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The Journal of Neuroscience, October 1, 2001, 21(19):7742-7750
Expression of Period Genes: Rhythmic and Nonrhythmic
Compartments of the Suprachiasmatic Nucleus Pacemaker
Toshiyuki
Hamada1,
Joseph
LeSauter2,
Judith M.
Venuti3, and
Rae
Silver1, 2, 3
1 Department of Psychology, Columbia University, New
York, New York 10027, 2 Department of Psychology, Barnard
College, New York, New York 10027, and 3 Department of
Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia
University, New York, New York 10032
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ABSTRACT |
The mammalian circadian clock lying in the suprachiasmatic nucleus
(SCN) controls daily rhythms and synchronizes the organism to its
environment. In all organisms studied, circadian timekeeping is
cell-autonomous, and rhythmicity is thought to be generated by a
feedback loop involving clock proteins that inhibit transcription of
their own genes. In the present study, we examined how these cellular
properties are organized within the SCN tissue to produce rhythmicity
and photic entrainment. The results show that the SCN has two
compartments regulating Period genes
Per1, Per2, and Per3 mRNA
expression differentially. One compartment shows endogenous rhythmicity
in Per1, Per2, and Per3
mRNA expression. The other compartment does not have rhythmic mRNA
expression but has gated light-induced Per1 and
Per2 and high levels of endogenous nonrhythmic Per3 mRNA expression. These results reveal the
occurrence of differential regulation of clock genes in two distinct
SCN regions and suggest a potential mechanism for producing functional
differences in distinct SCN subregions.
Key words:
suprachiasmatic nuclei; circadian rhythms; clock gene; Per1; Per2; Per3 Bma1; calbindinD28K; vasopressin; Fos; light pulse; oscillator; pacemaker
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INTRODUCTION |
Circadian regulation of physiology
and behavior has been observed in organisms from cyanobacteria to
mammals. The discovery of mammalian homologs of nonmammalian clock
genes has focused attention on the conservation of circadian mechanisms
in evolution (Dunlap, 1999 ). Molecular models of circadian oscillators
in mammals are based on findings in Drosophila and
Neurospora (for review, see Young 1998; Dunlap 1999). In
these models, rhythmicity is produced by a cell-based negative
transcriptional feedback loop, in which expression of the putative
clock genes is suppressed by their own protein products, and
entrainment results from their modification by light-induced signals.
Although many clock genes are conserved, there are distinct species
differences, as noted in the reviews cited above. Mice have three
Period genes (mPer1, mPer2, and
mPer3), whereas Drosophila has one
(dPer). Although the expression of dPer is
rhythmic, dPer is not acutely responsive to light, as are
mPer1 and mPer2 mRNAs. These data suggest that certain features of entrainment and oscillation may be unique to mammals.
A model explaining the mammalian circadian system will require
understanding the circadian clock in the suprachiasmatic nucleus (SCN)
at both the cellular and tissue levels. There is substantial evidence
that SCN cells are autonomous oscillators. Individual dispersed SCN
cells exhibit a circadian rhythm of electrical activity (Welsh et al.,
1995; Liu et al., 1997; Herzog et al., 1998), rhythmically secrete
vasopressin (VP; Murakami et al., 1991; Watanabe et al., 1993), and
restore rhythmicity in locomotor activity to SCN-ablated hamsters
(Ralph et al., 1990; Silver et al., 1990). These results demonstrate
the cell-autonomous nature of the SCN oscillators but cannot account
for entrainment. Mechanisms of entrainment in the mammalian SCN have
been addressed only at the tissue level. Photic cues are the most
important entraining signals, and it is well established
that the greatest density of retinal fibers travels to an
area of the SCN termed the "ventrolateral" or "core" region and
are more sparse in the area called the "dorsomedial" or "shell"
region (Leak and Moore, 1996; Miller et al., 1996; Moore, 1996).
These reports indicate that SCN cells are neither functionally nor
regionally homogeneous.
The present study explores how cell-autonomous oscillators are
organized within SCN tissue to produce rhythmicity and photic entrainment. We examine the localization of light-induced and endogenously rhythmic expression of Per1, Per2, and
Per3 mRNAs using calbindinD28K
(CalB), FOS, and VP mRNA or protein as markers. The results show that
the regulation of clock genes is not uniform among SCN cells.
Expression of rhythmic and light-induced Per mRNA is
regionally specific. Endogenous rhythmicity in Per1 and Per2 mRNA expression was primarily restricted to the VP
region of the SCN, whereas light-induced Per1 and
Per2 mRNA expression occurred primarily in the CalB region,
where rhythmic Per1 and Per2 mRNA expression was
not detectable. Interestingly, Per3 mRNA was strongly
expressed in the CalB region but was not rhythmic in this area. In the
VP region, Per3 mRNA expression showed low-amplitude rhythmicity. These data suggest that two compartments, one rhythmic and
the other nonrhythmic, constitute the SCN pacemaker and form the basis
of functional differences within the SCN.
