differential effects of constant light on circadian clock resetting by

DIFFERENTIAL EFFECTS OF CONSTANT LIGHT ON CIRCADIAN

CLOCK RESETTING BY PHOTIC AND NONPHOTIC STIMULI

IN SYRIAN HAMSTERS

by

Glenn J. Landry

B.A., Simon Fraser University, 1999

THESIS SUBMITTED IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF ARTS

in the

Department of Psychology

Faculty of Arts and Social Sciences

O Glenn J. Landry 2006

SIMON FRASER UNIVERSITY

Spring 2006

All rights reserved. This work may not be reproduced in whole or part, by photocopy or

other means, without permission of the author.

APPROVAL

Name: Glenn J. Landry

Degree: Master of Arts (Psychology)

Title of Thesis: Differential Effects: of Constant Light on Circadian Clock Resetting by Photic and Nonphotic Stimuli in Syrian Hamsters

Examining Committee:

Chair: Dr. Cathy McFarland Professor, Department of Psychology

Dr. Ralph E. Mistlberger Senior Supervisor Professor, Department of Psychology

Dr. Neil V. Watson Associate Professor, Department of Psychology

Dr. J. David Glass External Examiner Professor, Department of Biological Sciences Kent State University

Date Approved: DEC . a /XOS

SIMON FRASER IUNIVERSITY

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(a) Human research ethics approval from the Simon Fraser University Office of Research Ethics,

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ABSTRACT

Circadian rhythms in Syrian hamsters can be phase shifted by behavioural

arousal during the usual rest phase of the circadian rest-activity cycle. Phase shifts can

be greatly potentiated by prior exposure to constant light for 2 cycles. This coulld reflect

nonphotic input pathway specific changes to the circadian system, or it could be caused

by decreased pacemaker amplitude. If the latter, then phase shifts to any stimulus,

including those activating the photic input pathway, should be potentiated. This

hypothesis was tested by measuring phase shifts induced by microinjections of NMDA

(500nl, 10mM) into the SCN area of hamsters exposed to constant light (LL) or dark

(DD) for 2 days. NMDA induced significant phase delays mimicking those of light

exposure early in the night. The magnitude of these shifts did not differ by prior lighting

condition, suggesting that LL potentiation of nonphotic shifts reflects changes specific to

the nonphotic input pathway.

DEDICATIION

To my father, the most generous and selfless person I've ever known. And to my

mother, who through her persistence and patience, awakened in me a desire to never

stop learning.

ACKNOWLEDGEMENTS

This work would not have been possible without the support of many people. Dr.

Ralph Mistlberger provided guidance and mentorship, thank you. My committee

members, Dr. Neil Watson and Dr. J. David Glass were generous with their time, thank

you. In addition, I'd like to thank the staff of the Simon Fraser Animal Care Facility for

their assistance and technical support. Peter Cheng in the Department of Psychology

also provided valuable technical assistance.

My friends and fellow students provided much needed support. In particular, I'd

like to thank Jessica Green for assisting during surgeries, Ian Webb for his help setting

up data collection, and Mike Pollock for helping with placement surgeries.

I'd like to thank my parents, George & Marlene Bacon, and in-laws, Gord &

Christine Kline, for their endless support and encouragement. My success in this journey

would not have been possible without their generosity.

Finally, I want to thank my best friend, partner, and wife, Catherine Kline, for her

incredible understanding and encouragement. Your many contributions to this project

may not be listed here, but know that I am truly grateful for all that you do.

This research was supported by operating grants from NSERC Canada, to Dr.

Ralph Mistlberger.

TABLE OF COINTENTS

Approval ............................................................................................................................ ii ... Abstract ............................................................................................................................ . H I

Dedication ........................................................................................................................ iv Acknowledgements ........................................................................................................... v Table of Contents ............................................................................................................. vi List of Figures ................................................................................................................... vii

.................................................................................................... Introduction .............. 1 Photic Signal Transduction ............................................................................................... 1

Glutamate and the RHT ........................................................................................ 2 SCN Physiology and Putative Molecular Genetics ................................................ 3 Photic Signalling ..................................................................................................... 5

Nonphotic Signal Transduction .......................................................................................... 8 Phase Response Curves ........................................................................................ 8 Nonphotic Signalling ............................................................................................ 11

Interactions Between Photic and Nonphotic Stirnuli ........................................................ 12 Other Modulating Factors ................................................................................................. 12 Are Clock Parameters Modulated by Constant Light? .................................................. 14

Period .................................................................................................................. 14 Amplitude ............................................................................................................ 15

Limit Cycle Models of Circadian Oscillators ..................................................................... 17

Methods ........................................................................................................................ 20 Animals. Housing. and Apparatus ................................................................................... 20 Surgery ............................................................................................................................ 20 Experimental Protocols ................................................................................................... 21

Effect of LL on Activity-Induced Phase Shifts ...................................................... 21 Effect of LL on NMDA-Induced Phase Shifts ....................................................... 21

Histology ......................................................................................................................... 23 Data Analysis .................................................................................................................. 23

Results ........................................................................................................................... 24 Two Days of LL Potentiates Phase Shifts to Novel Wheel-Induced Running ................ 24 Two Days of LL Does Not Potentiate Phase Shifts to NMDA ........................................ 27 No Effect of Time in LL on NMDA-Induced Phase Shifts ................................................ 30

......................................................................................................................... Histology 31

Discussion ...................................................................................................................... 33

References ....................................................................................................................... 38

LIST OF FIGURES

Figure 1 : Novel Wheel-Induced Phase Shifts ............................................................ 25

........................... Figure 2: Effect of Lighting on Novel Wheel-Induced Phase Shifts 26

Figure 3: Effect of LL on Photic Shifts ....................................................................... 27

Figure 4: Actograms .................................................................................................. 28

Figure 5: Activity Onsets in DD and LL ...................................................................... 29

Figure 6: Activity Onsets after 13 Days in LL ............................................................. 31

Figure 7: Histological Confirmation of Cannula Placement ....................................... 32

Differential Effects of Constant Light on Circadian Clock Resetting 1

INTRODUCTION

All organisms are faced with the challenge of synchronizing their behaviour and

internal physiology to a rhythmically changing external environment dictated by the 24h

solar cycle. These cycles in behaviour (e.g., sleeplwake) and physiology (e.g., core body

temperature, circulating hormone levels, and gene transcription) are known as

circadian (approximately a day) rhythms. Synchrony with the external environm~ent is not

simply reactive, but instead involves an organism's ability to anticipate changes in its

environment. A genetically determined biological clock makes this critically adaptive

anticipation possible. In mammals, the suprachiasmatic nucleus (SCN) acts as the

primary pacemaker, or clock, for biological and behavioural rhythms (see van Ekeveldt,

2000, for a lengthy, wide ranging review of the circadian time-keeping system).

Synchronization, or entrainment, to an environmental cycle requires the ability to receive

and process time of day information from an organism's surroundings, thereby allowing

the organism to make the necessary periodic adjustments. There are two distinct

systems through which the mammalian SCN receives environmental cues, the photic

and nonphotic pathways, both of which are reviewed in the following pages.

