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The Journal of Neuroscience, July 1, 2001, 21(13):4883-4890
Dissociation between Light-Induced Phase Shift of the Circadian
Rhythm and Clock Gene Expression in Mice Lacking the Pituitary
Adenylate Cyclase Activating Polypeptide Type 1 Receptor
Jens
Hannibal1,
Francoise
Jamen2,
Harriette
S.
Nielsen1,
Laurant
Journot2,
Philippe
Brabet2, and
Jan
Fahrenkrug1
1 Department of Clinical Biochemistry, Bispebjerg
Hospital, University of Copenhagen, DK-2400 Copenhagen, Denmark, and
2 Unité Propre de Recherche 9023, Centre National de
la Recherche Scientifique, 34094 Montpellier Cedex 5, France
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ABSTRACT |
The circadian clock located in the suprachiasmatic nucleus (SCN)
organizes autonomic and behavioral rhythms into a near 24 hr time that
is adjusted daily to the solar cycle via a direct projection from the
retina, the retinohypothalamic tract (RHT). This neuronal pathway
costores the neurotransmitters PACAP and glutamate, which seem to be
important for light-induced resetting of the clock. At the molecular
level the clock genes mPer1 and mPer2 are
believed to be target for the light signaling to the clock. In this
study, we investigated the possible role of PACAP-type 1 receptor
signaling in light-induced resetting of the behavioral rhythm and
light-induced clock gene expression in the SCN. Light stimulation at
early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice
(PAC1 /)
compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and
c-fos gene expression. Light stimulation at late night
induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and
PAC1/ mice.
However, in contrast to the phase advance seen in wild-type mice,
PAC1/ mice
responded with phase delays after photic stimulation. These data
indicate that PAC1 receptor signaling participates in
the gating control of photic sensitivity of the clock and suggest that
mPer1, mPer2, and c-fos
are of less importance for light-induced phase shifts at night.
Key words:
retinohypothalamic tract; suprachiasmatic nucleus; clock
genes; knock-out; mouse; light entrainment
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INTRODUCTION |
In mammals, autonomic, endocrine,
and behavioral rhythms are generated by a master clock located in the
hypothalamic suprachiasmatic nuclei (SCN) (Klein et al., 1991 ). To
maintain synchrony with the solar day-night cycle, the clock is
adjusted (entrained) by light through a monosynaptic pathway
originating from a subset of retinal ganglion cells, the
retinohypothalamic tract (RHT) (Moore and Lenn, 1972; Johnson et al.,
1988; Levine et al., 1991; Moore et al., 1995). Our knowledge on the
molecular machinery driving the endogenous clock has until recently
been limited, but identification of components of the "molecular
clock" participating in autonomous transcriptional/translational
feedback loops have shed light on parts of this complex regulatory
mechanism (for review, see Dunlap, 1999; King and Takahashi, 2000). How
light entrains the clock is, however, not fully understood. The clock genes mPer1 and mPer2, which show circadian
expression within the SCN, have been attributed a role in light-induced
resetting of the mammalian circadian clock caused by rapid induction of both genes after light stimulation at night (Albrecht et al., 1997;
Shigeyoshi et al., 1997; Zylka et al., 1998; Akiyama et al., 1999; Yan
et al., 1999; Field et al., 2000). A support for this notion is the
observation that the use of antisense oligonucleotide blocks
mPer1 mRNA transcription, light-induced phase shift of running wheel activity, and glutamate-induced phase shift of neuronal firing activity (Akiyama et al., 1999).
Pituitary adenylate cyclase activating polypeptide (PACAP), a member of
the vasoactive intestinal polypeptide (VIP)-secretin-glucagon family
of neuropeptides (Arimura, 1998), is located in a small population of
retinal ganglion cells constituting the RHT (Hannibal et al., 2001).
PACAP is colocalized with glutamate in the RHT (Hannibal et al., 2001).
In nanomolar concentrations, PACAP phase shifts the endogenous rhythm
and induces per gene expression in the SCN similar to light
(Harrington et al., 1999; Nielsen et al., 2001) whereas micromolar
concentrations of PACAP modulate glutamate-induced phase shift (Chen et
al., 1999) and Per1 and Per2 gene expression
(Nielsen et al., 2001). Three types of high-affinity G-protein-coupled
receptors for PACAP have been cloned: the PACAP preferring type 1 (PAC1) receptor (Spengler et al, 1993) and two VIP/PACAP
preferring type 2 (VPAC1 and VPAC2) receptors (Harmar et al., 1998).
