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The Journal of Neuroscience, January 15, 1999, 19(2):828-835
Rapid Resetting of the Mammalian Circadian Clock
Jonathan D.
Best,
Elizabeth S.
Maywood,
Karen L.
Smith, and
Michael H.
Hastings
Department of Anatomy, University of Cambridge, Cambridge CB2 3DY,
United Kingdom
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ABSTRACT |
The suprachiasmatic nuclei (SCN) contain the principal circadian
clock governing overt daily rhythms of physiology and behavior. The
endogenous circadian cycle is entrained to the light/dark via direct
glutamatergic retinal afferents to the SCN. To understand the molecular
basis of entrainment, it is first necessary to define how rapidly the
clock is reset by a light pulse. We used a two-pulse paradigm, in
combination with cellular and behavioral analyses of SCN function, to
explore the speed of resetting of the circadian oscillator in Syrian
hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and
respond to light pulses presented 1 or 2 hr apart. Analysis of the
phase shifts of the circadian wheel-running activity rhythm of hamsters
presented with single or double pulses demonstrated that resetting of
the oscillator occurred within 2 hr. This was the case for both
delaying and advancing phase shifts. Examination of delaying shifts in
the mouse showed resetting within 2 hr and in addition showed that
resetting is not completed within 1 hr of a light pulse. These results
establish the temporal window within which to define the primary
molecular mechanisms of circadian resetting in the mammal.
Key words:
suprachiasmatic nucleus; circadian rhythms; entrainment; immediate-early genes; light; CREB; circadian clock
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INTRODUCTION |
Circadian rhythms are fundamental to
the behavior and physiology of all higher organisms (Pittendrigh, 1960 ;
Aschoff, 1981), expressed at the level of organism, tissue, and cell
(Welsh et al., 1995; Plautz et al., 1997; Balsabore et al., 1998). The
recent identification of putative clock genes in mammals (Sun et al., 1997; Tei et al., 1997) has provided a major impetus to understanding the clock mechanism. The principal circadian oscillators of mammals are
the hypothalamic suprachiasmatic nuclei (SCN) (Klein et al., 1991).
Light acting via retinal afferents is the primary synchronizer (Pittendrigh and Daan, 1976; Czeisler, 1995). Although the
neurochemical basis of photic entrainment is well characterized (Nelson
and Takahashi, 1991; Kornhauser et al., 1996; Hastings, 1997), its molecular basis is not. To identify critical elements of the
entrainment mechanism, it is necessary to define the temporal window
over which resetting is completed. Responses to light that occur after this interval cannot cause resetting. Resetting of the clock is described by a phase response curve (PRC), which plots the magnitude and direction of the phase shifts of an overt rhythm as a function of
the circadian times at which light is presented. Typically, delays are
completed after one circadian cycle, whereas advance shifts can take
several cycles to be expressed in full (Pittendrigh and Daan, 1976).
However, this analysis can be misleading, because it is the position of
the PRC rather than the phase of any particular output rhythm that
provides a true reflection of oscillator phase. To determine the speed
of resetting, it is therefore necessary to define how quickly the PRC
is reset by light. This has been examined in lower organisms using a
"two-pulse" protocol, the rationale being that an initial pulse
causes a phase shift of the oscillator, and therefore it shifts the PRC
to a new phase. This new phase can then be mapped by examining the
response to "probe" pulses delivered shortly after the first.
Depending on the speed, direction, and magnitude of shifts induced by
the first pulse, the probe pulses will illuminate earlier (if delayed)
or later (if advanced) portions of the PRC than if the first pulse had
not been delivered. In Neurospora (Crosthwaite et al., 1995) and Drosophila (Pittendrigh, 1979), this has demonstrated
rapid (0.75 and 3 hr, respectively) resetting of the clock. A two-pulse protocol was used in the present study to determine whether photic resetting of the mammalian clock was equally rapid. Some models of the
mammalian clock suggest that exposure to light is followed by a
refractory period of 4-6 hr (Kronauer et al., 1996), which would
confound the two-pulse protocol. To confirm that light pulses separated
by 1 or 2 hr can be resolved by the mammalian circadian system, we
first examined the photic induction of c-fos (Kornhauser et
al., 1990; Rusak et al., 1990) and phosphorylation of the cAMP response element-binding protein (CREB) (Ginty et al., 1993) in the
retinorecipient SCN.
