………………………….…29

Fig 9: Effect of MK-801 on c-fos and egr1 expression………………………….31

Fig 10: Grid view of MK-801 early night c-fos expression……………………...32

Fig 11: Grid view of MK-801 late night c-fos expression……………………….33

Fig 12: Grid view of MK-801 late night egr1 expression………………………..34

Fig 13: Experiment 4 p-ERK expression………………………………………...36

LIST OF TABLES

PAGE

Table 1: Experiment 4 c-fos expression data……………………………………………27

Table 2: Experiment 4 egr1 expression data…………………………………………….30

Table 3: Experiment 4 p-ERK data……………………………………………………...37

ACKNOWLEDGEMENTS

I would like to start by thanking my advisor, Eric Mintz, for his guidance. Where my determination may have been lacking at times, his determination to see his students excel pushed me to complete my research. I have a profound respect for his advising style, as it has prepared me to enter the workforce with knowledge of the importance of self-motivation and holding myself accountable for my work and work ethic. I appreciate his patience, along with the past and present members of the lab (Erin Gilbert, Veronica

Porterfield, George Kallingal, and Linley Moreland), in teaching me the techniques I needed to succeed and never finding any question too ridiculous to answer.

I would also like to thank the members of my committee for believing that I could finish my research in a year when I came to them after joining Dr. Mintz’s lab in January of 2008. A year and a half later they were ready to advise me as I prepared to defend my thesis. They have educated me in other areas of the field of Neuroscience with passion for their concentrations and, for that, I greatly admire them.

I will forever be grateful to my parents for forgiving me for deciding not to go to medical school and supporting my decision to join the Kent State University Biomedical

Sciences program. They have always had the utmost faith in my abilities (perhaps more so than I) and continue to be 100% supportive of my decisions in all aspects of my life, whether they agree with them or not. Without them, I would be nothing and I hope that they know how much I truly appreciate their opinions and advice.

vii

Lastly, I’d like to thank my fiancée, Brian, for encouraging me whenever I became overwhelmed or cranky. He refused to let me give up and pushed me to work hard and prove to myself that I could do it.

viii

Introduction:

Rhythms are present in almost every aspect of the environment; be it seasonal weather changes or fluctuations in day length. In order to survive it is necessary for mammals to be able to anticipate these rhythms and the patterns by which they fluctuate.

These mammals rely on environmental cues in order to synchronize their own circadian

(daily) rhythms and data collected to date implicate the suprachiasmatic nucleus (SCN) as the primary mammalian circadian clock.

The SCN is a group of approximately 20,000 neurons (Riley & Moore, 1977; van den Pol, 1980; Güldner, 1983), which resides in the hypothalamus adjacent to the third ventricle and just dorsal to the optic chiasm (Cassone et al, 1988). Destruction of the

SCN results in a loss of circadian control over endocrine, metabolic, and behavioral rhythms (Abe et al, 1979; Stephan & Zucker, 1972). Rhythms in a previously arrhythmic animal (due to lesioning of the SCN) are restored by transplanting small neural grafts from the suprachiasmatic region of a donor animal (Ralph et al, 1990) and, according to lesion studies done by Harrington et al (1993), it appears that rhythms are exhibited as long as 15-25% of the SCN remains intact. There are also clocks located peripherally in the body, such as those found in the liver and pancreas, that are partially dependent on the

SCN for the generation of rhythms (Damiola et al, 2000). Destruction of the SCN disrupts the expression of circadian genes in these peripheral clocks (Reddy et al, 2007).

Therefore the SCN uses environmental cues such as light and dark to synchronize

1 2

rhythms that control a variety of physiological and behavioral processes both centrally and peripherally.

Entrainment

The SCN relies on photic cues to synchronize (entrain) rhythms to the environment and photoreceptors located in the retina are responsible for mediating the entraining effect that light has on the SCN/entire mammalian circadian system. Retinal ganglion cells carry information to the SCN via two pathways. The first is through direct connection via the retinohypothalamic tract (RHT) and the second is indirectly via the intergeniculate leaflet (IGL) (Rusak & Boulos, 1981). Destruction of the IGL results in changes in amplitude of phase shifts resulting from photic stimulation but does not abolish them (Pickard et al, 1987). Destruction of the RHT results in an inability of light to entrain circadian rhythms (Johnson et al, 1988a) which supports a role of the RHT as the primary circuit necessary for photic entrainment.

Exposure to light can have significant effects on the behavior of an animal. One such effect is a phase shift, where the animal either begins/ends its activity period earlier or later than it did before the light exposure occurred. Whether a phase shift occurs or not depends on whether the light exposure occurs during the subjective night or the subjective day. Exposure to light during the subjective night will result in a phase shift of activity rhythms whereas a light pulse during the subjective day has no effect (Pittendrigh

& Daan, 1976). The direction of the phase shift elicited by exposure to light during the subjective night is also subject to variation depending on which part of the night the light 3

pulse occurs. A light pulse given during the early night will result in a phase delay while a light pulse given during the late night will result in a phase advance (Daan &

Pittendrigh, 1976). Entrainment to a new light cycle may take several days depending on how much the new light cycle deviates from the cycle the animal was previously entrained to.

Common Light-Induced Genes of the SCN

In addition to the behavioral phase shifts during the subjective night, light pulses also result in the expression of immediate early genes (IEGs) (Guido et al, 1999).

Immediate early genes are rapidly and transiently expressed in response to a variety of stimuli (for our purposes, to photic stimuli). A light pulse at just any time of the day will not necessarily result in the expression of IEGs in the SCN. Most IEGs will only be expressed during a time when light pulses are expected to cause phase shifts, (i.e. subjective night) (Rusak et al, 1990). Examples of IEGs that are expressed in response to photic stimulation include FBJ osteosarcoma oncogene (c-fos), early growth response protein 1 (egr1), and Jun oncogene (Jun).

To date, c-fos is the most studied and well characterized of the IEGs. It is induced by photic stimulation of the retinas (Rea, 1989) and its expression occurs mainly in retinorecipient areas of the SCN (Aronin et al, 1990). C-fos also exhibits its own spontaneous rhythms in the dorsomedial portion of the SCN independent of the effects of light (Sumová et al, 1998). It has been proposed that the increase in c-fos immunoreactivity is the result of an increase in Fos mRNA (Kornhauser et al, 1990). 4

Porterfield et al (2007) have shown that the increase in c-fos expression following a light pulse is approximately 30 fold. In hamsters, the degree to which c-fos expression is induced by photic stimulation is correlated with the behavioral phase shifting effects of light such that brighter light results in larger phase shifts and increased c-fos expression

(Lin et al, 1997). Also, when antisense oligonucleotides are used in rats to prevent the translation of c-fos protein, photic phase shifts are inhibited (Wollnik et al, 1995). Each of these pieces of evidence suggests a link between c-fos expression and light induced phase shifts. However, c-fos knockout mice are able to entrain to light dark cycles

(Honrado et al, 1996) suggesting that c-fos is not necessary for photic entrainment.

Egr1, another IEG, is a DNA-binding transcription factor and is also induced by light (Kornhauser et al, 1990). Like c-fos, light induced egr1 expression is localized in areas of retinal projection by the RHT (Johnson et al, 1988b). There are, however, some differences in expression between c-fos and egr1. First, light induced expression of Fos mRNA in the SCN peaks approximately 30 minutes following light exposure

(Kornhauser et al, 1990) whereas egr1 expression peaks approximately 45-60 minutes following light exposure (Lin et al, 1997). In mice, egr1 exhibits an increase in response to light during both the subjective day and subjective night (Dong et al, 2002) unlike c- fos which can only be induced by light during the subjective night (Rusak et al, 1990).

This daytime light induced egr1 expression follows a different pattern than that of c-fos and egr1 induced during the subjective night. Egr1 expression during the subjective day occurs in a small population of cells located at the lateral edges of the SCN (Dong et al,

2002) as opposed to throughout most of the SCN as occurs during the night. Egr1 is also 5

inducible at a much lower threshold of light intensity than c-fos and there does not appear to be a relationship between the threshold required for expression and the threshold required for a behavioral phase shift (Lin et al, 1997).