Both PAC1 (Hannibal et al., 1997) and VPAC2 (Lutz et al.,
1993) receptors are expressed in the SCN but so far, most findings
indicate that the PAC1 receptor is responsible for PACAP signaling to the circadian system (Hannibal et al., 1997; von Gall et
al., 1998; Kopp et al., 1999).
The present study shows that in mice lacking the PAC1
receptor, light stimulation causes a dissociation between photic phase shifting of the circadian rhythm and induction of clock gene expression in the SCN. Our findings indicate that PAC1 receptor
signaling participates in the gating control of photic sensitivity of
the clock and suggest that mPer1, mPer2, and
c-fos are of less importance for light-induced phase shifts
at night.
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MATERIALS AND METHODS |
Animals. Males from an
F1-F4-crossbred strain
(129/SvXC57BL6/J) of PAC1-receptor mutant mice were used
(Jamen et al., 2000). Animals were maintained in a 12 hr light/dark
(LD) cycle with food and water ad libitum. Experiments were
performed according to the principles of laboratory animal care (Law on
animal experiments in Denmark, publication number 382, June 10th, 1987).
Behavioral analysis. Mice (age 6-10 weeks at the beginning
of the experiments) were housed in individual cages equipped with a
running wheel in ventilated, light-tight chambers with controlled lightning. Wheel-running activity was monitored by an on-line personal
computer connected via a magnetic switch to the Minimitter Running
Wheel activity system (consisting of QA-4 activity input modules, DP-24
dataports, and Vital View data acquisition system, version 2.19;
MiniMitter Company, Sunriver, OR). Wheel revolutions were collected
continuously in 10 min bins. Animals were entrained to a 12 hr LD cycle
(lights on at 7:00 A.M., lights off at 7.00 P.M.) for at least 14 d. Free-running period ( ) was assessed during days 4-18 in constant
darkness (DD) using Active View software version 1.2 (MiniMitter
Company). To determine the light-induced phase shift of locomotor
activity, animals in their home cages were moved to another room and
exposed to a 30 min pulse of white light (>300 lux) at two different
circadian times (CTs) 16 and 23; n = 7-10 of each
genotype) at which CT12 was designated as activity onset. The CT times
were determined according to the equation by Daan and Pittendrigh
(1976a). Three wild-type and three
PAC1/
mice were also exposed to light at CT2, 6, 10, 14, and 20 to obtain a
full phase-response curve (PRC). Light-induced phase shift amplitude
was derived from regression lines drawn through the activity onset of
at least 7 d immediately before the day of stimulation and 7 d after reestablishment of steady-state circadian period after
stimulation (Chen et al., 1999).
In situ hybridization histochemistry. In situ hybridization
was performed as previously described (Hannibal et al., 1997). cRNA
probes were prepared using 33P-UTP and
cDNA of the rat PAC1 receptor (Hannibal et al., 1997), rPer1, and rPer2 probes (Nielsen et al., 2001).
The mouse c-fos cDNA (pTRI-c-fos/exon4-Mouse probe template)
obtained from Ambion (Austin, TX) was prepared using T3 polymerase
generating a cRNA probe of 279 bases. Control sections were hybridized
with the corresponding sense probes, which gave no specific signals.