A preliminary account of this study was presented at the 19th
International Summer School of Brain Research, Amsterdam, September 1995, and appeared in the conference proceedings (Hastings et al.,
1996).
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MATERIALS AND METHODS |
Animals and treatments. All procedures were conducted
under license, in accordance with the Animals (Scientific Procedures) Act of 1986. All reagents were obtained from Sigma (Poole, UK) unless
stated otherwise. Adult male Syrian hamsters (100 gm; Wrights of Essex,
Chelmsford, UK) or outbred mice [ICR(strain CD-1); Harlan Olac,
Bicester, UK] were housed individually in cages equipped with running
wheels, and food and water were available ad libitum. The
initial photoschedule was a 16/8 hr light/dark cycle [light, 50 or 200 µW/cm2; dim red light (DR),
<1 µW/cm2]. Rotation of the
wheel interrupted an infrared light beam and was recorded by a personal
computer (Viglen HD40V; Viglen Computers, London, UK) running
Dataquest IV software (Minimitter Co.). Data were collected in 10 (hamsters) or 6 (mice) min time bins. After 3 weeks, the activity
rhythms of the animals were entrained to the photoschedule, and the
animals were released into constant dim red light to free-run (DR:DR)
for at least 10 d. The onset of activity was defined as circadian
time 12 (CT12), estimated by fitting a line by eye through at
least five consecutive activity onsets on double-plotted actograms.
Exposure to a light pulse (50 or 200 µW/cm2)
involved moving the animal in its home cage into an adjacent room to be
placed under a fluorescent strip light. In the "dark pulse" control
procedure, the animal was transferred to the adjacent room, which
remained in dim red light. In some cases, animals received a second
light pulse 1 or 2 hr after the first pulse. Animals were killed
for immunocytochemistry, in situ hybridization, or
dissection of SCN tissue for Western blot, or they were allowed to
free-run for 7-10 d to determine the magnitude of any phase shift.
In situ hybridization. At the indicated time points,
hamsters free-running in DR:DR for 30 d were killed by cervical
dislocation, and the brains were removed and frozen immediately on dry
ice. Sections (16 µm) through the hypothalamus were cut with a
cryostat, and slides were stored at  20°C until use. The in
situ hybridization procedure was adapted from Young and Kuhar
(1986) and Young et al. (1986). The slides were hybridized
overnight at 37°C in buffer [deionized formamide, mix bed resin
(Bio-Rad), 2× SSC, 50× Denhardt's solution, yeast tRNA,
sterile water, and dextran sulfate] containing 1 × 107 cpm/ml 35S-labeled oligoprobe in a
humidified chamber (GGG AGG ATG ACG CCT CGT AGT CCG CGT TGA AAC CCG AGA
ACA TCA TGG) (Vendrell et al., 1992). On the next day, the sections
were washed in 1× SSC, dipped twice in distilled water at room
temperature, and then left to dry. They were then exposed for 3 weeks
at 4°C to Hyperfilm  max (Amersham, Arlington Heights, IL). The
intensity of the autoradiographic images of c-fos
hybridization in the SCN was assessed by computerized densitometry
using a Hamamatsu CCD (C3077) camera connected to an Apple
Macintosh IIci computer running NIH Image 1.49 image analysis software
(gift of Dr. Wayne Rashband, National Institutes of Health, Bethesda, MD).
Western blotting. Hamsters free-running in DR:DR for 30 d were killed 0, 2, or 4 hr after a light pulse delivered 1 hr after activity onset (CT13), brains were rapidly removed, and coronal slices
were cut through the anterior hypothalamus at the level of the SCN
using a brain matrix as a guide (Agar Scientific). Tissue from the SCN
was microdissected using a 14 gauge hypodermic needle and immediately
frozen on dry ice. Extraction of nuclear proteins was conducted at
4°C. Tissue punches were homogenized using a Dounce homogenizer in
400 µl of homogenization buffer containing: 10 mM
HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml
aprotinin, and 2 µg/ml pepstatin. The nuclear fraction was
precipitated by centrifugation for 2 min at 14,000 rpm, and the pellet
was resuspended in 36 µl of ice-cold extraction buffer (10 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM
EDTA, 5 mM DTT, 1 mM PMSF, 10 µg/ml
aprotinin, and 2 µg/ml pepstatin), and incubated on ice for 20 min.