Parts of this paper were presented previously at the seventh meeting of
the Society for Research in Biological Rhythms, 2000 (Amelia Island, FL).
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MATERIALS AND METHODS |
Animals and housing. Adult male hamsters
(Mesocricetus auratus) were given food and water ad
libitum. The animal colony room was kept on a 12 hr light/dark
cycle (LD), with light intensity of 600 lux. The testing rooms were
equipped with a white noise generator (91 dB sound pressure level) to
mask environmental noise. For animals housed in constant darkness (DD),
a dim red light (<1 lux; Delta 1, Dallas, TX) allowed for maintenance.
For studies of zeitgeber time (ZT), animals were maintained in LD for
at least 2 weeks before being killed. For studies of circadian time
(CT), animals were housed in LD and then placed in DD for at least 1 week before being killed. In this case, hamsters transferred to DD were
placed in cages equipped with running wheels (diameter, 16 cm), and
locomotor activity was monitored continuously using a computer-based
data acquisition system (Dataquest; Data Sciences, St Paul, MN).
All handling of animals was done in accordance with the Institutional
Animal Care and Use Committee guidelines of Columbia University.
Free-floating digoxigenin in situ
hybridization. Hamster Per1, Per2, and
Per3 cDNA fragments were PCR-amplified using the following
oligonucleotides: 5'-CGAGATGTGTTTCGGGGTG-3' and
5'-AGAGTGGTCAAAGGGCTGC-3' for Per1,
5'-TGCCGTGTCAGCGTTGGAA-3' and 5'-CGCTGGATGATGTCTGGCT-3' for
Per2, and 5'-GAAGAAGCCAAGCAGAGCC-3' and
5'-GGGAGAGCAGACAACAGAG-3' for Per3. Hamster CalB, VP, and
Bmal1 cDNA fragments were amplified by PCR using the
following oligonucleotides: 5'-CTGGAAGGAAAGGAGCTG-3' and
5'-GTATCCGTTGCCATCCTG-3' for CalB, 5'-AGTGTCTCCCCTGCGGCCC-3' and
5'-CAGCTGCGTGGCGTTGCTC-3' for VP, and 5'-GCAACCGCAAGAGGAAAGG-3' and
5'-AACAGGTGGAGGCGAAGTC-3' for Bmal1. These PCR products were then cloned into the pGEM-T Easy vector (Promega, Madison, WI) and
sequenced to verify their identity. Cloned partial hamster Per1 and Per3 show 93 and 75% homology to mouse
Per1 and Per3, respectively. Hamster
Per2 was 90% homologous to rat Per2. Hamster Bmal1, CalB, and VP were 96, 94, and 90% homologous to rat
Bmal1, CalB, and VP, respectively. Antisense and sense cRNA
probes (digoxigenin-labeled) were generated using the MEGAscript
in vitro transcription kit (Ambion, Austin, TX).
For in situ hybridization using digoxigenin (DIG) cRNA
probes in free-floating tissue, we examined 50-100 serial sections
(20-30 µm) through the hypothalamic region, including the entire
extent of the SCN, for each animal reported here. In situ
hybridization was performed as described previously (Hamada et al.,
1999), except for the use of free-floating sections in the present
study. Sections were photographed on Fuji 35 mm film, and color prints
were made. For quantification of optical density, images of brain
sections were captured using a CCD video camera (Sony XC77) attached to
a light microscope (BH-2; Olympus Optical, Tokyo, Japan). mRNA
expression was quantified by measuring stain density using the NIH
Image program version 1.61.
Measurements of relative optical density (ROD), assessing the mean gray
value per pixel of the measured area, were used to quantify the
intensity of the signal. Optical density of staining for Per1,
Per2, Per3, and Bmal1 mRNA were assessed in the CalB and VP regions, as defined in alternate sections stained for these peptides. To calculate the ROD, the background intensity of staining was subtracted from the intensity of staining in the SCN region of
interest. Background OD for Per1, Per2, Per3, and
Bmal1 mRNA was measured in the lateral hypothalamic area.
Background OD for CalB mRNA was measured in the VP area of the SCN.
This was done because that CalB immunoreactivity occurs in the
extra-SCN hypothalamic region, whereas the VP region of the SCN is
devoid of CalB mRNA and protein (Fig. 1A,B; Silver et
al., 1996).
Immunohistochemistry. Hamsters were heavily anesthetized
(pentobarbital, 200 mg/kg), and perfused intracardially with 150 ml of
0.9% saline followed by 300-400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Brains were post-fixed for 18-24 hr at 4°C and cryoprotected in 20% sucrose in 0.1 M phosphate buffer overnight. For immunocytochemistry,
sections (20-30 µm) were processed using the
avidin-biotin-immunoperoxidase method (Silver et al., 1996; LeSauter
et al., 1999a). The primary antibodies used were rabbit polyclonal FOS
(1:5000; Calbiochem, Cambridge, MA) and mouse monoclonal CalB
(1:20,000; Sigma, St. Louis, MO.).