Wild-type and PAC1
/ mice
(n = 6-8 in each group) kept in 12 hr LD were
decapitated at zeitgeber time (ZT)6, ZT17, and ZT24 (in dim red light,
<3 lux). For light-stimulation experiments, animals received a 30 min
pulse of white light (>300 lux) at ZT16 and ZT23 and were killed (in
dim red light, <3 lux) 60 min after the initiation of light exposure
(ZT17 and ZT24). We cut 12-µm-thick coronal brain sections through
the SCN from each animal on a cryostat on five consecutive
gelatin-coated slides. From each animal this included a total of 20 sections through the rostrocaudal SCN, representing four different
levels of the SCN on each slide. A slide from each animal was
hybridized under identical conditions with
33P-labeled rper1, rper2, and
c-fos antisense cRNA probes, respectively. After
hybridization and washing, the slides were exposed to Amersham Hyperfilm (Amersham, DK) for 1-2 weeks. Autoradiograms were quantified using an image analysis system (Q500MC Image Analysis System version 2.02A; Leica Cambridge, UK), and grain densities were converted to
disintegrations per minute per gram of wet weight using
14C standards as references (Amersham, DK)
as described previously (Hannibal et al., 1995a). The level of mRNA of
each animal was quantified corresponding to the retinorecipient zone of
the SCN by measuring positive hybridization signals in the bilateral
SCN, which included four sections through the rostrocaudal SCN, and the
calculated mean of these measurements from each of six to eight animals
was used to calculate the group mean and SEM. As reported, gene
expression of Per1 and Per2 is induced by light in this part of the SCN of rats and mice (Albrecht et al., 1997; Shigeyoshi et al., 1997; Yan et al., 1999). Measurements obtained from
the individual sections were corrected for nonspecific background by
subtracting grayscale values from neighboring areas (the optic chiasma)
considered free of positive hybridization. Thus, the sections served as
their own internal standards. The changes in expression between the
mRNA levels of the control groups and the stimulated groups were
presented in disintegrations per minute per gram according to the
14C standards.
Immunohistochemistry. PACAP immunoreactivity in the mouse
SCN was investigated in wild-type and
PAC1/
mice (n = 3 of each group). The mice were fixated by
perfusion through the heart at ZT6 in Stefani's fixative and
cryoprotected and cut in a cryostat in 40-µm-thick free-flowing
sections, as described previously (Hannibal et al., 1997). PACAP was
visualized using a specific monoclonal mouse anti-PACAP antibody
characterized previously (Hannibal et al., 1995b) and a mouse on
mouse immunodectection kit (Vector Laboratories, Burlingame, CA)
combined with ABC-tyramide-DAB staining (Fahrenkrug and Hannibal,
1998). As controls, sections were incubated with antibodies preabsorbed
with PACAP38 (20 µg/µl), which abolished all staining.
Statistical analysis. Data are presented as mean ± SEM. Mann-Whitney U test (using GrapPad Prism, version 3.0 software) was used to determine significant differences between values
from stimulated and control groups. p < 0.05 was
considered statistically significant.
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RESULTS |
PACAP immunoreactivity in the SCN of PAC1
receptor-deficient mice
Immunohistochemical analysis of SCN sections revealed that the
number, distribution, and staining intensity of PACAP-immunoreactive nerve fibers in the SCN of wild-type and
PAC1/
mice did not differ (Fig.
1a,b), indicating no gross
anatomical differences of the RHT between the wild-type and homozygous
littermates. As expected, the wild-type mice but not the
PAC1/
mice expressed PAC1 receptor mRNA in the SCN (Fig.
1c,d).
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Figure 1.
PACAP and PAC1 receptors in the
SCN. Photomicrographs showing PACAP-immunoreactive nerve fiber in the
SCN of wild-type (a) and PAC1
receptor-deficient mice
(PAC1/)
(b). In situ hybridization using a
33P-labeled rat cRNA PAC1 receptor probe on
a coronal brain section at the level of the SCN of wild-type
(c) and
PAC1/ mice
(d). Arrows indicate the positive
(c) and negative (d) signal
for PAC1 receptor expression in the SCN. 3v, Third
ventricle. Scale bars, 100 µm.
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Circadian rhythmicity in PAC1 receptor-deficient mice
Behavioral analysis of running-wheel activity showed that
wild-type, PAC1+/, and
PAC1/
mice were able to entrain to a 12 hr LD cycle. When transferred to
constant darkness, wild-type and
PAC1+/ mice displayed the same
period length (), whereas of the
PAC1/
mice was slightly, although significantly shorter, than the wild type
(23.3 ± 0.1 vs 23.7 ± 0.1 hr) (Fig.