Despite differences in time period for peak expression and the light intensity required to induce expression, it appears that c-fos and egr1 may be activated by similar mechanisms. When a light pulse is given during the subjective night, the p42/44 mitogen- activated protein kinase (MAPK) pathway is activated. The activation of this pathway occurs in a phase-restricted manner and parallels the effect that light has on IEG expression (Obrietan et al, 1998). The sequence of events involved in the MAPK cascade can be viewed in Figure one. Disruption of the MAPK pathway results in a disruption of light-induced phase shifts (Butcher et al, 2002). Butcher et al (2002) have also shown that activation of the MAPK pathway occurs within minutes of photic stimulation. This is before any increase in IEG mRNA expression is detected suggesting that activation of the

MAPK pathway may mediate the effect of light on IEG expression. Further supporting this idea are data presented by Dziema et al (2003) showing that blockage of MAPK activation results in a decrease or complete disruption of IEG expression, implicating

MAPK as a key intermediate between photic stimulation and transcriptional activation which is necessary to reset the circadian clock. 6

Glutamate

Tyrosine kinase autophosphorylation

GTP-bound Raf (active) Ras

CREB ERK MEK1 & phosphorylation/ (MAP MEK2 translocation to kinase) nucleus

Transcription of genes with cre elements in their promoters (i.e. light-inducible IEGs = activates and Per1)

Figure One: MAPK sequence of events as reviewed by Cobb (1999) and Porterfield et al

(2007).

This MAPK pathway also activates the period genes per1 (Travnickova-Bendova et al, 2002) and per2 (Cermakian et al, 2002). These genes play a role in one of two feedback loops that comprise the pacemaker mechanism of the SCN. The first loop is composed of Clock and Bmal1 which are both transcription factors. Clock is constitutively (Vitaterna et al, 1994) expressed while Bmal1 is expressed in a cyclic manner with its expression peaking toward the middle of the dark period (Ikeda &

Nomura, 1997). The protein products of these transcription factors, Clock and Bmal1 act as transcriptional activators for resetting of the circadian clock (Gekakis et al, 1998). The second loop acts as a negative feedback on the Clock/Bmal1 transcription activation with cryptochrome proteins Cry1 and Cry2 inhibiting Clock/Bmal1 (Kume et al, 1999). Per1 and per2 are part of the second, negatively regulated, loop and have the ability to inhibit

Clock/Bmal1 activated transcription (Jin et al, 1999). Per2 is also thought to act as a stabilizer of the Cry proteins by forming a Per2/Cry complex to prevent the ubiquitination of the two proteins (Isojima et al, 2003).

Studies using mutant mice have been done to further elucidate the role that period genes play in regulation of the circadian clock. Double mutant animals for both Per1 and

Per2 are completely arrhythmic (Zheng et al, 2001). This indicates that the two genes play a critical role in maintenance of circadian rhythms. From a behavioral standpoint,

Per1 null mutants exhibit persistent circadian rhythms but a shorter free-running period than wildtype controls (Cermakian et al, 2001). Per2 null mutants are able to exhibit rhythms with a shorter period for a couple of days in constant dark but eventually lose that rhythmicity (Zheng et al, 1999). 8

The SCN is composed of a dorsomedial region and a ventrolateral region.

Located dorsomedially are vasopressin-expressing cells while the ventrolateral region is composed of gastrin-releasing peptide-expressing cells, calbindin-expressing cells, vasoactive intestinal polypeptide-expressing cells, and cap cells (neurons where p-ERK phosphorylation rhythm is retinally driven) (Antle and Silver, 2005). Phase-delaying light pulses result in the expression of both Per1 and Per2 in the ventrolateral area of the SCN, followed by later expression of only Per2 in the dorsomedial SCN. Phase-advancing light pulses induce Per1 but not Per2 expression that begins in the ventrolateral SCN and later travels to the dorsomedial SCN (reviewed by Antle & Silver, 2005). It is therefore likely that the spread of Per2 expression to the dorsomedial parts of the SCN is responsible for phase delays while spread of Per1 to this area is responsible for phase advances. All of this indicates that Per1 and Per2 play separate but important roles in the regulation of circadian rhythms.

Glutamate

Based on its localization in RHT terminals in the SCN, glutamate is thought to be a likely candidate for the transmission of photic information to the SCN (Ebling, 1996).

This idea is further supported by data linking glutamate release to light exposure as well as light induced behavioral phase shifts. 3H-glutamate is released from the SCN after stimulation of the optic nerve (Liou et al, 1986). When glutamate or NMDA are applied to hypothalamic slices, it induces a phase shift in neuronal firing rhythm that mimics that which results from light exposure (Ding et al, 1994). Mintz et al (1999) further 9

demonstrated that NMDA receptor activation is a necessary step in the transduction of photic information to the SCN by showing that NMDA application to the SCN in vivo results in a light-like phase-response curve. Each of these findings supports the idea that glutamate is involved in the transmission of photic information to the SCN as well as behavioral phase-shifting.

5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-

801) is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist that is thought to act through blockade of an NMDA receptor-gated Ca2+ channel (Huettner &

Bean, 1988). The demonstration that MK-801 blocks the phase-shifting effects of light in both hamsters (Colwell et al, 1990) and mice (Colwell et al, 1991) indicates that NMDA receptors likely play a key role in circadian behavior.

MK-801 has been shown to inhibit the light-induced expression of c-fos in early subjective night (Abe et al, 1991; Vuillez et al, 1998). Blockage of MAPK activation results in a decrease or complete disruption of IEG expression, including c-fos (Dziema et al, 203). Glutamate signaling through NMDA receptors results in the phosphorylation and subsequent activation of the MAPK cascade (Bading & Greenburg, 1991). It is therefore a reasonable hypothesis that MK-801 inhibition of NMDA receptors is resulting in decreased activation of the MAPK cascade, resulting in a decrease in c-fos expression.

Goals

Vuillez et al (1998) demonstrated a pattern of MK-801 induced inhibition of c-fos in the ventral portion of the SCN as well as an upregulation of c-fos in the dorsal portion 10

of the SCN in hamsters. We will examine this expression and its implications in light- induced phase-shifting behavior in mice with the hypothesis that there will be more general attenuation of c-fos expression as opposed to just in the ventral areas of the SCN due to a more diffuse pattern of RHT innervation found in the mouse versus that of the hamster (Johnson et al 1988a; Cassone et al 1988).

A potential mechanism for MK-801-induced decrease in light-induced c-fos expression is via a down regulation of MAPK cascade activation. While there are several differences in the expression patterns of c-fos and egr1, both were attenuated by disruption of the MAPK cascade (Dziema et al, 2003). Based on these findings, we hypothesize that egr1 will show the same changes in pattern of expression in response to

MK-801 as those of c-fos. It is also hypothesized that the actions of MK-801 on egr1 and c-fos are the result of inhibition of NMDA receptors resulting in decreased activation of the MAPK cascade. We will further attempt to ascertain whether these changes in expression differ between phase delaying and phase advancing light pulses.

Figure two illustrates the light-induced effects in the SCN discussed thus far in this paper. The SCN subregions of the mouse are not as anatomically well defined as those of the hamster (Antle & Silver, 2005). It is the overall goal of this study to determine whether regional specificity of IEG expression in the mouse follows the same pattern exhibited by the hamster and to ascertain whether these are general properties of the rodent SCN or are specific to the hamster. We hope to establish a clearer picture of the pathways governing gene expression and expression patterns in the SCN both during 11

the early and the late night as well as establish links between genes (c-fos and egr1) potentially governed by the same pathways.

glutamate MAPK Transcription Activation

During During Late Night Early Night

Phase ? Advance (reset clock) Phase Delay (reset clock) Egr2, Egr3, Nr4a1, Per1, Per2, Dusp1, Rrad, Pim3, LKF4, Gadd45b, BtG2, Tiparp, Jun egr1 c-fos

= inhibits Mk801 = facilitates

Figure Two: Summary of light induced effects in the suprachiasmatic nucleus. It is unclear whether genes induced by light are the result of or cause of clock resetting. These genes are expressed as a result of photic stimulation in the early night, but we do not know what the expression patterns are, if any, of these genes as a result of late night exposure to light. We are not claiming that MK-801 may act directly on egr1 or c-fos to inhibit them but rather MK-801 acting on upstream processes may result in the inhibition of these genes as an end product.