Photic Signal Transduction

The parameters of light that are critical for entrainment include wavelength,

intensity, duration, and the timing of onsets and offsets. The clock responds to light in a

phase dependent manner, with phase delays early in the 'subjective night' (the active

phase for nocturnal mammals housed in constant conditions) and phase advances late

in the subjective night. In the 'subjective day' (the phase in constant conditions during


which nocturnal mammals are inactive or sleeping), light does not shift the clock (Daan &

Pittendrigh, 1976). In mammals, the SCN receives photic input directly from the retina,

primarily through the retino-hypothalamic tract (RHT), via photosensitive melanopsin-

containing retinal ganglion cells, which are distinct from the rods and cones of the visual

system.

Glutamate and the RHT

The excitatory amino acid, glutamate (GLU), is the primary RHT neurotransmitter

communicating photic information to the SCN (Ebling, 1996). GLU was targeted as a

potential RHT neurotransmitter following a series of studies that (1) found GLU receptors

in the SCN (Nishino & Koizumi, 1977; Shibata et a/., 1986); (2) located GLU in the RHT

(Castel eta/., 1993; de Vries et a/., 1993; van den Pol, 1991); (3) determined that GLU

was released following optic nerve stimulation (Liou et a/., 1986); (4) showed GLU

release in the SCN via optic nerve stimulation resulted in phase dependent resetting,

mimicking the effects of light (de Vries et a/., 19194); and (5) established that prior bath

application of Kynurenate, an excitatory amino acid (EAA) antagonist, blocked phase

shifts to optic nerve stimulation (Cahill & Menaker, 1987). GLU's role in photic :signalling

was clouded, however, by results from an in vivo study showing that GLU injedions

directly into the SCN resulted in phase shifts that differed from those to light (Meijer et

a/., 1988). These GLU injections resulted in large phase advances during the nnid-

subjective day and only very small delays at night, a pattern similar to shifts induced by

nonphotic stimuli or dark pulses. Moreover, the phase advances were significantly larger

in constant light (LL) than in constant dark (DD). Subsequent in vivo studies, hlowever,

confirmed the importance of GLU in photic resetting when GLU antagonists were shown


to block photic shifts (Colwell et a/., 1991; Colwell & Menaker, 1992; Rea ef a/., 1993).

The issue was finally resolved when studies targeting the SCN using GLU, or the GLU

agonist N-methyl-D-aspartate (NMDA), reported phase dependent resetting mimicking

the effects of light on the clock, both in vitro (Ding ef a/., 1994; Shibata ef a/., 1994) and

in vivo (Mintz & Albers, 1997; Mintz ef a/., 1999). The one conflicting result (Meijer et a/.,

1 994) remains unexplained.

SCN Physiology and Putative Molecular Genetics

Having established the role of GLU and the RHT in photic signal transduction,

and before exploring the mechanisms of photic entrainment, it may be worthwhile to

review SCN physiology and the putative molecidar genetic determinants of circadian

rhythmicity in mammals. Until very recently, the SCN was thought to be organized rather

simply, partitioned both spatially and pharmacollogically into two segments: (1) a core

component located in the ventrolateral SCN (vISCN) comprised of neurons producing

vasoactive intestinal polypeptide (VIP) or gastrin releasing peptide (GRP) colocalized

with GABAergic neurons, and (2) a dorsomedial shell (dmSCN) surrounding thc = core

with a large population of vasopressin (VP) secreting neurons, also colocalized with

GABAergic neurons (Moore ef a/., 2002; van Esseveldt eta/., 2000). Functionally, the

dmSCN is characterized by strong autonomous circadian rhythms in gene expression

with only limited light response, while the reverse is true for the vISCN. As will be

discussed later, this view of SCN organization is likely far too simplistic, but serves well

now for this introduction to signal transduction. In part because the vlSCN is richly

retinorecipient, while the dmSCN is not, it is believed that the vlSCN is the primary


receiver of environmental inputs, whereas the dmSCN is thought to be more involved in

signal output from the clock to other areas of thle brain.

A number of genes expressed in the SCN have now been identified, but a gene's

expression in the SCN does not necessarily qualify it as a clock gene. A clock gene

plays a critical role in the generation of circadian rhythms, whereas clock controlled

genes (CCGs) are simply outputs of the clock. Generally, to be considered a clock gene,

the gene must satisfy the following criteria (Aromson etal., 1994): (1) the absence of the

gene or the protein translated from it must lead to arrhythmicity; (2) there must be a

circadian rhythm in transcriptionltranslation of the gene in the absence of environmental

time cues (Zeitgebers); (3) Zeitgebers must phase shift this rhythm; and finally, (4) this

rhythm must be phase shifted by manipulation of protein levels. Several mammalian

clock genes have been identified, including three homologs of the Drosophila period

gene (perl, per2, and per3), the Drosophila cycle homolog (bmall), two cryptochrome

homologs (cry1 and cry2), and clock.

The fundamental characteristics of all k~nown circadian oscillators are the same;

from single cell cyanobacteria to complex multi-cellular mammals, the clock gene system

involves positive elements driving gene transcription with the translated protein products

negatively feeding back on their own transcription. In the mouse, for example, clock and

bmall form a promoter complex driving the expression of clock and clock controlled

genes. PERI-3 proteins, phosphorylated via casein kinase IE (CKIE), form heterodimers

with CRY1 and CRY2 proteins in the cytoplasm, allowing nuclear trans location^ and

negative feedback on gene transcription (Dunlap, 1999; Okamura et al., 2002; Preitner

etal., 2002; Reppert & Weaver, 2001). This mechanism results in a self-sustained

oscillation of clock and clock controlled gene e:wpression. Of particular importance to


signal transduction pathways and the mechanism of entrainment are per? and per2 gene

expression. For both genes, messenger RNA (nnRNA) levels begin to rise from a mid-

evening to late night nadir, to a mid-to-late afternoon peak. The challenge of entrainment

for an organism is to match the period of its endogenous clock (tau) with the period of its

external environment (T), which requires changes in clock gene expression in

accordance with external photic and nonphotic signalling. Steady-state entrainrnent is

achieved when the daily phase-shift (A@) matches the difference between tau and T.

These genetic determinants of circadian rhythm~icity and the mechanisms of entrainment

will be examined in greater detail below.

Photic Signalling

With this understanding of clock physiology and molecular genetics, we can now

continue our review of photic signalling. The effiects of GLU on the clock are gated in

part via a circadian rhythm in expression of two classes of GLU receptors: NMDA and

non-NMDA receptors (Gillette & Mitchell, 2002; Schurov eta/., 1999). Following light

exposure during phases that shift the clock (subjective night), GLU release in the vlSCN

initially activates the non-NMDA receptors, amino-methyl proprionic acid (AMPA) and

Kainate (KA), depolarizing the cell and eventually removing Mg2' blockage of NMDA

receptors. Removal of this ~ g ~ ' block releases NMDA receptors leading to an iinflux of

calcium (ca2'), which initiates a second messenger cascade ultimately producing a

phase shift of circadian clock gene cycles.

One effect of ca2' influx is the activation of nitric oxide synthase (NOS),

producing nitric oxide (NO) and stimulating two distinct signalling pathways gated by

circadian phase. In the early subjective night NO activates an as yet undefined pathway


involving ryanodine receptors, ultimately resulting in phase delays of the clock.