Delineation of distinct SCN regions. We used
immunoreactivity or in situ hybridization for CalB, VP, and
light-induced FOS to demarcate distinct regions of the SCN. To examine
the localization of Per1, Per2, Per3, CalB, and VP mRNA,
alternate free-floating sections were processed for DIG in
situ hybridization and immunochemistry.
Various terms have been used previously to delineate distinct SCN
regions. We use the descriptive terms "CalB region" and "VP
region" to demarcate nonoverlapping regions of the hamster SCN (Fig.
1A), with explicit recognition that SCN regions
delineated by these markers contain other cell types. We concur with
the characterization of the rodent SCN as heterogeneous on the basis of
the peptidergic content of its neuronal populations and their projections. Further analysis will be required to determine how the SCN
regions characterized by their clock gene expression in the present
study correspond to those previously defined as ventrolateral and
dorsomedial or core and shell in hamster, mouse, and rat (Miller et
al., 1996; Moore, 1996; Moore and Silver, 1998; Leak et al., 1999).
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RESULTS |
Rhythmic expression of Per1 and Per2 mRNA
occurs outside the calbindinD28K region
By examining alternate sections by in situ
hybridization and immunochemistry, we characterized the regional
distribution of Per1, Per2, and VP mRNA and CalB protein in
coronal sections from the rostral through the central (anterior and
posterior portions) and caudal quadrants of the SCN. Peak expression of
Per1 mRNA in whole SCN occurs around CT4 and ZT4, whereas
Per2 mRNA in whole SCN peaks at CT8-10 and ZT10-12 in the
hamster (Maywood et al., 1999; Horikawa et al., 2000, Moriya et al.,
2000; Yokota et al. 2000). Figure
1A shows
photomicrographs of brain sections harvested at CT4 and stained for
CalB taken through the extent of the SCN. As previously reported
(Silver et al., 1996), CalB is highly restricted to the central
posterior SCN. Photomicrographs of Per1 mRNA at CT4 and CalB
protein in adjacent sections indicate that Per1 mRNA is
expressed throughout most of the SCN but not in the CalB-positive region. This point is highlighted in an enlarged view showing an
overlay of Per1 mRNA and CalB taken from adjacent sections at ZT4 (Fig. 1B). There is little or no detectable
overlap in the distribution of cells expressing Per mRNA and
CalB protein (Fig. 1B). Photomicrographs from animals
killed at ZT4 indicate that the expression and distribution of
Per1 mRNA is similar under both LD and DD. The localization
of Per2 mRNA does not differ from that of Per1
mRNA under LD (data not shown) and DD (Fig. 1A),
although the expression level of Per2 mRNA is much
higher.
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Figure 1.
Photomicrographs depicting the localization of
endogenously rhythmic (A, B) and light-induced
(C-F) expression of Per1, Per2,
and Per3 mRNA with respect to other known markers of the
SCN, namely, CalB, VP, and FOS. Asterisks denote
adjacent sections. A, The columns show
coronal sections of each SCN quadrant from rostral to caudal from the
same animal. At circadian times when Per1 and
Per2 mRNA expression peaks (CT4, CT8,
respectively), both signals are localized to the VP region of the SCN,
and there is little expression (background level) in the CalB region of
the central SCN, posterior aspect. This is highlighted in higher-power
photomicrographs (B) in which the image of the
Per1mRNA at ZT4 is captured in Adobe
Photoshop, converted to a red signal, and superimposed
in an overlay on the image of the adjacent section immunoreacted for
CalB. The last two columns of A show
Per3 mRNA at two circadian times, CT5 and
CT20.5. Comparison of expression in each SCN quadrant at
these times reveals weak Per3 rhythmic expression
outside the CalB region. In contrast to Per1,
Per2, and VP mRNA, Per3 mRNA is strongly
expressed in the CalB region at both CT5 and CT20.5 but is not
rhythmic. C, D, Comparison of responses
in the presence and absence of light
[Light(+) vs
Light( )] indicates that photic input
induces Per1 (C) and
Per2 (D) mRNA expression in the
CalB region. The light-induced response can be seen during subjective
night (CT20.5, Per1; CT21,
Per2) but not during subjective day
(CT5.5, Per1; CT10,
Per2). Note that light-induced Per1 and
Per2 mRNA and rhythmic Per1 and
Per2 mRNA are expressed in different SCN compartments.