Methods:

Animals

Male and female C57BL/6J mice were bred in house and individually housed in a

12:12 light/dark cycle. Male Syrian hamsters were also bred in house and individually housed in a 14:10 light/dark cycle. All animals were fed ad libitum. All procedures were approved by the Kent State University Institutional Animal Care and Use Committee.

Experiment 1: The Effects of MK-801 on Light-Induced C-fos Immunoreactivity in the Hamster SCN.

Eight male Syrian hamsters were treated with either an intraperitoneal injection of

(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)

(5mg/kg; TOCRIS Bioscience, Ellisville, MO) or saline/vehicle (0.2ml) 15 minutes prior to a 15 minute, 300 lux light pulse. Light pulses occurred at zeitgeber time (ZT) 13. A zeitgeber is an environmental cue, such as the onset of daylight or changes in temperature, which helps to regulate the cycles of an organism's biological clock. With photic entrainment, lights off is defined as ZT 12. Animals were sacrificed one hour following the onset of the light pulse. They were deeply anaesthetized with sodium pentobarbital (200mg/kg) in dim red light. Each animal was perfused transcardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Brains were post-fixed in a 4% paraformaldehyde solution overnight.

13 14

Experiment 2: The Effects of MK-801 on Light-Induced C-fos Immunoreactivity in the Mouse SCN.

Twelve male C57BL/6J mice were treated with either MK-801 (2.5mg/kg) or saline/vehicle (0.2ml) 15 minutes prior to a 15 minute, 300 lux light pulse occurring at

ZT 13. Animals were sacrificed one hour following the onset of the light pulse. They were deeply anaesthetized with sodium pentobarbital (200mg/kg) in dim red light. Each animal was perfused transcardially with PBS followed by 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Brains were post-fixed in 4% paraformaldehyde overnight.

Experiment 3: Assessment of MK-801’s Effect on Light-Induced Phase Shifts.

Nine male mice were housed in 12:12 light/dark for 14 days, by which time consistent wheel-running patterns were established. On day 15, mice were injected with saline (0.2ml, n = 5) or MK-801 (2.5mg/kg, n = 4) 15 minutes before ZT 13 and then given a 15 minute, 200 lux light pulse at ZT 13. The light schedule was then changed to constant dark for 14 days following treatment to establish a post light pulse running pattern. Following those 14 days, the light schedule was returned to 12:12 light/dark and the experiment was repeated with those animals who received saline in the first trial receiving MK-801 in the second and vice versa. Amplitude of circadian phase shifts was assessed using Clocklab software (Actimetrics). Shifts were calculated by fitting a line between days one and seven prior to treatment as well as a line between days four and ten following treatment and determining the distance (in hours) between where line one and line two cross at day one following the phase shift. 15

Experiment 4: The Effects of MK-801 on Immediate-Early Gene and p-ERK

Immunoreactivity Following a Light Pulse in the Early and Late Night

30 male and female mice were treated with either MK-801 (2.5mg/kg) or saline/vehicle (0.2ml) 15 minutes prior to a 15 minute, 300 lux light pulse. Light pulses occurred at either ZT 13 (saline n = 7; MK-801 n = 7) or ZT 21 (saline n = 8; MK-801 n

= 8). Animals were sacrificed one hour following the onset of the light pulse. Mice were anesthetized and sacrificed following the protocol in experiment 2.

C-Fos, Egr1, & p-ERK Immunohistochemistry

40 m coronal sections of SCN were sliced using a vibratome. Sections were incubated overnight in rabbit anti-c-fos (1:5000; Santa Cruz Biotechnology, Santa Cruz,

CA), rabbit anti-egr1 (1:15,000; Santa Cruz) or rabbit anti-phospho-42/44 MAPK

(1:1,000; Cell Signaling Technology, Beverly, MA) in antisera diluent (PBS + 1% normal donkey serum + 0.3% Triton X-100) at room temperature. This was followed by one hour of incubation in biotinylated donkey anti-rabbit secondary antibody (1:500; Jackson

Laboratory, Bar Harbor, Maine) in antisera diluent followed by ABC reagent (Elite

Vector Kit, Vector laboratories, Burglingame, CA) for one hour. Sections were placed in

DAB substrate (Vector kit) using nickel intensification for 2-10 minutes until staining was sufficient. Fos-immunoreactive (Fos-ir), Egr1-ir, and p-ERK-ir cells were counted manually using Image J software. Prior to counting, SCN sections for each animal were separated into “front”, “middle”, or “back” sections based on how rostral or caudal their position was in the SCN. 16

Data Analysis

For experiments one and two data were analyzed using two-way ANOVAs (with treatment and SCN location as factors). For experiment four a three-way ANOVA was used (with treatment, sex, and SCN location as factors). Tukey-Kramer post hoc tests were performed to determine where an interaction was occurring. NCSS statistical software was used to perform all ANOVA and post hoc analyses. For experiments one and two, the SCN was divided into a dorsal 2/3 portion and a ventral 1/3 portion (Figure

3). For experiment three, a paired t-test was conducted using Microsoft Excel software.

For experiment four, the SCN was divided into grids for cell counts. Corresponding grids

(Figure 3) from each half of the SCN were summed and treated as one grid before an

ANOVA was performed.

ventricle

rd 3

Dorsal 2/3

Ventral 1/3

Optic Chiasm b)

1 2 3 3 2 1

4 5 6 6 5 4

7 8 9 9 8 7

Figure Three: (a)An example of the dorsal 2/3 vs. ventral 1/3 division of the SCN. This division was chosen in order to achieve a more direct comparison with the results of other studies. (b)Representation of grid numbers for each half of the SCN. This SCN division was chosen in order to get a closer look at more specific areas of the SCN. Results:

Experiment 1: The Effects of MK-801 on Light-Induced C-fos Immunoreactivity in the Hamster SCN.

In this experiment, the effects of MK-801 on light-induced c-fos expression in the

Syrian hamster SCN were evaluated. MK-801 did not alter the total number of cells expressing c-fos immunoreactivity in the SCN (Figure 4a) in response to a light pulse in the early night (p = 0.23, Mean ± Standard Error of the Mean for saline = 236.4 ± 31.8 and MK-801 = 183.6 ± 26.6). However when the dorsal 2/3 and ventral 1/3 of the SCN were analyzed as separate entities, there was a near significant decrease (p = 0.07, saline

= 73.4 ± 7.3, MK-801 = 55.4 ± 6.1) in c-fos expression following MK-801 treatment in the ventral 1/3. There was no effect of MK-801 on c-fos immunoreactivity in the dorsal

2/3 of the hamster SCN (p = 0.33, saline = 163.0 ± 26, MK-801 = 128.3 ± 21.

18 19

a) b)

Total SCN c-fos Expression Dorsal SCN c-fos Expression 300 300

250 250

200 200

150 150

100 100

Avg Number of CellsNumber Avg of Avg Number of CellsNumber Avg of 50 50

0 0 Saline MK-801 Saline MK-801 c)

Ventral SCN c-fos Expression

140

120

100

40 Avg Number of CellsNumber Avg of

0 Saline MK-801 Figure Four: (a) There was no significant effect of MK-801 on the overall c-fos expression (p = 0.23) or (b) on the dorsal 2/3 (p = 0.33) of the Syrian Hamster SCN. (c)The effect of MK-801 on c-fos expression in the ventral 1/3 of the hamster SCN was not significant (p=0.07). Error bars represent the standard error of the mean.

Experiment 2: The Effects of MK-801 on Light-Induced C-fos Immunoreactivity in the Mouse SCN.