Conversely, in the late subjective night NO triggers a pathway involving guanylyl

cyclase (GC), cyclic GMP (cGMP), and protein b a s e G, leading to phase

advances (Gillette & Mitchell, 2002). Another effect of increased intracellular calcium

involves calmodulin (CaM) activation, thereby initiating CaM-dependent kinases and the

phosphorylation of cyclic AMP (CAMP) response element-binding

protein (CREB) (Schurov et al., 1999). Phosphorylated CREB (pCREB) then binds to

CAMP response element (CRE) on various prornoter sites on genes, inducing expression

of the immediate early genes c-fos, jun-B, and egr-I. Immediate early gene (IEG)

induction by light at night (Kornhauser etal., 1990; Rusak etal., 1990) is correlated with

light induction of perl expression (Albrecht et a/., 1997; Field etal., 2000; Shigeyoshi et

a/., 1997), perhaps providing the necessary entrainment connection between the

environmental photic signal and the biological c.lock. Though IEG induction by light

capable of shifting the clock precedes light-induced increases in perl , causality has not

been established. Nevertheless, in the case of , ~ e r I , the speed, magnitude, and

sensitivity of light-induced gene expression is correlated with the extent of clock

resetting, consistent with light induction of perl playing a critical role in photic

resetting (Shigeyoshi etal., 1997).

A plausible mechanism of photic resetting can be summarised as follows: the

circadian rhythm of perl mRNA levels in the SCN decrease in the early evening;

therefore light exposure at that phase, which increases perl mRNA levels and thus

delays the decrease in mRNA, results in a phase delay of the circadian rhythm.

Conversely, light exposure in the late night, wh~en perl levels are increasing, results in


further increase in perl causing the clock to phase advance. However, as was

mentioned earlier, this view of SCN organization and function is likely too simplistic.

Silver and colleagues have recently proposed a cellular model of the SCN

circadian pacemaker that at least initially promises to be controversial, but

compelling (Antle etal., 2003). Their model assigns distinct functions to SCN

subregions; a core region is conceptualized as a 'gate' for environmental inputs, and a

shell region as the circadian oscillator (Antle & !Silver, 2005). In summary, they have

shown that the hamster SCN is comprised of a dorsomedial shell of VP expressing cells,

and a core with four distinct cell groups: (1) VIP cells in the ventral most region; (2) a

cluster of calbindin (CalB) containing cells dorsal to the VIP cells; (3) a set of GRP-

activated cells forming a cap over the CalB cell!;; and (4) a cluster of GRP expressing

cells that overlap the CalB and "cap" cells. Examination of photic signalling within the

hamster SCN identifies GRP as the output signal of retinorecipient CalB cells, \~h i ch

activates cap cells (Antle et a/., 2005). Support for the gate and oscillator concept comes

from temporal and spatial expression patterns of canonical clock genes and

CCGs (Hamada etal., 2004b). Following a light: pulse, perl expression occurs first in the

retinorecipient core of the SCN (within 60min of light exposure), after which perl

expression is seen in the shell (90min after the light pulse). Taken together, these

findings fit nicely with functional differences between the core and shell of the SCN

discussed earlier. The non-rhythmic, light sensi'tive core gates environmental cues, and

communicates photic entrainment signals to the light-insensitive, oscillating shell via

GRP signalling.

To recap, photic signal transduction likely involves a complex cascade of events,

from GLU activation of non-NMDA and NMDA receptors in the SCN core, to CalB-driven

Differential Effects of Constan~t Light on Circadian Clock Resetting 8

GRP activation of cap cells, thereby increasing ,per expression in rhythmic cells of the

SCN shell, ultimately resulting in phase dependent advances or delays of the clock.

Nonphotic Signal Transduct ion

Light is likely the principle zeitgeber for entrainment of the mammalian c;lock, but

nonphotic stimuli have also been shown to phase shift the clock (e.g., behavioural

arousal, serotonin, neuropeptide Y, GABA, ben.zodiazepines, and melatonin) (Hastings

eta/., 1998). For a number of species the circadian pacemaker has been shown to

entrain to nonphotic stimuli, under constant conditions in the absence of a lightldark (LD)

cycle (Edgar & Dement, 1991 ; Marchant & Mistlberger, 1996; Mistlberger, 199'1 ; Reebs

& Mrosovsky, 1989). While photic signalling is rnediated via a single pathway, the RHT,

nonphotic signalling is likely to involve at least two afferent pathways to the SCN: (1) a

neuropeptide Y (NPY) input from the intergeniculate leaflet (IGL) of the thalamus, via the

geniculo-hypothalamic tract (GHT); and (2) a serotonin (5HT) input direct from the

median raphe nuclei. In addition, the dorsal raphe has efferent connections to the IGL,

which may provide an indirect 5HT input to the SCN, via modulation of NPY output.

Phase Response Curves

For both photic and nonphotic stimuli we can construct a phase response

curve (PRC) by plotting the magnitude and direction of resultant phase shifts as a

function of the circadian phase of stimulus onset. As reviewed earlier, the PRC: to light is

defined by delays early in the subjective night, advances in the late subjective night, and

no response during the mid-subjective day. The first nonphotic PRC was actually

produced by a photic manipulation. Hamsters free-running in LL were exposed to 2-6h of


darkness. This dark pulse PRC was characterized by delays in the late subjective night

and advances beginning in the late subjective day and extending into the early

subjective night (Boulos & Rusak, 1982).

Phase shifts to dark pulses were originally interpreted as the mirror image of

shifts to light pulses. Subsequent studies showed, however, that the critical variable may

not be darkness per se, but rather the locomoter activity stimulated by dark (Mrosovsky

& Salmon, 1987; Reebs et a/., 1989; Reebs & Mrosovsky, 1989; Van Reeth & 1-urek,

1989). Initially, Mrosovsky & Salmon (1987) established that activity stimulated by 3h

confinement to a novel wheel greatly accelerated entrainment to an LD cycle advanced

by 8h. Reebs & Mrosovsky (1 989) showed that dark pulses weren't necessary .to

produce phase shifts. Their study defined the PRC to novel wheel-induced activity,

which is characterized by advances in the mid-subjective day, no response in tlhe early

subjective night, and small delays in the mid-to-late subjective night. Despite subtle

differences in the dark pulse and activity PRC1s, studies showing that shifts to dark

pulses could be blocked by restricting activity, either by confinement to a small nest box

(Reebs et a/., 1989) or by restraint (Van Reeth I& Turek, 1989), suggested that dark

pulse shifts were the result of behavioural activation or some correlate thereof. Thus, this

type of PRC came to be known as the activity or exercise PRC.

Intensity of activity, as measured by the number of wheel revolutions during

novel wheel confinement, was later shown to be an excellent predictor of the magnitude

of phase shifts, defined by a sigmoidal relationship (Janik & Mrosovsky, 1993). Wheel

counts less than 4000 resulted in small advances or no response at all, whereas

maximal advances were observed when wheel counts exceeded 5000. Howevler, the

issue was once again clouded by more recent studies showing that exercise or intense


activity is not necessary, since sleep deprivation by gentle handling produces an

exercise type PRC (Antle & Mistlberger, 2000; Mistlberger etal., 2003; Mistlberger etal.,

2002). Could it be that hamsters failed to shift in prior novel wheel manipulatior~s

because the hamsters slept in the wheels interrnittently and thus had lower wheel

counts? Is sustained wakefulness sufficient to explain the exercise type PRC? Alas, the

answer is not that simple since other procedures capable of sustaining wakefulness fail

to produce exercise type phase shifts. EEG cor~firmed wakefulness can be achieved

without activity using either a restraint tube or confinement to a pedestal over water, but

neither procedure results in phase shifts (Mistlberger etal., 2003). Both procedures

induce stress, however, and thus these results suggest that either some level of

locomoter activity is necessary, or something albout these stress paradigms inhibits

shifting. The nonphotic picture becomes murkier still, given that restraint has its own

PRC, characterised by delays in the early subjective night, but no advances in the

subjective day (Van Reeth etal., 1991). To date, the critical stimulus mediating the

nonphotic shifts described above is yet to be determined.