E, Data shown in C and D
highlighted in a high-power photomicrograph and overlay in which the
image of the Per2 mRNA is captured in Adobe Photoshop,
converted to a red signal, and superimposed in an
overlay on the image of the adjacent section immunoreacted for CalB
protein. The time of killing is given at the top of each
column. Light(+),
Presentation of a light pulse (600 lux for 30 min);
light(), no light pulse. Each
column shows sections from two central SCN
quadrants [anterior (Ant) and posterior
(Post)] of the same animal. F,
Photomicrographs showing the localization of light-induced FOS,
Per3, and CalB mRNA in the central anterior and
posterior aspects of the SCN. It is clear that light induces FOS in the
CalB region of the SCN. Comparison of Per3 at
CT5 in A and at CT20.5 in
F indicates that light does not affect the expression of either
Per3 or CalB mRNA.
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Expression of VP mRNA was examined at CT8, when it peaks (Jin et al.,
1999). Some VP mRNA can be seen in each quadrant of the SCN, but its
distribution does not overlap with that of CalB protein (Fig.
1A). Comparison of mRNA expression for
Per1 and Per2 in sections adjacent to those
showing VP mRNA at two circadian times indicates that rhythmic
Per1 (Fig. 1, A vs C) and
Per2 (Fig. 1, A vs D) mRNAs are
expressed in the VP region.
Quantitative analysis (Fig.
2A) of the ROD in each
quadrant of the SCN outside the CalB area shows marked differences in
Per1 and Per2 mRNA expression during subjective
day and night (Fig. 2A). In contrast, within the CalB
area, expression of Per1 and Per2 mRNA is at low,
background levels evident at all circadian times (Fig.
2B).
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Figure 2.
Quantification of the expression of
Per genes in distinct SCN regions for each quadrant in
the presence (+) and absence () of light. Values shown are mean ± SEM; n = 3~4 hamsters per time point.
*p < 0.05; **p < 0.01;
***p < 0.001, Student's t test).
A, In the VP area, ROD measurements of
Per1 and Per2 mRNA at times of peak and
trough expression reveal rhythmicity in each SCN quadrant.
B, In the CalB region, comparison of circadian day and
night for Per1 and Per2 mRNA in the
absence of light indicates no detectable change in either mRNA. On the
other hand, marked light-induced Per1 and
Per2 mRNA occurs in the CalB region of the SCN.
C, ROD measurements for Per3 mRNA in each
SCN region indicate differences between subjective day and night in the
VP region of the rostral and central SCN. Note that Per3
mRNA is strongly expressed but is not rhythmic in the CalB region of
the central posterior SCN in both subjective day and night.
D, Expression of Per3 and CalB mRNA is
not affected by a light pulse given during the subjective night.
Ant, Anterior; Post, posterior.
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Light-induced and rhythmic Per1 and
Per2 mRNA are expressed in different SCN compartments
The hamster SCN receives photic input primarily in the CalB
region. This is based on both tract-tracing (Moore and Silver, 1998)
electron microscopic studies (Bryant et al., 2000) and FOS induction
after a light pulse (Hastings et al., 1996; Silver et al., 1996). The
present results show that rhythmic Per1 and Per2 mRNAs are not detectable in this region (Fig. 1A,B).
In the mouse, however, Per1 and Per2 are reported
to have an important role in light-induced phase shifts (Shigeyoshi et
al., 1997; Akiyama et al., 1999; Wakamatsu et al., 2001), and
Per1 is induced by light in the
ventromedial SCN (Shigeyoshi et al., 1997). We therefore examined the
regional distribution of light-induced Per1 and
Per2 mRNAs. Hamsters were exposed to a 30 min light pulse at
suitable circadian times and killed 1.5-2 hr later. Control animals
were not exposed to light but were otherwise treated identically.
Photomicrographs through the two central quadrants of the SCN reveal
that a light pulse during the subjective day did not induce
Per1 or Per2 mRNA expression (Fig.
1C,D). Control animals not exposed to a light pulse are
shown in Figure 1A. In contrast, during late
subjective night, there was strong light induction of Per1
and Per2 mRNA, highly concentrated in the CalB region.
Further evidence of the overlap in their distribution is shown in
adjacent sections of Per2 mRNA and CalB protein at high
power (Fig. 1E). This is different from the area that
expresses endogenously rhythmic Per1 and Per2 mRNAs. Quantification of the results shows significant light-induced Per1 and Per2 mRNA during subjective night but
not during subjective day in the CalB region (Fig.
2B).
Per3 mRNA expression is high but is neither rhythmic
nor affected by a light pulse in the calbindinD28K
region
The pattern of Per3 mRNA expression differs
substantially from that of Per1, Per2, and VP mRNA (Figs.
1A, 2C,D). Endogenous Per3 mRNA
is expressed in both the VP and CalB regions (Fig.
1A). There is circadian expression of Per3
mRNA in the rostral and central VP region of the SCN (CT4-5 vs
CT20.5-22). The amplitude of the Per3 mRNA rhythm is less
than that of Per1 and Per2 mRNA in this area. In
contrast, there is no detectable circadian expression of
Per3 mRNA within the CalB region, where rhythmic Per1,
Per2, and VP mRNA expression is also not detectable (Fig.