In this experiment, the effects of MK-801 on light-induced c-fos expression in the

SCN of the C57BL/6J mouse were evaluated. Administration of MK-801 prior to a light pulse had no significant effect on the total number of cells expressing c-fos immunoreactivity (p = 0.11, saline = 192.9 ± 17.9, MK-801 = 153.3 ± 16.3) or on immunoreactivity in the dorsal 2/3 (p = 0.59, saline = 123.4 ± 11.9, MK801 = 113.9 ± 13) of the mouse SCN as evidenced by figure 5a,b. MK-801 treatment did result in a significant decrease (p = 0.00098, saline = 69.5 ± 7.2, MK-801 = 39.4 ± 3.7) in c-fos expression in the ventral 1/3 of the mouse SCN.

a) b)

Total SCN c-fos Expression Dorsal SCN c-fos Expression 300 300

250 250

200 200

150 150

100 100

Avg Number of CellsNumber Avg of Avg Number of CellsNumber Avg of 50 50

0 0 Saline MK-801 Saline MK-801 c)

Ventral SCN c-fos Expression

140

120

100

60 *

40 Avg Number of CellsNumber Avg of

0 Saline MK-801 Figure Five: (a)MK-801 had no significant effect on the overall c-fos expression (p = 0.11) or (b) c-fos expression in the dorsal 2/3 of the mouse SCN (p = 0.59). (c)MK-801 treatment significantly decreased c-fos expression in the ventral 1/3 of the mouse SCN (p = 0.00098). * denotes significance. Error bars represent the standard error of the mean.

Experiment 3: Assessment of MK-801’s Effect on Light-Induced Phase Shifts.

The effects of MK-801 on light-induced behavioral phase shifts in mice were evaluated in the early (phase delaying) portion of the night for this experiment. Vehicle treated animals exhibited an average shift of -0.70h and the MK-801 animals shifted an average of -0.66h. A paired t-test indicated that there was no significant difference in behavioral phase shifts between vehicle-treated and MK-801-treated animals (t = 0.87, saline = -0.70 ± 0.19, MK-801 = -0.66 ± 0.18). MK-801 did not attenuate the phase delay in running rhythm caused by a light pulse in the early night. Figure six depicts representative actograms of both treatment groups.

a) Vehicle b) MK-801

c) Vehicle d) MK-801

Figure Six: Actograms of vehicle treated animals with a (a) -0.93h and (c) -1.37h shift and MK-801 treated animals with a (b) -0.98h and (d) – 1.05h shift. Circles indicate administration of a light pulse. Injection occurred 15mins prior to the light pulse. There was no significant difference between the behavioral phase shifts of the two groups (t = 0.87, standard error = ±0.22).

Experiment 4: Effect of MK-801 on Mouse c-fos Immunoreactivity Following a

Light Pulse in the Early and Late Night

For this experiment, the effects of MK-801 on light-induced c-fos immunoreactivity in the SCN were evaluated during both the early and late night for both males and females. The total number of mouse SCN cells expressing c-fos immunoreactivity in the early night was not affected by treatment with MK-801 prior to a light pulse (p = 0.38, saline = 391.9 ± 39.3, MK-801 = 346.3 ± 36.0) (Figure 7a, Table 1).

However c-fos expression was significantly decreased by MK-801 in grids 8 (p = 0.04, saline = 108.3 ± 8.8, MK-801 = 82.2 ± 7.6) and 9 (p = 0.04, saline = 42.2 ±5.5, MK-801 =

29.1 ± 3.1) which correspond to the ventral portion of the SCN (Figure 7b, 10) as seen in experiment two. Administration of MK-801 prior to a light pulse in the late night resulted in a significant decrease in the total number of c-fos expressing cells in the SCN (p =

0.026, saline = 470.4 ± 51.3, MK-801 = 353.3 ± 29.3) (Figure 7a, 9). Those grids which exhibited significant decreases in c-fos immunoreactivity following MK-801 treatment in the late night included grids 2 (p = 0.004, saline = 29.4 ± 6.3, MK-801 = 16.0 ± 2.9), 8 (p

= 0.04, saline = 134.8 ±17.3, MK-801 = 99.1±10.1), and 9 (p = 0.006, saline = 41.8 ± 6.3,

MK-801 = 24.6 ± 3.1) (Figure 11). Grid 2 does not correspond to an area within the SCN but rather is located just dorsal to the SCN. It was analyzed, along with other grids not corresponding to a location within the SCN, for effects outside the SCN that may be spreading to or from the SCN itself. Grids 1, 3, 4, and 5, correspond to the dorsal portion of the SCN as well as areas just outside of the SCN while grids 6 and 7 are located more ventrally in and around the SCN. C-fos immunoreactivity in these grids did not change 25

significantly in the late night (Figure 7c, Table 1). There was no sex difference in the effect of MK-801 on c-fos expression detected in either the early (p = 0.14) or late (p =

0.48) night. There were no interaction effects between area (front, middle, back) and

MK-801 treatment in the early (p = 0.66) or late (p = 0.73) night.

a) b)

C-Fos Response to Mk-801 Saline C-Fos Early Night Grid Comparisons Saline Mk801 Mk-801 600 160

140 500 120 400 * 100 * 300 80

60 200

Avg Number of CellsNumber Avg of 40 * 100 Avg Number of Cells Number of Avg Cells 20

0 0 Early Night Late Night 1 2 3 4 5 6 7 8 9 Time Point Grid c)

C-Fos Late Night Grid Comparisons Saline Mk-801 160

140

120 * 100

40 Avg Number of CellsNumber Avg of * 20 *

0 1 2 3 4 5 6 7 8 9 Grid Figure Seven: Comparison of early vs late night MK-801 treatment on c-fos expression in mice. (a)MK-801 significantly reduced total c-fos expression in the late night (p = 0.026) as well as (b) grids 8 (p = 0.04) and 9 (p = 0.04) in the early night and (c)grids 2 (p = 0.004), 8 (p = 0.04), and 9 (p = 0.006) in the late night. Grid 2 is not located within the SCN. Grids 8 and 9 correspond to the ventral portion of the SCN. * denotes significance. Error bars represent the standard error of the mean.

Mean ± Standard Error Grid p-value of the Mean Table One: Means and Saline MK-801 standard errors for grid 1 0.98 2.5 ± 0.7 2.5 ± 1.1 assessment of c-fos 2 0.56 15.9 ± 3.2 18.5 ± 5.4 immunoreactivity in the

3 0.48 6.7 ± 2.8 3.9 ± 1.6 early and late night. 4 0.55 29.7 ± 4.8 34.0 ± 6.0 *indicates significance. 5 0.86 103.9 ± 10.3 104.9 ± 10.4

6 0.10 43.1 ± 7.3 29.5 ± 5.3 EarlyNight 7 0.87 39.8 ± 5.2 41.6 ± 5.8 8 0.04* 108.3 ± 8.8 82.2 ± 7.6 9 0.04* 42.2 ± 5.5 29.1 ± 3.1

1 0.11 5.4 ± 1.7 2.6 ± 1.1

2 0.004* 29.4 ± 6.3 16 ± 2.9

3 0.10 7.7 ± 2.0 3.5 ± 1.2

4 0.07 36.1 ± 5.4 22.4 ± 5.6 5 0.27 133.5 ± 16.7 113.5 ± 11.9

6 0.74 40.2 ± 5.8 36.4 ± 6.0 Late Night Late 7 0.20 41.6 ± 4.8 35.2 ± 5.1 8 0.04* 134.8 ± 17.3 99.1 ± 10.1 9 0.006* 41.8 ± 6.3 24.6 ± 3.1

Experiment 4 Part 2: Effect of MK-801 on Mouse egr1 Immunoreactivity Following a Light Pulse in the Early and Late Night

The effects of MK-801 on light-induced egr1 immunoreactivity in the SCN were evaluated during both the early and late night for both males and females for this experiment. As with c-fos, the total number of egr1 immunoreactive cells in the SCN during the early night was not affected by treatment with MK-801 (p = 0.44, saline =

265.2 ± 37.9, MK-801 198 = ± 38.5) but late night treatment of MK-801 resulted in a significant decrease in egr1 expression (p = 0.036, saline = 397.6 ± 44.7, MK-801 = 268.7

± 30.4) (Figure 8a, 9). Grid comparisons of egr1 expression in the early night showed no significant differences between vehicle and MK-801 treated animals (Figure 8b, Table 2).