Analysis of the relationships between photic and nonphotic phase response

curves provides some insight, however (Rosenwasser & Dwyer, 2001). Rosen\~asser's

findings suggest that the PRC's to light and act~ivity are virtually identical in shape, but

with opposite phasing of sensitivity, indicating that photic and nonphotic shifting may

involve a shared mechanism, at least at some level. We will return to this idea later. With

respect to the dark pulse PRC, Rosenwasser's results point toward a more cornplex

function involving the summation of the activity PRC and a mirror image of the photic

PRC. Thus it would seem that the dark pulse PRC is neither photic, nor nonphotic, but

rather is a hybrid of the two. For convenience, from this point forward the term


'nonphotic' will be used in reference to the PRC produced by stimulated running or sleep

deprivation by gentle handling.

Nonphotic Signalling

The evidence supporting the importance of the IGL and NPY in nonphotic shifting

is clear (Challet et a/., 1996; Harrington, 1997; lvlarchant & Mistlberger, 1996; Marchant

et a/., 1997; Maywood et a/,, 1997). Activation of the IGL is both necessary and sufficient

to induce nonphotic resetting. Unfortunately the 5HT picture is far more

contradictory (Mistlberger et a/., 2000). 5HT levels have a strong positive correlation with

behavioural activation throughout all clock phases, however, to date it is not clear that

5HT is necessary or sufficient to reliably alter clock phase. Species differences or subtle

changes in methodology may be the key to contradictory 5HT findings. Or perhaps the

signalling mechanisms vary for different nonphotic cues. Nevertheless, nonphc~tic signal

induction likely involves opening potassium (K':) channels, thereby hyperpolarizing the

cell and activating protein kinase C or protein kinase A cascades for NPY or 5HT,

respectively (Hall et a/., 1999; Jiang et a/., 1995; Prosser et a/., 1994). Thus, nonphotic

signal transduction appears to suppress activation of the SCN core, while photic

signalling does the reverse. These findings are supported by examination of cfos and

per? expression following nonphotic resetting, \~h ich indicates that cfos is suppressed by

sleep deprivation (Antle & Mistlberger, 2000) and that nonphotic resetting is the direct

result of decreased per? expression in the SCbI (Hamada etal., 2004a; Maywood &

Mrosovsky). However, recent findings suggest that light exposure and melatonin

administration block 5HT resetting without blocking the 5HT-induced down-regulation of

per? or per2 mRNA levels in the SCN (Caldelars etal., 2005). Thus it is important to point


out that per is likely just one component of a vely complex mechanism probably

involving several redundancies.

Interactions Between Photic and Nonphotic Stimuli

Given that photic resetting involves increased per expression, while nor~photic

resetting suppresses per expression, it is not surprising that these zeitgebers interact in

complex ways. In addition, because the terminall fields of all three major projections to

the SCN (the RHT, GHT, and median raphe) arje colocalized in the core, this overlap is

suggestive of synaptic contacts between the three input pathways (Morin, 1994., 1999;

Morin eta/., 1992). Thus, interconnectivity provides the means for interaction, while

regulation of per expression is likely the medium (Challet et a/., 2003). An example of

this interaction is found in the behavioural inhibition of photic resetting, which appears to

involve 5HT modulation of photic regulation (Mistlberger & Antle, 1998). However, NPY

likely plays a role as well, since NPY differentiallly suppresses photic induced per1 and

per2 mRNA (Brewer et a/., 2002). Interestingly, the reverse interaction is possible:

nonphotic advances to activity in the mid-subjective day can be blocked by a subsequent

light pulse, even though light alone at this time would not shift the clock (Joy & Turek,

1992; Mrosovsky, 1991). Taken together, these findings suggest that photic and

nonphotic stimuli combine in a complex manner, resulting in non-linear interactions.

Other Modulating Factors

While GLU has been shown to be the primary neurotransmitter of the pliotic

pathway, other transmitters have been localized in the RHT including substance P (SP)

and pituitary adenylate cyclase-activating peptide (PACAP) (Hannibal, 2002). The

Differential Effects of Constant Light on Circadian Clock Rese1:ting 13

literature on SP is contradictory and it is not yet clear whether SP is intrinsic to or an

afferent neurotransmitter of the SCN, nonetheless it is likely a modulator of photic

signalling (Hannibal, 2002). In contrast, the PACAP research clearly indicates its direct

role in modulating the effects of GLU on clock phase (Chen etal., 1999; Hannibal etal.,

1997; Kopp etal., 2001). PACAP appears to have both dose and phase depenldent

effects on clock function. While very low concer~tration levels (< I nM) result in photic-like

phase shifts at night, likely through NMDA receptor potentiation, higher concentrations (>

10nM) result in nonphotic effects including phase advances in the subjective day and

inhibition of GLU-induced phase advances in the late subjective night (Chen etal., 1999;

Harrington et a/., 1999). At this higher concentration, however, PACAP potentiated GLU-

induced phase delays in the early subjective night. The dose dependent interaction of

PACAP and GLU is thought to involve modulation of SCN per gene expression: nM

concentrations induce per expression, while pM concentrations block GLU-induced per

expression (Hannibal, 2002).

As discussed earlier, light can block nonphotic resetting. In addition,

environmental light history, in particular the duration of the photic component of'the LD

cycle, has been shown to modulate nonphotic phase shifts (Knoch et a/., 2004;

Mistlberger et a/., 2002). Hamsters housed in L1- vs. LD, showed strongly poten~tiated

phase advances to sleep deprivation (SD), novel wheel-induced activity, and in,jections

of the 5HT1~,7 agonist, &OH-DPAT, during the mid-subjective day. Interestingly, the

effect of LL on these nonphotic stimuli was transient: potentiation was observed after

1-3 days in LL, but not after more than 10 days in LL. The mechanism by which1 LL

potentiates nonphotic resetting is undetermined, but at least two possibilities exist. The

first possibility is that strong resetting in LL is specific to the nonphotic pathway. LL may


somehow increase receptor sensitivity to 5HT and/or NPY. Another possibility is that the

phase resetting potentiation observed following brief LL exposure is the result of reduced

pacemaker amplitude. Limit cycle models of circadian oscillators predict that as the

amplitude of cycling decreases, the size of phase shifts to phase resetting stimuli will

increase, and there is empirical evidence consistent with this prediction (Johnston et a/.,

chapter in Dunlap textbook; Jewett et a/., 1991). Limit cycle models and their

implications for the amplitude hypothesis of LL potentiation of nonphotic resetting will be

discussed in greater detail below.

Are Clock Parameters Modulated by Constant Light?