The mixture was then centrifuged at 14,000 rpm for 2 min, and the
supernatant was collected and stored at 40°C. Extracts (10 µl)
were separated on a 3% stacking-12% SDS- polyacrylamide gel and
electrotransferred at 70 V for 2 hr to nitrocellulose. The blots were
blocked for 60 min at room temperature in 4% nonfat milk (NFM) in PBS
and then incubated overnight in 2.5% NFM in PBS blocking buffer
containing c-fos antisera (Santa Cruz Biotechnology, Santa Cruz, CA).
The blot was washed for 30 min in 2.5% NFM and 1:200 Tween 20 in PBS and then incubated for 60 min in 1:2000 biotinylated anti-rabbit antibody (Amersham). The blots were then washed in 2.5% NFM in PBS for
15 min, developed with the ECL+ system (Amersham), and exposed
to film.
Immunocytochemistry. After being allowed to free-run in
DR:DR for 10 d, mice were exposed to a light pulse or a control
dark pulse, and at intervals after the pulse, were given an overdose of
anesthetic (Euthatal; Rhone-Merieux, Harlow, UK) and perfused via the aorta with fixative (4% paraformaldehyde in PBS). Brains were
removed, post-fixed for 4 hr, and then transferred to 20% sucrose
solution cryoprotectant, before sectioning at 40 µm on a freezing
microtome. Immunocytochemistry for Ser133
phospho-CREB was conducted on free-floating sections using a specific
antiserum (New England Biolabs, Beverly, MA) at 1:1000 dilution and
visualized with Vectastain ABC kit (Vector Laboratories, Burlingame,
CA) and diaminobenzidine as chromogen. The number of
immunoreactive nuclei in the SCN was determined using the NIH Image program.
Statistical analyses. Differences between experimental
groups were analyzed using ANOVA, followed by post
hoc Dunnett's t tests.
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RESULTS |
Transcriptional responses confirm that the SCN can resolve paired
light pulses 1 or 2 hr apart
To test whether the SCN can resolve individual light pulses
presented 1 or 2 hr apart in a paired-pulse paradigm, we examined the
photic induction of c-fos and phosphorylation of CREB in the retinorecipient SCN. As reported previously (Kornhauser et al., 1990),
the induction of c-fos mRNA in the SCN of Syrian hamster by
a single light pulse (50 µW/cm2) was rapid and
transient. In animals exposed to a control dark pulse during subjective
night (CT13), there was no c-fos mRNA hybridization signal
in the SCN (Fig. 1a). Thirty
min after exposure to light at CT13 (15 min, 50 µW/cm2), there was a robust induction of
c-fos mRNA in the SCN (Fig. 1b) that was not
detectable 1 hr after the light pulse (Fig. 1c). Quantification of the relative intensity of the hybridization signal
revealed a significant effect of treatment (ANOVA; F = 9.7; p < 0.01), with levels being significantly
elevated relative to dark control tissue 15-30 min (0.1 ± 1.9 vs
29.5 ± 4.8 gray scale units) after a single pulse but not
significantly above background 1 (3.9 ± 1.9) to 3 (3.1 ± 0.1) hr after the pulse. In animals that received a double-pulse
protocol, a very strong c-fos mRNA hybridization signal was
evident in the SCN 30 min after the second pulse (28.1 ± 3.9 gray
scale units) (Fig. 1d). This corresponded to 2.5 hr after
the first pulse, a time when the signal arising from the first pulse
would have dissipated. Repeated induction of c-fos was
confirmed by Western blot analysis of protein extracts of SCN. In
animals free-running in DR and exposed to dark pulses at CT13, the
c-fos signal was very weak (Fig. 1e, lane 3).
However, 2 hr after a light pulse at CT13, a strong c-fos band was
evident (Fig. 1e, lane 4),
and by 4 hr after the pulse at CT13, levels of c-fos had declined to
those seen in dark-treated controls (lane 5). However,
in animals that were killed 4 hr after the first pulse but had received
a second pulse 2 hr after the first, there was a very strong c-fos
signal (Fig. 1e, lane 6), comparable
to that seen in animals sampled 2 hr after a single pulse. The repeated
induction of c-fos demonstrates that the circadian system is
able to resolve light pulses 2 hr apart.
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Figure 1.