1A,B). Per3 mRNA expression in the CalB
region is constant and high (Fig. 2C). Notably, this is the
region where light induces Per1 and Per2 mRNA
(Fig. 1C-E). Per3 mRNA, however, is not
detectably induced by light during subjective night in any part of the
central SCN (Figs. 1F, 2D).
Quantification confirms the weak but significant (p < 0.05, Student's t test)
rhythmic expression of Per3 mRNA in the VP region (Fig.
2D).
Bmal1 mRNA expression occurs outside the
calbindinD28K region
To further define the regulatory mechanisms that underlie the
differential regulation of the Per genes in the two SCN
regions, we used the expression of the clock gene Bmal1 as a
marker to study its relation to light-induced FOS protein expression
with the following rationale: Most light-induced expression of FOS is
concentrated in the CalB area rather than in the VP region of the SCN
(Fig. 1F; Hastings et al., 1996; Silver et al.,
1996). BMAL1 and CLOCK are important regulators of rhythmic VP and
Per1 gene expression in the SCN (Jin et al., 1999). To
determine whether there is differential expression of the regulatory
gene Bmal1 in distinct SCN regions, we compared Bmal1,
Per3, and CalB mRNA expression in each SCN quadrant. Results
indicate that at its peak, the Bmal1 mRNA signal is strong
in the VP region in the rostral and central anterior SCN, but it is
very weakly expressed in the central posterior CalB region (Fig.
3). In contrast, Per3 mRNA is
very highly expressed in the CalB region at CT16. Light-induced FOS
also occurs here (Fig. 1F).
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Figure 3.
Expression of Bmal1 and
Per3 mRNA occur in different regions. Photomicrographs
show the localization of Bmal1 mRNA with respect to
Per3 and CalB mRNA in each SCN quadrant of the SCN. (For
CT16, each row shows serial sections from the same
animal.) Expression of Bmal1 mRNA is stronger outside
the CalB region of the SCN, whereas Per3 mRNA expression
is high within this region. Comparison between CT4 and CT16 shows that
Bmal1 mRNA is expressed rhythmically outside the CalB
region. Ant, Anterior; Post,
posterior.
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DISCUSSION |
The identification and analysis of clock genes have been very
successful in demonstrating how a cellular oscillator can be built both
mechanistically (Young, 1998; Dunlap, 1999; King and Takahashi, 2000)
and formally (Goldbeter, 1995). In mammals, the mechanism of cellular
oscillation is likely to be similar to the Drosophila model,
although some components of the oscillator are different (Shearman et
al., 2000b). The Drosophila model, however, does not speak
to the mechanism of photic entrainment in mammals. The present analysis
uses the information derived from molecular events to understand SCN
organization at the tissue level. The results indicate that
understanding mechanisms of entrainment in mammals will require
knowledge of SCN circuitry. The present studies of cellular
organization of the SCN reveal novel aspects of the mammalian circadian
clock organization that set the parameters for further studies of
mechanisms at the level of the cell, the tissue, and the organism as a whole.
First, cells in one subregion of the hamster SCN do not oscillate with
respect to Per production. The relationship between nonoscillating and oscillating cells is shown schematically in Figure
4. The commonly used tripartite
organization of the SCN, with an input, clock, and output, is retained.
The key feature is the segregation of the SCN clock into two distinct
elements, shown as a small nonrhythmic region and a larger rhythmic
region expressing the clock genes. It is noteworthy that cells in the nonrhythmic CalB region are easily missed, because they form a small
proportion of the SCN population. In the rat, each SCN has ~8000-10,000 cells (van den Pol, 1980); assuming that the hamster SCN has a comparable number of cells, the CalB subregion constitutes no
more than 10% of the nucleus (Silver et al., 1996). In the hamster the
CalB region is restricted to the posterior aspect of the central region
of the nucleus (Fig. 1). In other species, cells with comparable
properties may be more dispersed among other SCN neurons and difficult
to localize.
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Figure 4.
Model of rhythmicity and photic
entrainment at the tissue level, comprising two functionally different
SCN compartments. As is widely accepted, the circadian system has three
primary components: input, SCN, and output. A,
Model of rhythmicity. In both Light:dark
and Constant dark conditions, there are two distinct
regions of the SCN showing the same pattern of Per gene
expression. In the hamster SCN, these regions can readily be delineated
by cells containing CalB and VP respectively, as indicated in Materials
and Methods. These two compartments constitute the SCN pacemaker. In
the CalB region, rhythmic expression of Per1 and
Per2 mRNA are not detectable (SCN,
left). Another characteristic of this area is that CalB
and Per3 mRNA are highly expressed but not rhythmic.