The state of an oscillator can be definedl by three parameters: (1) its current

phase (a particular point in the cycle, i.e., activity onset); (2) its period (tau, the rate at

which the oscillator cycles); and (3) its amplitude (the distance from peak of os~cillation to

its mid-point, the point at which there would be no oscillation at all - called the point of

singularity, where the oscillator has stopped and the state variables are completely

steady) (Lakin-Thomas, 1995). Acute changes in light alter phase, as described in the

PRC section above. The effect of light on the period and amplitude of an oscillator are

discussed below.

Period

Now known as Aschoffs rule, tau of nocturnal and diurnal organisms is

differentially dependent on light intensity (Aschoff, 1960): tau lengthens for nocturnals

housed in high versus low intensity light, while tau shortens for diurnals under the same

conditions. In addition, the length of the light cycle (photoperiod) in LD alters tau in a


history dependent manner, called after-effects (Cambras et a/., 2000; DeCoursley, 1961;

Joshi & Gore, 1999; Pittendrigh & Takamura, 1989). Photoperiodic effects on tau are

similar to those of light intensity on tau: as the duration of light increases, tau in~creases

or decreases for nocturnals or diurnals, respectively. In nocturnals previously entrained

to LD, tau modulation is strikingly evident during the initial days following release into LL,

with progressive lengthening of tau each day for several days, until the rhythm stabilizes

with a significantly increased tau (Aschoff, 1960; Pittendrigh & Daan, 1976b).

Amplitude

While direct measures of pacemaker amplitude are not yet available, a number of

clock outputs have been used in an attempt to assess amplitude changes in LL..

Because the examples listed below involve cloc:k outputs (hands of the clock), however,

one should keep in mind that these hands may be reflective of clock state, but are not

the actual clock. The important point here is that it is possible to modulate or ewen stop

clock output, without necessarily altering parameters of the clock itself. In the case of tau

modulation by LL, it is clear that the frequency of the clock has changed, rather than just

its outputs. However, with respect to amplitude, while the hands are clearly darnped in

LL, it is as yet unclear whether that suppressiorl extends to the source of rhythmicity.

In nocturnals, LL has perhaps its most profound effect on the intensity and

amplitude of clock outputs, seen most clearly in the circadian resuactivity rhythm

(Aschoff, 1960; Eastman & Rechtschaffen, 1983; Redlin, 2001), but also observed in

circadian rhythms of temperature (Eastman & Flechtschaffen, 1983), melatonin (Redlin,

2001), SCN neuronal discharge (Mason, 1991), and protein expression (Moriya etal.,

2000; Sudo etal., 2003). In every case, LL strongly dampens both the intensity and


amplitude of these clock outputs, providing classic examples of a phenomenon called

masking (Aschoff, 1960). For example, activity can be used as an indirect measurement

of clock state, but LL strongly suppresses activity, masking it, and rendering it less

effective as a marker of clock function.

At this point, returning briefly to SCN organization will help illuminate the

mechanism by which LL likely disrupts rhythms in nocturnals. The SCN is comprised of

bilateral nuclei containing -10,000 neurons each (Van den Pol, 1980). Within these

nuclei are a large population of autonomous single cell oscillators, forming the :SCN shell

(Antle & Silver, 2005; Welsh etal,, 1995). These single cell oscillators are mutuially

coupled via synaptic connections (Guldner & Wolff, 1996), gap junctions (Van den Pol,

1980; Welsh & Reppert, l996), and diffusible substances (Bos & Mirmiran, 1990; Welsh

etal., 1995). In nocturnals, LL disrupts the coupling of these single cell oscillators and as

they drift out of phase from each other, output signal amplitude decreases, eventually

resulting in arrhythmicity. Similarly, splitting is the result of decoupling of the left and right

SCN (de la lglesia et a/., 2000; Pickard & Turek, 1982). Thus amplitude could be

suppressed in LL by phase dispersion of a popidation of oscillating clock cells, I.e., an

extracellur effect, or perhaps LL suppresses the amplitude of each SCN neuron as well,

an intracellular effect.

As discussed above, one way to assess amplitude is to look for cellular and

molecular correlates of pacemaker amplitude, but we are as yet unsure what these may

be. Over the years with the advance of technololgy and refinement of circadian models of

clock function, studies have navigated upstream closer to the source, but have not yet

reached the circadian clock. Fortunately, it may be possible to answer the amplitude


question without actually tapping into the clock. Theoretical modelling of the pacemaker

as a limit cycle oscillator provides the shortcut.

Limit Cycle Models of Circadian Oscillators

Beginning with a simple limit cycle oscillator, the two dimensional preda~tor-prey

model of population oscillation provides a good example (Johnson eta/., 2004). The

dynamics of population interdependence are easy to understand: as prey become more

plentiful, the predator population can grow, but with increased numbers of predlators

comes a decrease in prey population, which in turn decreases predator population,

ultimately allowing the cycle to begin anew with1 a subsequent growth in the prey

population. Assuming external factors remain constant, the result is a self-sustained

oscillation in predator-prey populations. However, if external factors (e.g., rain or

drought) change environmental parameters causing an increase or decrease in prey

population, for example, the system adjusts with an increase or decrease in predator

population, respectively. This simple limit cycle model can be applied to circadian

oscillation in gene expression. For example, in much the same way that prey availability

drives increases in predators, per mRNA drives levels of PER protein, and just as

predators feedback on prey population, increased PER levels suppress per mFiNA

expression. The phase response of population levels to external factors translates nicely

as well: we can think of increases in population as phase advances and decreases in

population as phase delays. Computer simulations confirm that this simplistic rnodel

works well when describing small adjustments, such as the phase shifts typically

required for entrainment to the 24h solar cycle (Lakin-Thomas, 1995; Winfree, 1980,

1987). However, assuming the phase resetting stimulus strength is held constant across


conditions, this model fails to account for strongly potentiated resetting of circadian

rhythms.

There are, however, studies that report very large phase shifts, known iaS Type 0

resetting. When comparing strong versus weak resetting, if we produce a phase

transition curve by plotting new phase as a function of initial phase, the slopes are 0 and

1, respectively. Hence, strong resetting is designated Type 0 and weak resetting is

designated Type 1 (Winfree, 1980). Examples of Type 0 resetting in hamsters ,were

described earlier (Knoch et a/., 2004). Type 0 resetting has also been observed in

humans (Jewett eta/., 1991) as well as invertebrates and plants (Engelmann 8;

Johnsson, 1978; Shaw & Brody, 2000; Winfree, 1973). With the increasing evidence of

Type 0 resetting, revisions to limit cycle models of circadian oscillators were necessary.

The simple limit cycle model, for example, could not explain LL potentiation of nonphotic

resetting, if we assume the resetting strength of each nonphotic stimulus remalined the

same under both lighting conditions.

As discussed earlier, an oscillator has three parameters, phase, period, and

amplitude. In the case of the simple limit cycle oscillator, period and amplitude are fixed,

and phase is the only parameter that varies over time. Lakin-Thomas (1995) defines

phase as a state variable, explaining that simple oscillators are 'phase only systems',

meaning that in order to describe the state of the system completely, we need only know

its phase. Thinking of phase as a circle, the system moves along the circle but cannot

move inside or outside of the circle. Thus for a simple oscillator, the diameter of the

circle (amplitude) remains constant. As noted above, a system with only one state

variable (i.e., phase) fails to explain Type 0 resetting, but adding a second stat~e variable

(i.e., amplitude) allows for strong resetting. According to the non-simple limit cycle


model, even when strength of the resetting stimulus is held constant, decreased

pacemaker amplitude will theoretically result in strongly potentiated, Type 0 resetting

(Lakin-Thomas, 1995). In addition, this non-simple limit cycle model allows for i3

precisely timed stimulus of the appropriate magnitude to instantaneously stop the

circadian clock, which makes this model particularly attractive given the reported effects

of light on behavioural rhythms (Engelmann & Johnsson, 1978; Jewett eta/. , 1991).