Photic induction of c-fos in
hamster SCN by single and paired light pulses. Autoradiograms of
coronal sections of hypothalamus of hamster SCN produced by in
situ hybridization for c-fos mRNA after exposure
to darkness (15 min, <1 µW/cm2) or light pulses
(15 min, 50 µW/cm2) during early subjective night.
a, Thirty minutes after exposure to a control dark pulse
at CT13. 3V, Third ventricle; oc, optic
chiasm. b, Thirty minutes after start of a light pulse
at CT13. Note hybridization signal in bilateral SCN. c,
One hour after start of a light pulse at CT13. Note hybridization
signal is lost. d, Thirty minutes after start of second
pulse in a double-pulse protocol. Note reinduction of
c-fos in SCN. Scale bar, 500 µm. e,
Western blots probed for c-fos. Lanes 1,
2, Negative and positive (metrazole-treated) controls
from extracts of hamster cerebral cortex samples; lanes
3-6, tissue extracts from pooled SCN (5 animals per sample);
lane 3, dark pulse control, weak signal; lane
4, 2 hr after light pulse at CT13, strong induction;
lane 5, 4 hr after light pulse at CT13, signal lost;
lane 6, 2 hr after second pulse, 4 hr after first pulse
(note strong reinduction).
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To test whether the SCN can resolve pulses separated by 1 hr, mice
free-running in dim red light received a light pulse at CT13 and a
second either 1 or 2 hr later. Phosphorylation of CREB was used as the
index of response, because its transient nature would provide the
necessary resolution. Dark-pulsed controls had low to undetectable
levels of phospho-CREB (Fig.
2a) nuclei in the
retinorecipient SCN. Within 7.5 min of a light pulse, the number of
phospho-CREB-ir nuclei was maximal (Fig. 2b,c).
The peak was transient, and levels declined to baseline within 1 hr. Presentation of a second pulse 1 or 2 hr after the first was followed by a second wave of phospho-CREB induction, detectable after 10 min
(Fig. 2c). These results confirm that the SCN can resolve light pulses separated by 1 or 2 hr.
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Figure 2.
Photic induction of phospho-CREB in mouse SCN by
single and paired light pulses (15 min duration). Coronal sections of
SCN reveal low expression in dark-pulsed control animals
(a) but extensive induction in retinorecipient
SCN of animals 10 min after start of a light pulse (200 µW/cm2) at CT13 (b).
c, Time course for phospho-CREB induction in mouse SCN
(mean ± SEM) shows rapid and transient response, with reinduction
by second pulses presented 1 or 2 hr after initial pulse
(arrows).
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For both advance and delay shifts, the circadian clock of the
Syrian hamster is reset within 2 hr of a light pulse
Exposure to light during subjective day or exposure to dark pulses
during subjective night had no effect on the circadian rhythm of
wheel-running of Syrian hamsters. Exposure to single pulses of light in
early subjective night caused phase delays of the free-running activity
rhythm, whereas pulses delivered later in subjective night advanced the
rhythm (Figs. 3,
4a). Overall, animals pulsed
at CT15 showed no significant shifts; this circadian phase represents
the crossover point of the PRC (Fig. 4a). Animals that
received double-light pulses continued to exhibit stable free-running
rhythms (Fig. 3b-d). Presentation of the first light pulse
at CT13 and the second two hr later was followed by large phase delays
in the free-running rhythm (Fig. 3b). When the first of the
two light pulses was given at CT20 and the second was given 2 hr later,
the subsequent phase advance was comparable to that observed after a
single light pulse at CT20 (Fig. 3c). In contrast, when the
first light pulse was administered at CT18 and the second was presented
2 hr later, it was followed by a phase advance larger than that
anticipated after a single pulse at CT18 (Fig. 3d).
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Figure 3.
Representative double-plotted actograms of
wheel-running activity of Syrian hamsters held in continuous dim red
light and exposed to brief pulses of light (15 min,
asterisks) at one pulse only at CT13
(a), paired pulses first at CT13 and a second 2 hr later (b), at CT20 and a second 2 hr later
(c), and at CT18 and a second 2 hr later
(d). Lines on left
side indicate phase shift of activity onset. Pulses in
a, b, 15 min, 50 µW/cm2; pulses in c,
d, 15 min, 200 µW/cm2.