Late night grid comparisons revealed significant decreases in egr1 expression for grids 1

(p = 0.011, saline = 3.1 ± 0.9, MK-801 = 0.5 ± 0.3), 2 (p = 0.0056, saline = 22.8 ± 4.8,

MK-801 = 6.6 ± 2.1), 3 (p = 0.04, saline = 5.6 ± 1.5, MK-801 = 1.5 ± 0.9), 4 (p = 0.047, saline = 24.9 ± 4.5, MK-801 = 11.2 ± 3.9), and 5 (p = 0.066, saline = 123.1 ± 15.6, MK-

801 = 84.4 ± 10.8) which are located dorsally in the SCN as well as just dorsal to the

SCN itself (Figure 12). Grids 6-9, located in the ventral area of the SCN, were not significantly altered by MK-801 treatment in the late night (Figure 8c, Table 2). There was no sex difference detected in the effect of MK-801 on egr1 immunoreactivity in either the early (p = 0.92) or late (p = 0.82) night. There were also no interaction effects between area of the SCN and MK-801 treatment in the early (p = 0.95) or late (p = 0.76) night.

a) b)

Egr1 Response to Mk-801 Saline Egr1 Early Night Grid Comparisons Saline Mk801 Mk-801 600 160

140 500 120 400 100 * 300 80

200 60 Avg Number of CellsNumber Avg of 40

Avg Number of Cells Number of Avg Cells 100 20

0 0 Early Night Late Night 1 2 3 4 5 6 7 8 9 Time Point Grid c)

Egr1 Late Night Grid Comparisons Saline MK-801 160

140

120

100 *

Avg Number of CellsNumber Avg of 40

20 * * 0 * * 1 2 3 4 5 6 7 8 9 Grid Figure Eight: Comparison of early vs late night MK-801 treatment on egr1 expression in mice. (a)MK-801 significantly reduced total egr1 expression in the late night (p = 0.036). (a,b)There was no effect of MK-801 on egr1 in the early night. (c)Egr1 expression in grids 1 (p = 0.011), 2 (p = 0.0056), 3 (p = 0.04), 4 (p = 0.047), and 5 (p = 0.066) of the dorsal SCN were all decreased by MK-801 treatment in the late night. *denotes significance. Error bars represent the standard error of the mean.

Mean ± Standard Error Table Two: Means and Grid p-value of the Mean standard errors for grid Saline MK-801 assessment of egr1 1 0.96 1.0 ± 0.4 1.4 ± 0.9 immunoreactivity in the 2 0.75 10.4 ± 3.4 9.0 ± 5.6 early and late night. *

3 0.59 1.5 ± 0.9 0.4 ± 0.4 indicates significance. 4 0.34 11.9 ± 2.3 15.2 ± 3.9 5 0.88 86.0 ± 13.0 79.2 ± 20.3

6 0.14 28.4 ± 5.8 11.2 ± 4.2 EarlyNight 7 0.96 15.7 ± 4.3 14.2 ± 4.7 8 0.28 87.1 ± 12.9 57.2 ± 6.1 9 0.18 23.2 ± 4.7 10.2 ± 2.1 1 0.011* 3.1 ± 0.9 0.5 ± 0.3 2 0.0056* 22.8 ± 4.8 6.6 ± 2.1 3 0.04* 5.6 ± 1.5 1.5 ± 0.9

4 0.047* 24.9 ± 4.5 11.2 ± 3.9 5 0.066* 123.1 ± 15.6 84.4 ± 10.8

6 0.32 39.6 ± 6.1 29.6 ± 6.6 Late Night Late 7 0.31 30.6 ± 5.5 20.8 ± 6.5 8 0.14 112.7 ± 13.2 86.8 ± 11.3 9 0.44 35.3 ± 7.1 27.3 ± 5.8

C-fos/Saline C-fos/MK-801 Figure Nine: Effect of MK-801 on c-fos and egr1 expression in the late night. The expression of both IEGs is markedly Egr1/Saline Egr1/MK-801 reduced as a result of MK-801 treatment prior to a light pulse.

Saline MK-801 Figure Ten: Grid view of MK-801 inhibition of c-fos expression in grids 8 and 9 in the early night in mice.

8 9 9 8 8 9 9 8

Figure Eleven: Grid

Saline MK-801 view of MK-801 inhibition of c-fos expression in grids 8 and 9 in the late night for mice.

8 8 9 9 8 9 9 8

Figure Twelve: Late Saline night treatment of 1 2 3 3 2 1 MK-801 prior to a light pulse results in inhibition of egr1 4 5 6 6 5 4 immunoreactivity in grids 1-5 of the mouse SCN. MK-801

1 2 3 3 2 1

4 5 6 6 5 4

Experiment 4 Part 3: Effect of MK-801 on Mouse p-ERK Immunoreactivity

Following a Light Pulse in the Early and Late Night

Finally, p-ERK immunoreactivity in the SCN was evaluated during both the early and late night for both males and females following MK-801 treatment prior to a light pulse. MK-801 had no significant effect on the expression of p-ERK in either the early night (p = 0.11, saline = 399.7 ± 32.7, MK-801 = 288.8 ± 60.9) or late night (p = 0.67, saline = 538.4 ± 91.2, MK-801 = 398.4 ± 58.2) time points (Figure 13a). There was also no effect of MK-801 on any of the specific grids in the early night or the late night

(Figure 13b,c; Table 3). No sex difference was detected in the effect of MK-801 on p-

ERK immunoreactivity in either the early (p = 0.31) or late (p = 0.64) night. There were also no interaction effects between area of the SCN and MK-801 treatment in the early (p

= 0.51) or late (p = 0.06) night.

a) b)

p-ERK Response to MK-801 p-ERK Early Night Grid Comparisons Saline Saline MK-801 MK-801 600 160

140 500 120

400 100

300 80 60 200 40

100 AverageDensity Staining of 20 AverageDensity Staining of 0 0 1 2 3 4 5 6 7 8 9 Early Night Late Night Grid c)

p-ERK Late Night Grid Comparisons Saline MK-801 160

140

120

100

AverageDensity Staining of 20

0 1 2 3 4 5 6 7 8 9 Grid Figure Thirteen: (a)MK-801 had no overall effect on p-ERK expression in the early or late night. (b,c) There was also no effect of MK-801 on any of the individual grids in the early or late night. Error bars represent the standard error of the mean.

Mean ± Standard Error Grid p-value of the Mean Table Three: Means and Saline MK-801 standard errors for grid 1 0.53 3.9 ± 3.2 7.5 ± 2.9 assessment of p-ERK staining 2 0.24 22.8 ± 8.3 38.6 ± 8.9 density in the early and late night.

3 0.29 24.8 ± 10.9 53.4 ± 13.5

4 0.32 19.7 ± 5.3 31.0 ± 8.0 5 0.06 55.2 ± 6.8 74.5 ± 11.0

6 0.10 39.9 ± 8.6 65.5 ± 10.4 EarlyNight 7 0.98 33.0 ± 6.8 32.8 ± 5.3 8 0.86 44.6 ± 7.5 46.3 ± 6.4

9 0.99 33.2 ± 6.9 32.9 ± 6.4 1 0.48 11.9 ± 6.8 8.0 ± 1.4 2 0.80 52.1 ± 13.5 49.9 ± 7.9 3 0.28 41.2 ± 11.7 52.1 ± 11.3

4 0.33 45.6 ± 10.0 33.2 ± 6.0 5 0.99 86.5 ± 14.5 86.5 ± 9.9 6 0.16 66.7 ± 8.8 83.1 ± 14.0 Late Night Late 7 0.30 57.9 ± 10.8 43.9 ± 6.9 8 0.61 80.8 ± 10.5 72.4 ± 11.6 9 0.96 62.1 ± 9.0 60.6 ± 9.9

Discussion:

Abe et al (1991) and Vuillez et al (1998) found that, in Syrian hamsters, MK-801 inhibits the light-induced expression of c-fos in early subjective night. There was also a pattern of expression exhibited where c-fos was increased in the dorsal regions of the

SCN and decreased in the ventral regions (Vuillez et al, 1998). To our knowledge, this work has only been done in hamsters and, because the subregions of the mouse SCN are not as anatomically well defined as those of the hamster (Antle & Silver, 2005), we were interested in whether a C57BL/6J mouse would respond to MK-801 with a similar c-fos expression pattern as that of the Syrian hamster. It is unknown where in the SCN the

NMDA receptors that are affected by MK-801 are located, so based on the c-fos attenuation patterns exhibited by Vuillez et al (1998) it was thought that those receptors may be found in the ventral portion of the SCN which coincides with the location of the most dense RHT innervation (Morin et al, 2006). Hamster RHT terminals are more highly concentrated in the ventral SCN whereas mouse RHT terminals, while still located in large quantities in the ventral SCN, can be found throughout the SCN (Johnson et al,

1988b; Cassone et al, 1988; Morin et al, 2006). Based on these differing patterns of RHT innervation between the hamster and mouse, we hypothesized that c-fos in the mouse would be inhibited in the SCN by MK-801 but that there would be a more uniform attenuation instead of the dorsal excitation and ventral inhibition found in the hamster.