Endogenous rhythmic expression of clock genes (Per1, Per2,
Per3, and Bmal1 mRNA) and VP mRNA occurs in a
large number of SCN cells restricted to the VP region (SCN,
right). B. Model of photic entrainment. Gated
light induced Per1 and Per2 mRNA and FOS
expression. A light pulse during the day (top) has no
effect on the expression of Per1 and Per2
mRNA or on FOS. A light pulse during the night (bottom)
induces Per1 and Per2 mRNA and FOS
expression in the CalB region. Importantly, some genes in this region
are not induced by light pulse during the night (Per3,
CalB). See Discussion for the proposed mechanism of entrainment.
Ant, Anterior; Post, posterior.
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As shown in the model, clock gene oscillation is a property of some but
not all SCN cells. One SCN region, marked by VP mRNA in this study,
shows rhythmic expression of Per1, Per2, and Per3 mRNA. It has been reported that spontaneous expression of Jun-B and FOS
occur in the dorsomedial region of hamster SCN (Guido et al., 1999).
The model also shows that the other SCN region, marked by CalB cells,
has time-gated light-induced Per1 and Per2 mRNA
and FOS protein and constant high expression of Per3 mRNA. The key concept emerging from the present results is this separation of
the nonrhythmic and rhythmic compartments of the SCN expressing clock
genes, along with the demonstration that responses of nonrhythmic cells
are themselves gated by the circadian system (Fig.
4A,B). Photic information from the retina reaches the
CalB region of the SCN directly (Bryant et al., 2000). This SCN
compartment is not rhythmic in Per mRNA (Fig. 2), CalB mRNA
(data not shown), or CalB protein expression (LeSauter et al., 1999a).
At appropriate times of day, in the presence of a light pulse, photic
input induces Per1 and Per2 mRNA (Fig.
4B). These results are consistent with many reports
in hamsters of light-induced responses concentrated in the CalB region,
including induction of FOS (Silver et al., 1996; Fig. 3D),
vgf (Wisor and Takahashi, 1997), and egr-3
(Morris et al., 1998).
An important question is how (and whether) phase-setting information,
reflected in high expression of light-induced Per1 and Per2, travels from the CalB compartment to other SCN
regions. One possibility is that the VP region itself receives direct
retinal input. Tract-tracing studies, which label retinofugal pathways, indicate that retinal fibers occupy a large region of the SCN, although
they are most dense in the CalB (i.e., ventrolateral or core) region
(Pickard and Silverman 1981; Johnson et al.; 1988; Youngstrom et al.,
1991; Miller et al., 1996; Silver et al., 1996). Synaptic connections
made by retinal fibers in the hamster have been documented only for
CalB (Bryant et al., 2000) and gastrin-related peptide, which is
located in the CalB region (Aioun et al., 1998). It seems unlikely that
photic input directly resets rhythmic Per1 and
Per2 expression in the VP region, given the sparse
expression of these mRNAs after a light pulse (Fig. 1C,D).
The relatively sparse induction of FOS in this region after photic
input also argues against this possibility (Kornhauser et al., 1990;
Rea, 1992; but see Guido et al., 1999). The alternative possibility is
that Per1 and Per2 mRNA expression in the VP
region are produced indirectly, via information relayed from the CalB
region. It has been shown in anatomical studies of rats that
information travels selectively within the SCN, from the
retinorecipient to the nonretinorecipient regions (Moore 1996; Leak et
al., 1999), even though available evidence on intra-SCN circuitry
suggests that SCN neurons make hundreds of intranuclear synapses and
appositions in rats and hamsters (Guldner 1976; van den Pol and Gorcs,
1986; Daikoku et al., 1992; Romijn et al., 1997; Jacomy et
al., 1999; LeSauter et al., 1999b). In support of this hypothesis, in
SCN cell culture, vasoactive intestinal polypeptide (VIP) induces phase
shifts of VP secretion in a light-like phase shift (Watanabe et al.,
2000). Importantly, gastrin-related peptide (Moore and Silver, 1998) strongly induces a light pulse-type phase shift in hamster locomotor activity (Piggins et al., 1995).
The absence of rhythmic expression of clock genes in the CalB region
begs the question of why lesions of the CalB region, which spare other
compartments of the SCN, result in loss of circadian rhythms in
hamsters (LeSauter and Silver, 1999). One possibility is that there is
a population of pacemaker cells, yet to be identified, lying near but
not in the CalB region, with rhythmic Per expression. An
alternative possibility is that in the absence of the cell group with
gated expression of clock genes, oscillators in extra-SCN brain areas
drift out of synchrony. One puzzling feature of SCN organization is the
fact that efferents from the VP region and CalB regions both reach
common target areas in the hamster brain (LeSauter et al., 1999b).
Although the function of these overlapping pathways remains to be
examined directly, such results indicate how phase information might travel.
Another feature suggested by our data, that the regulation of clock
genes is not uniform among SCN cells, is counter to current models of
circadian regulation of Per genes (Dunlap 1999; Shearman et
al., 2000b). In rat and mouse, the Per1 promoter contains
both an E-box (CACGTG) and a cAMP response element (CRE; Hida et al., 2000; Yamaguchi et al., 2000). VP also has an E-box and CRE sequence in
its promoter, and circadian expression of VP is thought to be
controlled by BMAL1 and CLOCK protein binding to the E-box (Jin et al.,
1999). Bmal1 has an important role in activating E-box-dependent rhythmic Per1 and VP mRNA expression.