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Figure 4.
a, PRCs (mean ± SEM) of the
circadian activity rhythm of Syrian hamsters held in continuous dim red
light and exposed to a brief pulse of light (15 min) of 50 (filled symbols; n = 84) or
200 (open symbols; n = 45)
µW/cm2. The dotted line about the
abscissa is the SEM for control dark pulses (mean shift, 0.01 hr;
n = 15). b, Predicted and observed
resetting to paired pulses in delaying phase of PRC. Shifts to
individual pulses (15 or 30 min, 50 µW/cm2)
delivered at CT13, CT14, or CT15. NO, Predicted shift if
no resetting within 2 hr of first pulse; YES, predicted
composite shift if delay arising from pulse at CT13 is completed before
second pulse is given; OBS, observed data.
n = 11-25; n = 99. **p < 0.01, pairwise comparison. c,
d, Predicted and observed resetting to paired pulses in
advancing phase of PRC. Shifts to individual pulses (15 min, 200 µW/cm2) delivered at CT18, CT20, CT22, CT23, or
CT24. NO, Predicted shift if no resetting within 2 hr of
first pulse; YES, predicted composite shift if advance
arising from pulse at CT20 or CT18 is completed before second pulse is
given; OBS, observed data. n = 6-11; n = 74. **p < 0.01, pairwise comparison.
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Analysis of the grouped data for single and double pulses presented
between CT13 and CT15 revealed a significant overall effect of
treatment (F = 14.3; p < 0.01). The
prediction was that if the circadian clock had not delayed before the
second light pulse was presented, the PRC would retain its phase and
the second pulse would fall at CT15, and so the response to the
double-pulse treatment would be a composite of the delay associated
with light at CT13 and the nonsignificant shift associated with light
at CT15. This is indicated in Figure 4b, NO
(i.e., no shift within 2 hr). This composite value would not be
significantly different from the delay to light at CT13 alone.
Alternatively, if the PRC had been shifted within 2 hr of the first
pulse at CT13, the second pulse would fall not at CT15 but at CT14.
This would produce a composite shift to the double-pulse protocol,
representing the delay associated with light at CT13 and at
approximately CT14. This composite shift would be significantly greater
than that seen after light at CT13 alone (Fig. 4b,
YES). Post hoc comparison revealed that the
observed shifts (Fig. 4b, OBS) after the
double-pulse procedure were significantly greater than those after
light at CT13 alone. The increased shifts could not be attributed to
the longer total exposure to light associated with two pulses, because
exposure to light for 30 min did not cause a significant increase in
the size of the delay.
For phase advances, there was also a significant effect of treatment
(F = 13.2; p < 0.01). Single pulses at
CT20 and CT22 caused large phase advances of ~2 hr, whereas light at
CT24 caused only a small advance not significantly different from that
seen after a dark pulse. The prediction was that if the circadian clock had not advanced before the second light pulse was presented, the
response to the double-pulse treatment would be a composite of the
advance associated with light at CT20 and the shift associated with
light at CT22. This is indicated in Figure 4c, NO
(i.e., no shift within 2 hr). Alternatively, resetting within 2 hr of the first pulse at CT20 would cause a composite shift, representing the
advance associated with light at CT20 and the minimal shift associated
with light at CT24 (i.e., CT20 + 2 hr advance + 2 hr interval). This
composite shift would not be significantly greater than that seen after
light at CT20 alone (Fig. 4c, YES). The observed shifts (Fig. 4c, OBS) were significantly
different from the NO, but not the YES,
prediction (Figs. 3c, 4c).
To demonstrate that large composite advances can be induced by the
double-light pulse protocol, in a further experiment, animals received
a light pulse at CT18, followed by a second pulse 2 hr later.
Regardless of how rapidly the clock reset to the first pulse, this
ensured that the second pulse would fall in the advance portion of the
PRC, and so it should induce a further advance, with the response to
the double-pulse protocol being significantly larger than the advance
to single pulses at CT18. The observed shifts (Fig. 3d)
confirmed this to be the case, with a significant difference between
the advances after two pulses and those receiving one at either CT18 or
CT20 (Fig. 4d).
In the current double-pulse protocol, the consequences of rapid
resetting were different for advance and delay shifts, insofar as
advances were not associated with large cumulative shifts, whereas the
delay shifts were. Nevertheless, the data from both types of shift are
consistent with resetting of the circadian clock within 2 hr of
exposure to light.