In summary, given that transient exposure to LL potentiates nonphotic shifts,

resulting in Type 0 like resetting, there are two equally plausible explanations: (1)

potentiation could be due to effects of LL that in some way hypersensitizes the clock to

nonphotic stimuli in a pathway specific manner; or (2) LL could suppress pacemaker

amplitude. If the latter, we would expect LL to potentiate phase shifts to any resetting

stimuli, including nonphotic and photic stimuli. TO test the amplitude suppression

hypothesis of LL potentiation, we examined phase shifts to NMDA, which activates the

photic input pathway, following two complete cycles in LL or DD. LL potentiation of both

photic and nonphotic resetting would provide st~rong evidence for the amplitude

hypothesis.


METHODS

Animals, Housing, and Apparatus

Adult male Syrian hamsters (90g) were obtained from Charles River (St.

Constant, PQ, Canada). Hamsters were group housed in a 14:lO LD cycle priolr to

surgery. Food (Rodents Lab Diet #5001; Purina Mills Inc., St Louis, MO, USA) and water

were available ad libitum throughout the study. After surgery hamsters were placed in

ventilated isolation cabinets under a 14:lO LD cycle (-1000:O lux), individually housed in

wire bottomed polypropylene cages (45 X 25 X 20cm) with 17cm running wheels.

Running wheel activity was recorded using a micro-switch connected through an

interface to a 486 PC (Activity Counting System, Simon Fraser University). Data were

collected in 10min bins and periodically downloaded to a Macintosh computer for

analysis using Circadia.

Surgery

Once hamsters reached the target weight for surgery (1 10-120g), they were

deeply anesthetized with i.p. injections (ketamine 135mg/kg, xylazine 25mg/kg, and

acepromazine 2mglkg) and placed in a stereotaxic frame. After bregma and lambda

were levelled, a 22 gauge guide cannula extending 9mm from the pedestal was

implanted at a 1 O0 angle toward midline. Target: co-ordinates for stereotaxic implants,

determined by prior placement surgeries targetiing the dorsal SCN, were 0.4mrn anterior

and 1.7mm lateral to bregma and 6.85mm ventral to the surface of the skull. The implant

was fixed to the skull using dental acrylic anchored by four jeweller's screws. To prevent


blockage or contamination, a dummy cannula was kept in the guide cannula and

removed only for injections. The dummy cannula as well as the 28-gauge injector

cannula protruded I mm from the guide cannula

Experimental Protocols

Effect of LL on Activity-Induced Phase Shifts

Previous studies have shown that prior exposure to 2-3 days of LL potentiates

phase shifts to nonphotic stimuli (Knoch eta/ . , 2004; Mistlberger et a/., 2002). To confirm

these findings, hamsters entrained to a 14:lO LD cycle (-1000:O lux) were explosed to

LL (-1000 lux), beginning at zeitgeber time (ZT) 12 for 2 days (by convention, ZT 12 is

dark onset). On the third day of LL, the lights wlere turned off -6h before the beginning of

the daily active phase (by convention, activity onset for nocturnals is designated

circadian time (CT) 12) and the animals were transported to and confined in a novel

running wheel for 3h. The hamsters were then returned to their home cages and left

undisturbed in DD (<llux) for 5-7 days. The hamsters were subsequently re-entrained to

the LD cycle for 7-10 days before the next manipulation. In the control conditio~n all

procedures remained the same except that hamsters were maintained in LD ra~ther than

LL prior to the onset of DD and the 3h wheel confinement test. A repeated measures

design counterbalanced for order was employed for all experiments.

Effect of LL on NMDA-Induced Phase Shiffs

SCN cannulated hamsters (N=18) were entrained to LD (14:lO) and then

released in LL (-10001ux) or DDred (<lOlux dim red light) for 12-15 days. To facilitate


microinjections, light levels in the DD condition were less than IOlux, equivalent to less

than 1 % of the light intensity during the light phase of the LD cycle. On the third day,

after two complete cycles in constant conditions, the hamsters received microirijections

of NMDA (500nl at 10mM in phosphate buffered saline; Sigma) administered at CT

13.5 k 15min, or vehicle alone, or no injection. For the injection conditions, hamsters

were removed briefly from their home cages, gently restrained to remove the dummy

cannula and insert the injector cannula, then placed in a clean holding area allowing the

hamster to move freely while the drug or vehicle alone was slowly injected using a

manual micro-syringe drive. Injections took I min to complete once the injector cannula

was inserted, and to minimize backflow the cannula was kept in place for an additional

minute following injection. The injector cannula was then removed, the dummy cannula

replaced, and finally the hamster was returned to its home cage. The lighting conditions

during injections were set to match those of the home cage lighting, either LL or DDred.

Following injections, hamsters remained in LL or DD for 9-1 2 days, after which they were

re-entrained to the LD cycle. Each hamster was tested 6 times, with conditions partially

counterbalanced for order within group (NMDA and no injection conditions were

counterbalanced, followed by vehicle controls).

A subset of hamsters (N=10) received NMDA (200nl) on days 3 and 13 of LL to

provide dose response information and to determine whether there are changes in the

magnitude of phase shifts over time in LL, as previously reported for arousal-induced

shifts (Knoch eta/., 2004).


Histology

The locations of the SCN cannulation implant sites were verified histologically.

The animals were euthanized with an overdose of sodium pentobarbitol, perfused

transcardially with phosphate buffered saline, their brains removed and placed in 10%

Formalin for 2 days at 4 OC and finally cryoprotected in a sucrose formalin solu'tion.

50-pm-thick frozen coronal sections were prepared by cryostat. Implant tract locations

were examined in sections mounted on glass sllides and stained with cresyl violet.

Data Analysis

The onset of nocturnal activity was used to measure phase shifts. Onsets were

defined as the first 10min bin in which wheel counts exceeded a threshold after more

than 240min in which activity did not exceed this level in any 10min bin. To

accommodate individual differences in the level of activity during LL, thresholds were set

for each hamster, but were consistent across tests within subjects. Onsets for 1:he day of

the manipulation as well as onsets for each day following the manipulation were

determined. The magnitude of phase shifts induced by NMDA was determined by

comparing onsets for drug vs. vehicle injections and drug vs. no injections within each of

the two lighting conditions. Statistical significance was evaluated using repeated

measures ANOVA and planned t-tests. Means in the text are reported + standard error.


RESUL.TS

Two Days of LL Potentiates Phase Shifts to Novel Wheel-Induced Running

Two days of LL prior to 3h novel wheel confinement (NWC) from CT 6-!3

significantly potentiated phase advances to NVVC by comparison with the standard LO

procedure (e.g., Fig. 1). Using a counterbalanced repeated measures design, hamsters

phase advanced an average of 97 + 33min following LD and 180 + 42min following

LL (83 + 15min; paired t(s, =5.6, p=0.001; Fig. 2A). Total wheel revolutions per 3h NWC

did not differ between conditions (204 f 295counts; paired t(6) =0.7, p=0.514; Fig. 2B).