Although the underlying mechanism is not yet known, rhythmicity in FOS and in CRE-mediated gene expression (using CRE- -galactosidase transgenic mice) has also been reported (Obrietan et al., 1999; Schwartz et al., 2000). The present studies show that the VP area is
rich in Bmal1 mRNA (Fig. 3), suggesting that the circadian expression of Per1, Per2, Per3, and VP
mRNA in this region occurs through the activation of E-boxes. In
contrast, in the CalB region, where the expression of BMAL1 is low,
Per1 and Per2 mRNA and FOS protein are strongly
light-induced. This suggests that Per1 and Per2
mRNA regulation in this region may be achieved through a light response
element such as the CRE. In further support of the notion that light
induction involves the CRE, expression of phosphorylated CRE binding
protein, a positive regulator of the c-fos gene, also occurs
in the region where light induces FOS protein (Schurov et al., 1999).
Last, Per3 mRNA is strongly expressed in the CalB region but
is not induced by light, suggesting that Per3 regulation in
this region is achieved by a distinct transcriptional system from that
of Per1 and Per2. These results suggest that cells expressing rhythmic Per1, Per2, Clock, and
Bmal1 mRNA in the VP region of the SCN are candidates for
principal oscillator cells.
Many previous reports indicate that the SCN may have functionally
distinct regions. In the rat and hamster, a substantial population of
VP-containing cells lies in the dorsomedial SCN, whereas VIP is located
mainly in the ventrolateral SCN (Card and Moore, 1984; Inouye and
Shibata, 1994; Miller et al., 1996; Moore, 1996; Moore and Silver,
1998). The ventrolateral region of the rat SCN has a dense region of
CalB expression (Arvanitogiannis et al., 2000), although these are not
as densely packed as in the hamster. The VP content of the SCN shows
circadian rhythmicity in both LD and DD and is not affected by a light
pulse (Inouye and Shibata, 1994). In contrast, the VIP content of the
SCN has diurnal variation in LD but not in DD in vivo.
Light-induced fos family genes are concentrated in the
ventrolateral region, whereas circadian rhythms of fos occur
primarily in the dorsomedial subdivision of the rat and hamster SCN
(Chambille et al., 1993; Guido et al., 1999; Schwartz et al., 2000). In
support of the notion that functional retinal input is highly localized
to part of the SCN, electrical stimulation of the optic nerve evokes
fast positive and late large negative waves in the ventrolateral but
not the dorsolateral SCN in a horizontal slice preparation of the rat
SCN (Shibata et al., 1984). These reports suggest that the SCN has two
functionally distinct regions, wherein one receives light information
and the other does not. Importantly, daily rhythms driven by the LD
cycle, such as SCN VIP content, do not involve an E-box-dependent
negative feedback loop. This is consistent with the present results
showing that there is no detectable rhythm in expression of
Per1 and Per2 mRNA in the CalB region.
Furthermore, in this region, Per1 and Per2 mRNA
expression are the same in both LD and DD (Figs.
1A,B, 4A). In support of this idea,
individual SCN neurons in homozygous Clock mutant mice are
arrhythmic in electrical activity, paralleling the effects on locomotor
activity in these animals (Herzog et al., 1998) and suggesting a role
for E-box-dependent mechanisms in SCN pacemaker function. The
occurrence of regional differences in E-box-dependent negative feedback
loops of clock genes and their products, shown in the present study,
seems to play an important role in regional differences within the SCN.
Our results also explain some of the behavioral phenotypes of various
circadian mutants. Per2 mutant mice, characterized by a
deletion mutation in the PAS domain of the Per2 gene, show
arrhythmic responses in constant darkness (Zheng et al., 1999). This is
consistent with our results showing that rhythmicity in Per2
occurs in SCN pacemakers (located in the VP region). Our results
suggest that regulation of light-induced clock genes is distinct from
that of endogenously occurring clock gene expression in the VP region. In this context, it is interesting that Clock mutant mice
(Vitaterna et al., 1994) and Mop3 (also known as
Bmal1) knock-out mice (Bunger et al., 2000) entrain but show
disrupted circadian rhythms of locomotor activity in DD. As might be
predicted from the present results, Clock mutant mice
express light-induced Per1 and Per2 mRNA
(Shearman and Weaver, 1999). Bmal1 and Clock are
both important for the regulation of rhythmicity (King and Takahashi,
2000). On the other hand, very low expression of CLOCK protein (van
Esseveldt et al., 2000) and Bmal1 mRNA (Fig. 4) in the CalB
region bring into question the role of these genes in light-induced
Per1 and Per2 mRNA expression. Finally, mice with
targeted disruption of the mPer3 gene had normal activity
rhythms, with a slightly shorter free-running period than wild-type
animals (Shearman et al., 2000a), consistent with our finding of low
Per3 mRNA expression in the rhythmic compartment.
| |
FOOTNOTES |
Received March 20, 2001; revised July 23, 2001; accepted July 23, 2001.