Delays of the circadian clock of the mouse are completed between 1 and 2 hr after a light pulse
Exposure to light during subjective night caused significant phase
delays, but not advances, of the circadian activity rhythm of mice
(Figs. 5a,
6a). The PRC could therefore
only be used to examine the speed of delay resetting. Moreover, the
slope of the delay portion of the PRC and the maximum size of phase
delays were not adequate to support a simple two-pulse paradigm; unlike the hamster, it was not possible to distinguish between resetting at
CT14 and CT15, which would be necessary to examine the speed of a 1 hr
delay after a single pulse at CT13. Consequently, a modification of the
protocol was that four light pulses were presented at intervals of 1 or
2 hr, the first pulse falling at CT13 or CT14, respectively. The PRC to
single pulses was then used to calculate the composite shifts expected
whether resetting did or did not occur in the interval between serial
pulses. Between CT13 and CT19, the PRC was linear
(r2 = 0.96; p < 0.01),
and so a regression function was fitted to the data so that predicted
phase shifts could be calculated by direct interpolation. As for the
previous study with hamsters, if delays were occurring rapidly, serial
pulses would continue to fall in the delay portion of the PRC.
Conversely, if resetting was slow, the later pulses would fall around
or beyond the crossover point. Consequently, rapid resetting predicted
larger cumulative delays than slow resetting. For example, for the 2 hr
series, if no resetting occurred at all, the cumulative delay would
represent the sum of shifts associated with light at CT13, CT15, CT17,
and CT19, which was predicted to be 1.65 hr (Fig. 6b,
NO). However, if the oscillator was reset within 2 hr of
every pulse, the cumulative delay would be at least 2.53 hr (Fig.
6b, YES). As anticipated from the hamster study,
the four serial 15 min pulses separated by 2 hr caused large delays
(Fig. 5c, 6b, OBS), significantly greater than single pulses at either CT13 or CT14 and significantly greater than predicted if resetting was not rapid (Fig. 6b).
The large shifts could not be attributed to a greater total exposure to
light, because they were considerably larger than those seen after a
single light pulse of 60 min at either CT13 or CT14 (Fig. 6b). These data are therefore consistent with rapid (<2 hr)
resetting of the clock.
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Figure 5.
Representative double-plotted actograms of
wheel-running activity of ICR(CD-1) mice held in continuous dim red
light and exposed to one or a series of four light pulses (200 µW/cm2; asterisks).
Lines on left side indicate phase shift
of activity onset. a, Single pulse (15 min) at CT13;
b, single pulse (1 hr) at CT13; c, series
of four pulses of 15 min presented at 2 hr intervals, first at
CT13.
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Figure 6.
a, PRC (mean ± SEM) of the
circadian activity rhythm of ICR(CD-1) mice held in continuous dim red
light and exposed to a brief pulse of light (15 min, 200 µW/cm2) (n = 153). The
dotted line about the abscissa is the SEM for control
dark pulses (mean shift, 0.05 hr; n = 15).
b, Predicted and observed resetting to single and serial
pulses (4 pulses, delivered at intervals of once every 1 hr or once
every 2 hr) in delaying phase of PRC. Shifts to individual pulses (15 or 60 min, 200 µW/cm2) delivered at CT13 or
CT14 as indicated. NO, Predicted shift after
four pulse series if resetting does not occur until final pulse is
delivered; YES, predicted composite shift if resetting
does occur in the interval of either 1 or 2 hr between pulses;
1-2, predicted shift if resetting occurs within 2 hr
but not within 1 hr; OBS, observed data.
n = 11-17; n = 73. **p < 0.01, pairwise comparison.
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For the series of pulses separated by 1 hr and starting at CT14, the
predicted cumulative delay if no resetting occurred over the 3 hr
between first and final pulse was 2.00 hr (Fig. 6b,
NO). If resetting did occur within 2 hr but not after 1 hr,
a cumulative delay of 2.39 hr was predicted (Fig. 6b,
1-2). Finally, if resetting did occur within 1 hr of
a light pulse, the predicted delay was 2.81 hr (Fig. 6b,
YES). The phase delays produced by serial pulses separated
by 1 hr (Fig. 6b, OBS) were larger than those
after single 15 min pulses at CT13 or CT14 but were not significantly greater than delays produced by a single 60 min pulse at CT13 or CT14.
Moreover, they were significantly smaller than those predicted for
resetting within 1 hr. In contrast, they were not significantly
different from the prediction of no resetting at all or resetting that
occurred between 1 and 2 hr after a pulse (Fig. 6b).
Finally, the composite shifts after four pulses once every 1 hr were
significantly less than those seen with four pulses every 2 hr, which
is also consistent with rapid resetting within 2 hr but not 1 hr.