38 39

We first ran our experiments on the hamster and, surprisingly, were unable to duplicate the results achieved by Abe et al (1991) and Vuillez et al (1998). We did not see a significant overall attenuation of c-fos expression by MK-801 nor was there visible increase in the dorsal areas of the SCN (figure 4). We did, however, see a near significant decrease in c-fos expression in the ventral areas of the SCN as a result of MK-801 treatment which lines up with the expected results based on previous work. It is unknown why we did not see the same patterns of expression as Vuillez et al (1998). We were unable to find discrepancies between their procedures and ours. It is possible that we did not detect an effect because our sample size was insufficient to detect an effect of this magnitude. This is evidenced by a near significant decrease in ventral c-fos expression which is similar to the ventral inhibition shown by Vuillez et al (1998).

Interestingly, while the results of experiment one did not turn out as expected, we found that administration of MK-801 prior to a light pulse resulted in decreased c-fos immunoreactivity in the ventral 1/3 of the SCN of our C57BL/6J mice. We had hypothesized that, due to a more diffuse RHT innervations pattern found in the mouse versus the hamster (Johnson et al 1988b; Cassone et al 1988; Morin et al, 2006), we would see a more general attenuation of c-fos expression across the SCN as opposed to just in the most ventral part of the SCN. However, as with the hamster, we found no significant overall decrease in c-fos expression in the SCN of these animals as well as no evidence of an increase in dorsal c-fos (figure 5). We did observe a significant decrease in c-fos expression in the ventral 1/3 of the mouse SCN which correlates well with the near significant ventral decrease observed in our hamsters and the pattern of ventral 40

inhibition exhibited by Vuillez et al (1998). It is interesting that, despite the differences in the retinal innervation of the SCNs of these two rodents, they exhibit a very similar response to the NMDA receptor antagonist MK-801.

Glutamate is localized in RHT terminals in the SCN and is released into the SCN after stimulation of the optic nerve (Liou et al, 1986). Therefore, we expected that MK-

801 would show its maximal suppressive effects on c-fos expression in the region of highest retinal terminal density. In the case of the hamster this appears to be true; we see a near significant decrease in c-fos expression in the ventral 1/3 of the SCN where the

RHT terminals/sites of glutamate release are located (Abrahamson & Moore, 2001). The

RHT terminals in the mouse are more widespread throughout the SCN and we would therefore expect to see a greater, more widespread knockdown of c-fos expression in the mouse but instead see the same pattern as exhibited in the hamster.

It has been shown that the release of glutamate into the SCN and the activation of

NMDA-type glutamate receptors results in the phase advances and delays associated with a light pulse (Mintz and Albers, 1997; Mintz et al, 1999). Despite other SCN subregions of the mouse not being as anatomically well defined as those of the hamster (Antle &

Silver, 2005), perhaps the reason for the unexpected similarity in the spatial distribution of light-induced c-fos expression in the SCN to MK-801 between the mice and hamsters is due to a similar organization of NMDA receptors within the SCN. There may be a higher volume of receptors located in the ventral 1/3 of the SCN versus the dorsal 2/3 in both rodents. It is therefore possible that, despite SCN structural differences, light exerts its effects in the same area of the SCN in both species. Also possible is the idea that light 41

affects the entire SCN but only certain parts (the ventral 1/3) contain MK-801 sensitive

NMDA receptors. Some of the effects of light may be mediated by non-NMDA receptors or perhaps by NMDA receptor subtypes that are less sensitive to MK-801.

It is also worth noting that the area of the SCN where we are seeing this ventral inhibition of c-fos immunoreactivity coincides with the area of the SCN occupied by VIP cells. VIP is a neuromodulator that has been implicated in intra-SCN communication

(Antle & Silver, 2005). When injected into the SCN, VIP produces photic-like phase shifts (Piggins et al, 1995). Perhaps there is some effect of MK-801 on the actions of these VIP cells which are key to photic communication within the SCN.

MK-801 has been shown to attenuate the phase shifting effects of light on wheel- running behavior in hamsters, rats, and the C57 mouse (Colwell et al, 1990; Rowe &

Kennaway, 1996; Colwell et al, 1991). To our knowledge, the effect of MK-801 on wheel running behavior has never been examined at a dosage of 2.5mg/kg. It was hypothesized that we would see a greater attenuation of phase shifting at this dosage than the highest dosage used in mice to date (1.5mg/kg) (Colwell et al, 1990). However, our results indicated that mice injected with 2.5mg/kg MK-801 prior to a light pulse exhibited the same degree of phase delay as those mice injected with vehicle.

We originally used a dose of MK-801 that was same as that used in hamster studies (5mg/kg body mass) and found that it was always fatal in the mice. That dosage was therefore reduced by half and the new dose had a 100% survival rate. The dosage of drug used in our protocol (2.5mg/kg) was greater than that used by Colwell et al (1991) whose maximum dosage was 1.5mg/kg. Perhaps there is a ceiling dosage that, once 42

surpassed, results in MK-801 no longer having an effect. There also may be counteracting pharmacological effects once the dose reaches that magnitude. The dosage we used resulted in seizures that, for every animal, lasted the duration of the light pulse and may have interfered with the actions of the drugs. Another major difference between our protocol and that of Colwell et al (1991) was that, in order to maintain consistency of treatment across experiments, we did not release our animals into constant dark 10 days prior to injection and light treatment. We instead followed the protocols outlined by

Rowe & Kennaway (1996), who performed the same behavioral experiment with rats, because they were most similar to the protocols of our c-fos dorsal/ventral studies.

Perhaps the time spent in constant dark is required for MK-801 to exert its effects properly on mice.

Our results, while not consistent with previous studies showing an inhibition of light-induced phase shifts by MK-801, are in line with the results of both experiments 2 and 4 where we see no significant overall effect of MK-801 on c-fos expression in the early night. While we do see an inhibition of c-fos in the ventral 1/3 of mice in the early night, we do not see the widespread inhibition of c-fos that is caused by MK-801 injection in the late night. The use of antisense oligonucleotides to prevent the translation of c-fos protein results in a widespread knockdown of c-fos and a subsequent inhibition of photic phase shifts (Wollnik et al, 1995). It seems that inhibition of c-fos in just the ventral portion of the SCN is not sufficient to inhibit phase shifts in the wheel running rhythms in mice. It would be interesting to perform our behavioral assessment in the late 43

night and see if the overall decrease in c-fos caused by MK-801 injection in the late night is associated with a decrease in the phase-shifting effects of light in mice.

The unexpected outcomes from our first three experiments warranted a more in depth approach to our IEG analysis. We used a standardized grid scheme (figure 3) composed of 9 grids that encompassed the entire SCN (grids 5, 6, 8, and 9) as well as some of the surrounding area (grids 1, 2, 3, 4, and 7) in order to get a more comprehensive idea of which specific locations in and around the SCN are being affected by MK-801 treatment. This allowed us to look at smaller divisions of the dorsal and ventral areas of the SCN to see if there were further effects that our dorsal/ventral analysis may have missed. In addition to looking at the effects of MK-801 on c-fos, egr1, and p-ERK in the early night, we also looked at its effects in the late night.