This research was supported by National Institutes of Health Grant
NS37919 (R.S.) and a grant from the Japan Society for the Promotion of
Science (T.H.). We thank Drs. Paul Hardin, Lance Kriegsfeld, and
William J. Schwartz for comments on drafts of this manuscript and Honor
Kirwan for technical assistance.
Correspondence should be addressed to Dr. Rae Silver, Columbia
University, Mail Code 5501, 1190 Amsterdam Avenue, Room 406, Schermerhorn Hall, New York, NY 10027. Email: QR{at}columbia.edu.
Dr. Venuti's present address: Department of Cell Biology and Anatomy,
Louisiana State University Medical Center, 1901 Perdido Street, New
Orleans, LA 70112.
| |
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L. Yan, N. C. Foley, J. M. Bobula, L. J. Kriegsfeld, and R. Silver
Two Antiphase Oscillations Occur in Each Suprachiasmatic Nucleus of Behaviorally Split Hamsters
J. Neurosci.,
September 28, 2005;
25(39):
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[Abstract]
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W. Nakamura, S. Yamazaki, N. N. Takasu, K. Mishima, and G. D. Block
Differential Response of Period 1 Expression within the Suprachiasmatic Nucleus
J. Neurosci.,
June 8, 2005;
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[Abstract]
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M. C. Antle, L. J. Kriegsfeld, and R. Silver
Signaling within the Master Clock of the Brain: Localized Activation of Mitogen-Activated Protein Kinase by Gastrin-Releasing Peptide
J. Neurosci.,
March 9, 2005;
25(10):
2447 - 2454.
[Abstract]
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F. Geier, S. Becker-Weimann, A. Kramer, and H. Herzel
Entrainment in a Model of the Mammalian Circadian Oscillator
J Biol Rhythms,
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83 - 93.
[Abstract]
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A. C. Jackson, G. L. Yao, and B. P. Bean
Mechanism of Spontaneous Firing in Dorsomedial Suprachiasmatic Nucleus Neurons
J. Neurosci.,
September 15, 2004;
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J. Itri, S. Michel, J. A. Waschek, and C. S. Colwell
Circadian Rhythm in Inhibitory Synaptic Transmission in the Mouse Suprachiasmatic Nucleus
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92(1):
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[Abstract]
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R. Brandstaetter
Circadian lessons from peripheral clocks: Is the time of the mammalian pacemaker up?
PNAS,
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Y. Tong, H. Guo, J. M. Brewer, H. Lee, M. N. Lehman, and E. L. Bittman
Expression of haPer1 and haBmal1 in Syrian Hamsters: Heterogeneity of Transcripts and Oscillations in the Periphery
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[Abstract]
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L. J. Kriegsfeld, J. LeSauter, and R. Silver
Targeted Microlesions Reveal Novel Organization of the Hamster Suprachiasmatic Nucleus
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March 10, 2004;
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I. N. Karatsoreos, L. Yan, J. LeSauter, and R. Silver
Phenotype Matters: Identification of Light-Responsive Cells in the Mouse Suprachiasmatic Nucleus
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H. S. Lee, H. J. Billings, and M. N. Lehman
The Suprachiasmatic Nucleus: A Clock of Multiple Components
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T. Hamada, J. LeSauter, M. Lokshin, M.-T. Romero, L. Yan, J. M. Venuti, and R. Silver
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M. C. Antle, D. K. Foley, N. C. Foley, and R. Silver
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H. Oster, S. Baeriswyl, G. T.J. van der Horst, and U. Albrecht
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[Abstract]
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A. N. Coogan and H. D. Piggins
Circadian and Photic Regulation of Phosphorylation of ERK1/2 and Elk-1 in the Suprachiasmatic Nuclei of the Syrian Hamster
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S. J. Kuhlman, R. Silver, J. Le Sauter, A. Bult-Ito, and D. G. McMahon
Phase Resetting Light Pulses Induce Per1 and Persistent Spike Activity in a Subpopulation of Biological Clock Neurons
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February 15, 2003;
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H. Arima, S. B. House, H. Gainer, and G. Aguilera
Neuronal Activity Is Required for the Circadian Rhythm of Vasopressin Gene Transcription in the Suprachiasmatic Nucleus in Vitro
Endocrinology,
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H. Oster, A. Yasui, G. T.J. van der Horst, and U. Albrecht
Disruption of mCry2 restores circadian rhythmicity in mPer2 mutant mice
Genes & Dev.,
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F. K. Stephan
The "Other" Circadian System: Food as a Zeitgeber
J Biol Rhythms,
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