These results confirm that LL at the intensity used in the present study potentiates

nonphotic phase shifts.

Figure 1:


Novel Wheel-Induced Phase Shifts

Description. Wheel running activity of a representative hamster illustrating phase shifts induced by a 3h bout of running stimulated by confir~ement to a novel wheel beginning 6h before the expected onset of the daily active period (beginning and ending of wheel confinement sessions are indicated by the 'v' markers). Each line represents a day, plotted in 10mir1 bins from left to right. Bins during which wheel running was registered are indicated by vertical deflections, producing a heavy bar during sustained bouts of spontaneous running at night (shading indicates lights off) or in the novel wheels. (A) Hamster exposed to LL for 2 full cycles prior to novel wheel confinement. (B) Hamster exposed to LD prior to wh~eel confinement.

Differential Effects of Constant: Light on Circadian Clock Resetting 26

Figure 2: Effect of Lighting on Novel Wheel-Induced Phase Shifts

Description. (A) Group mean phase shifts induced by a 3h bout of running stimulated by confinement to a novel wheel, in the LD and LL conditions (*conditions significantly differ at p=.001). (B) Group mean wheel revolutions during the 3h wheel confinement tests in the LD and LL conditions (no significant difference).


Two Days of LL Does Not Potentiate Phase Shifts to NMD.A

An omnibus F test on a repeated measures two-way anova, with lightin!g as the

first factor (LL vs. DD) and injection condition as the second (drug, vehicle, and no

injection), shows main effects for lighting (F(1,8):=8.7, p=0.018) and injection (F(2!,16, =22.4,

p=0.000), but no interaction between factors (F(2,16) =0.2, p=0.795).

Phase shifts in response to NMDA injections at CT13.5 on day 3 of DD were

assessed using repeated measures ANOVA with 3 injection conditions (DD+NIMDA,

DD+vehicle, and DD alone). Phase shifts differed significantly across conditions (F(2,16)

=16.6, p=0.000). Planned pairwise comparisons with Bonferroni correction show that

NMDA (500nl) injections induced significant phase delay shifts relative to the vehicle

control (52 f I I min; paired t(8) =5.1, p=0.004) a~nd to the no-injection control conditions

(49 f 10min; paired t(,) =4.8, p=0.003). Fig. 3 sliows the phase delays for all conditions.

These phase delays were clearly evident as acute displacements of activity onsets, and

appeared to be completed within one circadian cycle (e.g., Figs. 4 & 5A).

Figure 3:

Description. significant.

Effect of LL on Photic Shifts

LL+NMDA (500) vs. LL fiZB LL+NMDA (500) vs. Vehicle c;l DD+NMDA (500) vs,. DD

DD+NMDA (500) vs,. Vehicle LL+NMDA (200) vs. LL

ESi LL+NMDA (200) vs. Vehicle EE!4 13 days LL+NMDA 200 vs. LL

Group mean phase delays by condition. Differences between means isre not


The vehicle and no-injection control conditions iin DD did not differ significantly (3

f 10min; paired t(8) =0.3, p=1.00; Fig. 5A).

Figure 4: Actograms

Hours 12

Description. Wheel running activity of a representative hamster illustrating phase shifting effects of 500nl NMDA microinjections (indicated by diamond symbols) at circadian time 13.5 on day 3 of DD (panel B) or LL (panel D). Panels A and C illustrate activity in the no-injection control conditions in DD and LL, respectively. For plotting conventions, see Figure 1.

Phase shifts in response to NMDA injections at CT13.5 on day 3 of LL were

assessed using the procedure described above. Phase shifts differed significantly across

treatments (F(2,26) = I 3.2, p=0.000). Planned pailwise comparisons show that

NMDA (500nl) injections induced significant phase delay shifts relative to the vehicle

control (54 + 14min; paired t(,,)=4.3, p=0.005) and to the no-injection control conditions


(56 f 1 lmin; paired t(13)=4.6 p=0.000). Fig. 3 (p. 27) shows the phase delays for all

conditions. The NMDA-induced phase delays in LL were not so clearly evident in the

actograms because LL produced a characteristic shortening of the circadian period for a

few days, followed by a gradual lengthening (Fig. 4C & 4D). However, when group mean

activity onsets for each treatment condition were plotted for each day of LL, the phase

delay in the NMDA condition is strikingly eviden~t (Fig. 58). In LL, onsets for the first day

following treatment in the vehicle and no-injection control conditions did not differ

significantly (3 f 13min; paired t(13)=0.2, p= l .OC); Fig. 5B). There were no significant

differences between NMDA-induced phase shifts in LL and DD relative to vehicle

control (9 f 20min; paired t(s) =0.5, p=0.664) or to the no injection control (15 f 8min;

paired t(9) =1.9, p=0.086).

Figure 5: Activity Onsets in DD and LL

Time (min)


1

0 25 50 75 1100 125 150 175

Time (rnin)

Description. Group mean activity onsets (with standard errors) during DD (A) and LL (B) in the vehicle control (dashed lines, circles), no injection control (dotted lines, x's), and 500nl NMDA (solid lines, squares) microinjection conditions. Microinjections were done on day 3 (indicated by the arrows). Phase delay shifts are evident by the first cycle in each condition. In LL, there is a gradual modification of circadian period, characterized by an initial shortening followed by a lengthening. This was evident in the drug, vehicle control and no injection control conditions. The difference between the drug and control conditions was significant within conditions but not between conditions (see text).

No Effect of Time in LL on NMDA-l~nduced Phase Shifts

NMDA (200nl) injections at CT13.5 on days 3 and 13 of LL induced phase delay

shifts of 39 f 12min and 32 f Gmin, respectively, that were not significantly different

(mean difference 9 f 14min; paired t(9, =O.6, ~ 4 . 5 5 0 ; Fig. 6).


Figure 6: Activity Onsets after 13 Days in LL

1

Time (rnin)

Description. Group mean activity onsets (with standard errors) during LL in the injection control (dotted line, x's) and 200nl NMDA (solid line, squares) microinjection conditions. Microinjections were done on day 13 (indicated by tlhe arrow). The LL control onsets were calculated using a linear regression of the 5 onsets prior to injections. Phase delay shifts are evident by the first cycle following microinjections.

Histology

All cannula tips were within 45Opm from the SCN border, with an average

distance from the SCN of 131 f 36pm (e.g., Fig. 7). Distance from the SCN and

magnitude of phase shifts to NMDA (500nl) injelctions at CT13.5 on day 3 of LL show a

strong negative correlation (Pearson r = -.58, r2 =.33), suggesting that roughly 33% of

the variance in phase shifts can be explained by cannula placement.


Figure 7: Histological Confirmation of Cannula Placement

. - Description. Digital image of histological results illustrating a representative cannula placement. The cannula tip tract in this case came within 100pm of the dorsal border of the suprachiasmatic nucleus (SCN). Cells were stained using cresyl violet. 3V, third ventrical; SCN, suprachiasmatic nucleus; OX, optic chiasm.


DISCUSSION

Our findings confirm earlier reports of potentiated nonphotic resetting fclllowing 2

days in LL, with very similar increases despite methodological differences in behavioural

activation (Knoch eta/., 2004; Mistlberger et a/., 2002). Phase advances to 8-OH-DPAT

injections have been shown to increase in magnitude 3-fold by 2 days exposure to LL,

while advances to 3hr sleep deprivation (SD) in the mid-subjective day were doubled in

size. The latter manipulation more closely reflects the method of behavioural activation

in our study (3hr NWC), and thus, the 2-fold increase in advances observed in our study

suggests that SD and NWC are equivalent methods for induction of nonphotic resetting.