We first examined c-fos in the early night and found the same results as in experiment two. There was no overall significant difference in c-fos expression in the early night but there was a significant decrease in grids 8 and 9 (figure 7) which correspond to the ventral portions of the SCN. Our analysis of the effects of MK-801 on c-fos expression in the late night also showed a significant decrease in overall expression that we did not see in the early night. This indicates that there is stronger inhibition of c- fos by MK-801 in the late night than in the early night. It is unclear what might be causing this difference between early and late night.

It was hypothesized that egr1 would demonstrate the same patterns in response to

MK-801 as those of c-fos. We found that, as with c-fos, overall egr1 expression in the

SCN was not affected by MK-801 treatment in the early night. Also like c-fos, late night 44

treatment with MK-801 resulted in a significant decrease in overall egr1 expression

(figure 8). However when we looked at the individual grids we found very different patterns of expression between c-fos and egr1. Egr1 grids were not affected in the early night. In the late night we saw increases in grids 1, 2, 3, 4, and 5 which are all located either in the dorsal portion of the SCN or just dorsal to the SCN itself. This appears to be the exact opposite effect seen with c-fos expression in the late night where there were decreases in the ventral portions of the SCN.

Like c-fos, light induced egr1 expression occurs in areas where RHT terminals innervate the SCN (Johnson et al, 1988b) which was why we expected to see the effects of MK-801 exerted in these areas. However, the unexpected areas of egr1 suppression exhibited here further support the idea that MK-801 affected NMDA receptors that are not necessarily located near RHT terminals. It has been shown that, while RHT innervation occupies the vast majority of the mouse SCN there is a scarcity of RHT terminals in the most dorsomedial portion (Morin et al, 2006) which is the area where we are seeing the attenuation of egr1 immunoreactivity. It would seem that NMDA receptor activation in the dorsal portion of the SCN is both necessary and responsible for normal levels of egr1 expression while NMDA receptor activation in the ventral portion of the

SCN is required for normal levels of c-fos expression, regardless of RHT terminal location.

Blockage of MAPK activation results in a decrease or complete disruption of both c-fos and egr1 expression (Dziema et al, 2003) so our final hypothesis was that the actions of MK-801 on egr1 and c-fos brought on via inhibition of NMDA receptors 45

resulting in decreased activation of the MAPK cascade. In order to test this hypothesis we stained for the phosphorylated form of ERK (p-ERK), which is a marker for MAPK activation. We found that MK-801 had no effect on p-ERK in either the early night or the late night. Butcher et al (2003) showed that once a light pulse has ceased, p-ERK levels decay rapidly and decreased to approximately only 50% of the peak value after just 15 min. Our animals were sacrificed 45mins following the cessation of the light pulse and it is therefore likely that the p-ERK levels needed to determine a difference between the vehicle and MK-801 treated animals were no longer present. It will be necessary to assess p-ERK levels almost immediately following the light pulse in order to observe any real affect that MK-801 may be having on MAPK activation.

Overall Conclusions

In our attempts to better characterize the mechanisms governing IEG expression and the link to light-induced phase-changing behaviors through the use of the NMDA receptor antagonist MK-801, we have shown that the location of retinal innervation is not necessarily the site of activation for all immediate early genes. NMDA receptor activation in the dorsal areas of the SCN corresponds to egr1 expression and receptor activation in the ventral areas of the SCN corresponds to c-fos expression. However, c- fos attenuation in just the ventral portion of the SCN is not sufficient to cause a blockage of phase-shifting in the wheel running behavior of mice. Behavioral assessment following

MK-801 injection in the late night, which we have shown causes a more widespread knockdown of c-fos expression, will be required to determine if that is sufficient for a 46

blockage of phase shifts. It is still possible that a decrease in MAPK activation caused by an NMDA receptor antagonist is responsible for the decreases in IEG expression.

Analysis of p-ERK activation immediately after a light pulse will be necessary for further elucidation of this mechanism. References:

Abe, H., Rusak, B., & Robertson, H. (1991). Photic induction of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK- 801. Neuroscience Letters, 127, 9-12.

Abe, K., Kroning, J., Greer, M.A., & Critchlow, V. (1979). Effects of destruction of the suprachiasmatic nuclei on the circadian rhythms in plasma corticosterone, body temperature, feeding and plasma thyrotropin. Neuroendocrinology, 29, 119-131.

Abrahamson, E.E. & Moore, R.Y. (2001). Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections. Brain Research, 916, 172–191

Antle, M.C. & Silver, R. (2005). Orchestrating time: Arrangements of the brain circadian clock. Trends in Neurosciences, 28(3), 145-151.

Aronin, N., Sagar, S., Sharp, F., & Schwartz, W. (1990). Light regulates expression of a Fos-related protein in rat suprachiasmatic nucleus. Proceedings of the National Academy of Sciences, 87, 5959-5962.

Bading H, & Greenberg ME. (1991). Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science, 253, 912– 914.

Butcher, G.Q., Dziema, H., Collamore, M., Burgoon, P.W., & Obrietan, K. (2002). The p42/44 mitogen-activated protein kinase pathway couples photic input to circadian clock entrainment. Journal of Biological Chemistry, 277, 29519-29525.

Butcher, G.Q., Lee, B., & Obrietan, K. (2003). Temporal regulation of light- induced extracellular signal-regulated kinase activation in the suprachiasmatic nucleus. Journal of Neurophysiology, 90, 3854-3863.

Cassone V.M., Speh, J.C., Card, J.P., and Moore, R.Y. (1988). Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus. Journal of Biological Rhythms, 3, 71-91.

Cermakian, N., Monaco, L., Pando, M.P., Dierich, A., & Sassone-Corsi, P. (2001). Altered behavioral rhythms and clock gene expression in mice with a targeted mutation in the Period1 gene. The EMBO Journal, 20(15), 3967-3974.

47 48

Cermakian, N., Pando, M., Thompson, C., Pinchak, A., Selby, C., Guttierez, L., Wells, D., Cahill, G., Sancar, A., & Sassone-Corsi, P. (2002). Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases. Current Biology, 12, 844-848.

Cobb, M.H. (1999). Map Kinase pathways. Progress in Biophysics & Molecular Biology, 71, 479-500.

Colwell, C.S., Ralph, M.R., & Menaker, M. (1990). Do NMDA receptors mediate the effects of light on circadian behavior? Brain Research, 523, 117-120.

Colwell, C.S., Foster, R.G., & Menaker, M. (1991). NMDA receptor antagonists block the effects of light on circadian behavior in the mouse. Brain Research, 554, 105- 110.

Daan, S.A. & Pittendrigh, C.S. (1976). A function analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. Journal of Comparative Physiology, 106, 253-266.

Damiola, F., Le Minh, N., Preitner, N., Kornmann, B., Fleury-Olela, F., & Schibler, U. (2000). Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes and Development, 14, 2950-2961.

Ding, J.M., Chen, D., Webter, E.T., Faiman, L.E., Rea, M.A., & Gillete, M.U. (1994). Resetting the biological clock: nocturnal circadian shifts by glutamate and NO. Science, 266, 1713-1717.

Dziema, H., Oatis, B., Butcher, G.Q., Yates, R., Hoyt, K.R., & Obrietan, K. (2003). The ERK/MAP kinase pathway couples light to immediate-early gene expression in the suprachiasmatic nucleus. European Journal of Neuroscience, 17, 1617-1627.

Ebling, F.J.P. (1996). The role of glutamate in the photic regulation of the suprachiasmatic nucleus. Progress in Neurobiology, 50, 109-132.

Foster, R.G., Hudson, D., Riggs, A., Fiske, S., & Menaker, M. (1991). Circadian photoreception in the retinally degenerate mouse (rd/rd). Journal of Comparative Physiology A, 169, 39-50.

Gekakis, N., Staknis, D., Nguyen, H.B., Davis, F.C., Wilsbacher, L.D., King, D.P., Takahashi, J.S., & Weitz, C.J. (1998). Role of the CLOCK protein in the mammalian circadian mechanism. Science, 280 (5369), 1564-1569.