Together, the behavioral data from the mice indicate that delays of the
circadian clock take more than 1 hr but less than 2 hr to complete
after a light pulse.
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DISCUSSION |
Our behavioral and biochemical results demonstrate that the
circadian clock of the SCN of mammals can resolve light pulses separated by 1 and 2 hr. They also show that for both phase advances and phase delays, the oscillator is reset to a new phase within 2 hr of
exposure to light. Moreover, despite the SCN being able to resolve
light pulses presented 1 hr apart, the oscillator is not reset to a new
phase within 1 hr of a light pulse. The current study therefore
identifies the interval of 1-2 hr after presentation of a brief pulse
of light as the critical period during which the molecular events that
constitute resetting are completed. A mechanistic explanation of photic
entrainment should therefore focus on those light-induced responses of
the SCN that occur during this interval.
The PRCs generated in the current study are consistent with earlier
work. Whereas hamsters show both large delays and large advances, mice
typically exhibit small advance shifts (DeCoursey, 1964; Daan and
Pittendrigh, 1976; Elliott, 1981; Schwartz and Zimmerman, 1990; Nelson
and Takahashi, 1991; Grosse et al., 1995). Hamsters were therefore more
suitable to demonstrate equally rapid resetting in both directions.
Mice were also used in the current study to define the rate of delay
resetting, because the circadian and light-regulated expression of
putative clock genes are described more extensively in this species.
The assumptions behind the protocol, including additive phase shifts
and nondistortion of the PRC during resetting, are best fulfilled by
the type 1 resetting, characterized by relatively small (<3 hr)
shifts and a defined crossover point, as seen in the Syrian hamster and
mouse (Lakin-Thomas, 1995). Nevertheless, the current findings
complement studies in Drosophila (Pittendrigh, 1967, 1979)
and Neurospora (S. Dharmananda thesis cited in
Crosthwaite et al., 1995), showing that the oscillator was reset within
3 or 0.75 hr, respectively, although they exhibit type 0 resetting. In
double-pulse experiments with sparrows (Binkley and Mosher, 1987),
photic resetting was observed within 8 hr, but because 4 hr pulses were
used, the time course could not be defined more precisely. In a
different context examining the behavior of transient phase shifts
during advances of the hamster circadian system, Elliott and
Pittendrigh (1996) concluded that although full delays were completed
within one cycle and advances of behavioral rhythm were not completed
until ~6 d, detectable shifts of the oscillator could be observed
between 2 and 4 hr after a pulse, as reported here. Finally, a recent
study in wild-caught field mice (Mus booduga) has also
identified resetting of the oscillator to delaying pulses within 2 hr
(Sharma and Chandrashekaran, 1997), although advance resetting that
exhibits the most pronounced transients was not explored.
The molecular basis of the mammalian clock is unknown, and so the
recent cloning of mammalian homologs of the insect period gene is an important development (Sun et al., 1997; Tei et al., 1997).
Whatever their nature, it is clear that resetting mechanisms are
engaged very quickly, between 1 and 2 hr after a pulse. Thus, the rapid
photic induction of mPer1 and mPer2 expression,
which peaks after 1 hr and 2 hr, respectively (Albrecht et al., 1997; Shigeyoshi et al., 1997; Zylka et al., 1998), provides a molecular correlate of the overt resetting reported here. Increases in
their expression and subsequent abundance of their putative protein products may be the cause of resetting to a new phase. Such a model is
based on the Neurospora clock in which rapid resetting is
associated with equally rapid induction of the gene frq,
which has been shown to encode a state variable of the oscillation, i.e., the relative abundance of this transcript, and its protein products actually define circadian phase (Crosthwaite et al., 1995).
The report that light-induced advances remain sensitive to blockade by
inhibitors of protein synthesis for the 2-4 hr after a pulse (Zhang et
al., 1996) supports the possibility that the mammalian clock is based
on such an autoregulatory transcriptional cycle.
The rapid response of the SCN to light is dependent on glutamatergic
signaling by retinal ganglion cell afferents (Ebling et al., 1991;
Vindlacheruvu et al., 1992; Rea et al., 1993; Abe and Rusak, 1994).