With respect to the effect of intra-SCN NMDA injections in the early subjective

night, our findings clearly support earlier reports that NMDA produces photic-like

resetting (Mintz & Albers, 1997; Mintz et a/., 19!39). Behavioural observation of the

hamsters during injections documented the same sequence of behaviours reported

earlier. Hamsters became hyperactive within 20-30s of the injection. This hyperactivity

would continue for -60s, and would be followed by a quiescent period during which the

hamsters would clicklgrind their teeth. Analysis of the NMDA-induced phase delays

observed in our study, both in terms of magnitude and variability, confirm those reported

by Mintz. We conclude, therefore, that NMDA is an appropriate and effective stimulus for

the induction of photic-like resetting, even when the hamsters are housed in LL.

The mechanism by which LL potentiates nonphotic resetting is undetermined, but

as discussed in the introduction two possibilities exist: (1) changes specific to the

nonphotic input pathway(s); and (2) decreased pacemaker amplitude. In the case of the

former, candidate pathways include the 5HT projection from the median raphe to the


SCN, NPY projection from the IGL to the SCN, an indirect 5HT input to the SCN via

dorsal raphe modulation of NPY output from the IGL to the SCN, other direct nonphotic

afferents to the SCN, or further upstream. With regard to amplitude, limit cycle models

with an amplitude dimension can account for the LL-induced Type 0 nonphotic resetting

confirmed in our study (Johnson et a/., 2004; Lakin-Thomas, 1995). The mechanism by

which LL may decrease pacemaker amplitude is yet to be determined, but at least two

possibilities exist: (1) amplitude could correspolnd to the levels or range of oscillating

clock gene proteins within circadian clock cells (for clock gene review, see (Hastings &

Herzog, 2004); or (2) given that the SCN is coniprised of thousands of indeper~dently

oscillating neurons, amplitude could be a function of the degree to which these

oscillators are coupled. With regard to the former, transient exposure to LL has been

shown to suppress oscillation of canonical clock genes per1 and per2 in the SCN,

although this decreased amplitude has only been tracked for a few cycles (Sudo etal.,

2003; Sumova & Illnerova, 2005). As reviewed in the introduction, LL gradually dampens

overt activity and sleep-wake rhythms in nocturnals, in ways suggestive of decoupling of

multiple oscillators (Eastman & Rechtschaffen, 1983; Moriya etal., 2000; Pitte~ndrigh &

Daan, 1976a). Furthermore, LL may dampen circadian oscillations of SCN neuronal

firing rate (Mason, 1991; Shibata etal., 1984), though it is not clear whether this is the

result of arrhythmicity in single cells or desynchrony in a population of sampled cells

(Ohta et a/., 2005). Regardless, it is clear that 1-L significantly affects a number of

putative indices of pacemaker amplitude at both the cellular and systems levels.

The current study tested the amplitude hypothesis of LL-induced potentiation of

nonphotic resetting by examining the effects of LL on photic resetting. If LL potentiates

photic resetting in the same way it potentiates nonphotic resetting, a strong argument


can be made for a pathway non-specific mechanism such as decreased pacemaker

amplitude. If however, LL does not potentiate photic resetting, a weaker argument

supporting a nonphotic pathway specific mechanism can be made. Indeed, our findings

that photic resetting did not differ as a function lof prior lighting condition suggest the

latter instead of the former. Given the current findings, however, we can not rule out the

possibility that decreased pacemaker amplitude plays a role in LL-induced potentiation

of nonphotic resetting. It is conceivable, for example, that the phase resetting effect of

NMDA was in some way down-regulated as a result of exposure to LL so as to precisely

offset the functional consequences of decreased amplitude. In fact, increased

photoperiod has been shown to attenuate photic resetting in nocturnals (Evans, et al.,

2004). Their results showed that light-induced phase shifts are larger in hamsters

housed in LD 10:14 vs. 14:lO. Clearly LL represents the longest possible photoperiod,

while DD represents the shortest, and therefore we might expect attenuated NIVIDA-

induced resetting under LL compared to DD. Thus, our results support a nonphotic

specific mechanism for LL potentiation, but do not refute the amplitude hypothesis.

There are, however, a number of reasons to question the notion of LL-induced

decreases in NMDA sensitivity, which given our results is a requirement of the amplitude

hypothesis. First, to our knowledge there exists no evidence of LL-induced changes to

non-NMDA or NMDA receptors of the photic input pathway. Second, as reviewed in the

introduction LL has profound effects on circadian rhythms at the behavioural, cellular,

and molecular genetic levels. If the response of the SCN to photic input was greatly

attenuated by LL, then one would not expect LL exposure to result in behavioural

arrhythmicity, splitting of activity rhythms, suppression of neuronal firing rates, or

changes in per expression. And third, when animals are placed back into LD after LL,

Differential Effects of Constant: Light on Circadian Clock Resetting 36

they entrain very quickly (Eastman & Rechtschiaffen, 1983). Furthermore, our results

show phase shifts to NMDA are statistically equivalent following 3 and 13 days in LL,

whereas LL potentiates nonphotic resetting after 3, but not 13 days in LL, indicating that

any hypothetical damping of amplitude is transient. Taken together, all of the above

indicate the clock is still sensitive to light under LL, and thus LL-induced nonphotic

pathway specific changes are a more parsimonious explanation of LL-induced

potentiation of nonphotic resetting. Direct measurements of NMDA receptor number,

affinity, and function (e.g., electrophysiological responses of SCN neurons to NMDA)

across time in LL could help resolve this issue.

There is considerable evidence that photic and nonphotic inputs to the SCN

pacemaker are mutually antagonist. Light can bllock phase shifts to stimulated running in

the day, and stimulated running can block phase shifts to light at night (Maywood &

Mrosovsky, 2001; Mistlberger & Antle, 1998; Ralph & Mrosovsky, 1992). The siame

antagonist relationship holds at the neurochemical level, where the photic agonist GLU

is mutually inhibitory with the nonphotic agonists NPY and 5HT (Biello & Mrosovsky,

1995; Bobrzynska & Mrosovsky, 1998; Selim eta/. , 1993; Yannielli & Harringtom, 2004).

These reciprocal effects may reflect a convergence of photic and nonphotic inputs at the

level of second messenger pathways that regulate expression of one or more clock

genes (Maywood & Mrosovsky, 2001); e.g., dexras knockouts simultaneously attenuate

photic shifting and potentiate nonphotic shifting (Cheng et a/., 2004). However, photic

and nonphotic effects on the clock can be dissociated. As discussed earlier, the

magnitude of phase shifts to light is affected by prior photoperiod (shifts are smaller

following exposure to long vs. short days), but the magnitude of activity-induced shifts

has recently been shown to be unrelated to photoperiod (Evans et a/., 2004). The results


of the present study now demonstrate the converse; a manipulation that can strongly

potentiate nonphotic shifts does not affect a light input pathway to the clock. This is

significant, as it suggests that, in principle, pharmacological or other procedures can be

developed that would enable potentiation of one class of circadian clock inputs without

necessarily attenuating responses to another class of clock resetting inputs.


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