Guido, M.E., Goguen, D., De Guido, L., Robertson, H.A., & Rusak, B. (1999). Circadian and photic regulation of immediate-early gene expression in the hamster suprachiasmatic nucleus. Neuroscience, 90(2), 555-571.

Güldner, F.H. (1983) Numbers of neurons and astroglial cells in the suprachiasmatic nucleus of male and female rats. Experimental Brain Research, 50, 373-376.

Harrington, M.E., Rahmani, T., & Lee, C.A. (1993). Effects of damage to SCN neurons and efferent pathways on circadian activity rhythms of hamsters. Brain Research Bulletin, 30, 655–669.

Honrado, G.I., Johnson, R.S., Golombek, D.A., Spiegelman, B.M., Papaioannaou, V.E., & Ralph, M.R. (1996). The circadian system of c-fos deficient mice. Journal of Comparative Physiology, 178, 563-570.

Huettner, J.E. & Bean, B.P. (1988). Block of the N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: Selective binding to open channels. Proceedings of the National Academy of Sciences, 85, 1307-1311.

Ikeda, M. & Nomura, M. (1997). cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage. Biochemical and Biophysical Research Communications, 233(1), 258-264.

Isojima, Y., Okumura, N., & Nagai, K. (2003). Molecular mechanism of the mammalian circadian clock. Journal of Biochemistry, 134, 777-784.

Jin, X., Shearman, L.P., Weaver, D.R., Zylka, M.J., De Vries, G.J., & Reppert, S.M. (1999). A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell, 96(1), 57-68.

Johnson, R.F., Moore, R.Y., & Morin, L.P. (1988a). Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract. Brain Research, 460, 297-313.

Johnson, R.F., Morin, L.P., & Moore, R.Y. (1988b). Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin. Brain Research, 462, 301- 312.

Kennaway, D.J., Voultsios, A., Varcoe, T.J., & Moyer, R.W. (2001). Melatonin in mice: rhythms, response to light, adrenergic stimulation, and metabolism. American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology, 282, 358-365.

Kornhauser J.M., Nelson, D.E., Mayo, K.E., & Takahashi, J.S. (1990). Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus. Neuron, 5, 127-134.

Kume, K., Zylka, M.J., Sriram, S., Shearman, L.P., Weaver, D.R., Jin, X., Maywood, E.S., Hastings, M.H., & Reppert, S.M. (1999). mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell, 98(2), 183-205.

Larkin, J.E., Yokogawa, T., Heller, H.C., Franken, P., & Ruby, N.F. (2004). Homeostatic regulation of sleep in arrhythmic Siberian Hamsters. American Journal of Physiology, 287, R104-R111.

Liou, S.Y., Shibata, S., Iwasaki, K., & Ueki, S. (1986). Optic nerve stimulation-induced increase of release of 3H-glutamate and 3H-aspartate but not 3H-GABA from the suprachiasmatic nucleus in slices of rat hypothalamus. Brain Research Bulletin, 16, 527-531.

Lin, J.T., Kornhauser J.M., Singh, N.P., Mayo, K.E., & Takahashi, J.S. (1997). Visual sensitivities of nurr77(NGFI-B) and zif268(NGFI-A) induction in the suprachiasmatic nucleus are dissociated from c-fos induction and behavioral phase-shifting responses. Molecular Brain Research, 46, 303-310.

Mintz, E.M. & Albers, H.E. (1997). Microinjection of NMDA into the SCN region mimics the phase shifting effect of light in hamsters. Brain Research, 758, 245- 249.

Mintz, E.M., Marvel, C.L., Gillespie, C.F., Price, K.M., & Albers, H.E. (1999). Activation of NMDA receptors in the suprachiasmatic nucleus produces light-like phase shifts of the circadian clock in vivo. The Journal of Neuroscience, 19(12), 5124-5130.

Obrietan, K., Impey, S., & Storm, D.R. (1998). Light and circadian rhythmicity regulate MAP kinase activation in the suprachiasmatic nuclei. Nature Neuroscience, 1, 693-700.

Pickard, G.E., Ralph, M.R., & Menaker, M. (1987). The intergeniculate leaflet partially mediates effects of light on circadian rhythms. Journal of Biological Rhythms, 2(1), 35-56.

Pittendrigh, C.S. & Daan, S.A. (1976). A function analysis of circadian pacemakers in nocturnal rodents. IV. Entrainment: Pacemaker as a clock. Journal of Comparative Physiology, 106, 291-331.

Porterfield, V.P., Piontkivska, H., & Mintz, E.M. (2007). Identification of novel light- induced genes in the suprachiasmatic nucleus. BMC Neuroscience, 8(98).

Ralph, M.R., Foster, R.G., Davis, F.C., & Menaker, M. (1990). Transplanted suprachiasmatic nucleus determines circadian period. Science, 247, 975-978.

Rea, M. (1989). Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nucleus. Brain Research Bulletin, 23, 577-581.

Reppert, S.M. & Weaver, D.R. (2002). Coordination of circadian timing in mammals. Nature, 418, 935-941.

Reddy, A.B., Maywood, E.S., Karp, N.A., King, V.M., Inoue, Y., Gonzalez, F.J., Lilley, K.S., Kyriacou, C.P., & Hastings, M.H. (2007). Glucocorticoid signaling synchronizes the liver circadian transcriptome. Hepatology, 45, 1478-1488.

Rilev, J. N. & Moore, R. Y. (1977). Organization of the suprachiasmatic nucleus in the albino rat. Society for Neuroscience Abstract, 3: 355.

Rowe, S.A. & Kennaway, D.J. (1996). Effect of NMDA Receptor Blockade on Melatonin and Activity Rhythm Responses to a Light Pulse in Rats. Brain Research Bulletin, 41(6), 351-358.

Rusak, B. & Boulos, Z. (1981). Pathways for photic entrainment of mammalian circadian rhythms. Photochemistry and Photobiology, 34, 267-273.

Rusak, B., Robertson, H.A., Wisden, W., & Hunt, S.P. (1990). Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus. Science, 248, 1237-1240.

Stephan, F.K. & Zucker, I. (1972). Circadian Rhythms in Drinking Behavior and Locomotor Activity of Rats Are Eliminated by Hypothalamic Lesions. Proceedings of the National Academy of Sciences, 69(6), 1583-1586.

Sumová, A. Zde ka Trávní ková, Mikkelsen, J.D., & Illnerová, H. (1998). Spontaneous rhythm in c-Fos immunoreactivity in the dorsomedial part of the rat suprachiasmatic nucleus. Brain Research, 801, 254–258.

Travnickova-Bendova, Z., Cermakian, N., Reppert, S.M., & Sassone-Corsi, P. (2002). Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1 activity. Proceedings of the National Academy of Sciences, 99, 7728-7733.

van den Pol, A. N. (1980) The hypothalamic suprachiasmatic nucleus: Intrinsic anatomy. Journal of Comprehensive Neurology, 191, 66 l-702.

Vitaterna, M.H., King, D.P., Chang, A.M., Kornhauser, J.M., Lowrey, P.L., McDonald, J.D., Dove, W.F., Pinto, L.H., Turek, F.W., & Takahashi, J.S. (1994). Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science, 264(5159), 719-725.

Vuillez, P., Jacob, N., Teclemariam-Mesbah, R., Van Rossum, A., Viven-Roels, B., & Pévet, P. (1998). Effect of NMDA receptor antagonist MK-801 on light-induced Fos expression in the suprachiasmatic nuclei and on melatonin production in the Syrian Hamster. Journal of Neuroendocrinology, 10, 671-677.

Zheng, B., Larkin, D.W., Albrecht, U., Sun, Z.S., Sage, M., Eichele, G., Lee, C.C., & Bradley, A. (1999). The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature, 400(6740), 169-173.

Zheng, B., Albrecht, U., Kaasik, K., Sage, M., Lu, W., Vaishnav, S., Li, Q, Sun, Z.S., Eichele, G., Bradley, A., & Lee, C.C. (2001). Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell, 105(5), 683-694.