Light also causes rapid phosphorylation of CREB in retinorecipient SCN
neurons (Ginty et al., 1993), probably via NMDA-mediated calcium influx
(Kornhauser et al., 1996; Schurov et al., 1998). Phosphorylated CREB is
a positive regulator of the c-fos gene via the calcium
response element (Ginty et al., 1993; Hardingham et al., 1997;
Johnson et al., 1997), although its potential role in the induction of
mPer1 and mPer2 awaits clarification. The changes
in phospho-CREB expression are extremely rapid, occurring significantly
before increases in c-fos or mPer mRNA, although
the magnitude of the response is not related to the size or direction
of a phase shift. Moreover, in both hamsters and mice, it occurs after
presentation of a light pulse at all phases of circadian night but is
not induced by light during the subjective day (Ginty et al., 1993; von
Gall et al., 1998), closely matching the temporal induction of
mPer1 regardless of whether light advances or delays the
clock. If mPer1 does encode a state variable of the
oscillator, it should be induced repeatedly by serial resetting light
pulses. Moreover, the observation that photic induction of
mPer2 is a marker for delaying pulses (early subjective
night) but not advancing pulses (late subjective night) (Albrecht et
al., 1997; Zylka et al., 1998) suggests a novel means for mapping the
phase of the oscillator during serial resetting by using a molecular
index. If the inducibility of mPer2 is a marker for early
subjective night (delay phase of PRC), it should be possible to show
that serial pulses delivered in the delay zone hold the oscillator at
early subjective night, i.e., mPer2 induction is sustained.
This approach is comparable to using the onset and offset of photic
induction of c-fos to define the total limits of subjective
night (Mead et al., 1992; Grosse et al., 1995).
The immediate early gene c-fos is also a potential component
of the photic resetting pathway. Phase shifts are impaired in c-fos knock-out mice (Honrado et al., 1996), and central
infusion of antisense oligonucleotides to c-fos and
jun-B are reported to block light-induced resetting in the
rat (Wollnik et al., 1995). Induction of c-fos mRNA
by a light pulse peaks after 30 min and is undetectable after 60 min
(Kornhauser et al., 1990; Rusak et al., 1990; current study). Moreover,
the induction of c-fos protein, which was detectable after 7.5 min and
peaks after 1 to 2 hr (J. D. Best and M. H. Hastings
unpublished data), is simultaneous with the induction of
mPer1 mRNA, raising the possibility that c-fos contributes
to resetting by modulating expression of the mPer1 gene via
changes in AP-1 activity, which is known to occur after a light pulse
in subjective night (Kornhauser et al., 1992; Takeuchi et al., 1993;
Best and Hastings, unpublished data). In situ hybridization
and Western blots revealed that the second resetting pulse caused
de novo induction of c-fos, although the second
pulse was presented when c-fos protein levels were high. In other
contexts, it has been shown that c-fos protein can downregulate expression of the c-fos gene via upstream promoters
(Sassone-Corsi et al., 1988; Konig et al., 1989), although it is clear
that autoregulation of c-fos did not occur in the SCN.
Rapid resetting has also been reported in the hamster and rat in
response to nonphotic cues, such as behavioral arousal (Mead et
al., 1992) and injection of melatonin (Sumova and Illnerová, 1996). Nonphotic cues act via a mechanism distinct from phosphorylation of CREB and induction of c-fos (Mead et al., 1992; Sumova et
al., 1994) and are most potent during subjective day when expression of
mPer1, mPer2, and mPer3 is maximal,
suggesting a possible resetting mechanism based on suppression of these
genes and/or the activity of their products. The current demonstration
of rapid advance and delay resetting by light in the mammalian clock
provides the context for comparative analyses of the molecular basis of
resetting by different cues.
| |
FOOTNOTES |
Received Aug. 24, 1998; revised Oct. 19, 1998; accepted Oct. 29, 1998.
This work was supported by Wellcome Trust Project Grant 037667/Z/93 and
Biotechnology and Biological Sciences Research Council Studentships to
J.D.B. and K.L.S. We are grateful to J. Bashford, A. Newman, I. Bolton
(Audio-visual Media Group, Department of Anatomy, University of
Cambridge), C. Cardinal, and M. Carlson (Biomedical Services,
University of Cambridge) for excellent technical assistance, and to I. Schurov for advice on Western blot analyses.
Correspondence should be addressed to Dr. Michael H. Hastings, Reader
in Neuroscience, Department of Anatomy, University of Cambridge,
Downing Street, Cambridge CB2 3DY, United Kingdom.
| |
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Top
Abstract
Introduction