2a-d). The clock genes mPer1 and mPer2 are expressed in the SCN with a
clear circadian rhythm (Zylka et al., 1998). Consequently, we analyzed
the expression of mPer1 and mPer2 using
quantitative in situ hybridization at three circadian time
points (ZT6, ZT17, and ZT24) (Table 1). Except for a modest but statistically significant higher
mPer1 gene expression in
PAC1/
mice at ZT24 no differences in the level of mRNA for the per genes were observed between wild-type and
PAC1/
animals during LD conditions. No difference was observed in the regional distribution of per gene expression within the SCN
of wild-type and
PAC1/
animals examined at midsubjective day. At
this time point per gene expression level was uniformly high
in the entire SCN (CT6, data not shown).
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Figure 2.
Circadian rhythm of locomotor activity in the
PAC1 genotype mice. Wheel-running activity was
double-plotted according to the convention so that the data for each
day are represented both to the right and beneath that of the previous
day. Mice maintained in a 12 hr LD cycle (indicated by the top
black-white bar) were placed in constant darkness
(DD) conditions on the day indicated by the black
bar to the right in each figure. Free-running
period of locomotor activity in DD is exemplified by representative
free-running actograms of wild-type (a),
PAC1+/(b),
and PAC1/
animals (c). The calculated free-running periods
for each animal of the different genotypes in DD are shown in
d. In constant darkness, the free-running period ()
was significantly shortened in
PAC1/ mice
compared with wild-type (**p < 0.01, Mann-Whitney
U test).
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Table 1.
Expression of mPer1 and mPer2 genes
in the SCN of wild-type and PAC1/ mice kept in a 12 hr
light/dark cycle
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Photic stimulation at early night
We next examined the effect of exposure to a 30 min light pulse at
early night when light is known to induce phase delay (CT16) of the
circadian rhythm in mice (Daan and Pittendrigh, 1976a). PAC1/
mice showed a significantly larger phase delay in locomotor activity compared with the wild-type mice at CT16 (116.0 ± 18.8 vs
96.3 ± 10.3 min) (Figs. 3,
4a, 6). The behavior of
PAC1+/ mice and wild-type mice was
identical, and the heterozygous mice were not investigated further
(Figs. 4a, 5a).
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Figure 3.
The effects of a 30 min light pulse on the
circadian phase at two different circadian times (CT16 and CT23) in
wild-type and
PAC1/ animals
kept in constant darkness. Wheel-running activity was double-plotted
according to the convention so that the data for each day are
represented both to the right and beneath that of the previous day.
Representative free-running actograms from wild-type
(a) and
PAC1/ animals
(b) are shown. The animals were maintained in a
12 hr LD cycle, as indicated by the top black-white bar
and subsequently placed in constant darkness (DD)
conditions on the day indicated by the black bars to the
left in each figure. Light pulses indicated (by
arrows) at the two different time points (CT16 and CT23)
were given with at least a 10 d interval. Steady-state phase
shifts (in minutes) were determined by the drawn eye-fitted
lines.
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Figure 4.
The effects of photic stimulation at early
subjective night. A 30 min light pulse (>300 lux) administered at CT16
caused a phase shift of the circadian rhythm (a)
and gene expression of c-fos (b),
mPer1(c), and mPer2
(d) in the SCN of wild-type,
PAC1+/ (only in a)
and PAC1/
mice. Representative in situ hybridization signals for
c-fos, mPer1, and mPer2 in the SCN at
each time point are shown on the top of each
panel. Note the increased phase delay observed in
PAC1/ mice
compared with wild-type animals and the blunted light response of
c-fos, mPer1, and mPer2
gene expression in
PAC1/ mice.
Values are given as means ± SEM (n = 6-8
animals). *p < 0.05; **p < 0.01 (Mann-Whitney U test).
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Figure 5.
The effects of photic stimulation at late
subjective night. A 30 min light pulse (>300 lux) administrated at
CT23 causes a phase shift of the circadian rhythm
(a) and gene expression of c-fos
(b), mPer1
(c), and mPer2
(d) in the SCN of wild-type,
PAC1+/ (only in a)
and PAC1/
mice. Representative in situ hybridization signals for
c-fos, mPer1, and mPer2 in the SCN at
each time point are shown on the top of each
panel. Note the phase delay observed in
PAC1/ mice
compared with a phase advance seen in wild-type animals and the
light-induced inhibition of mPer2 gene expression in
PAC1/ mice.
Values are given as means ± SEM (n = 6-8
animals). *p < 0.05; **p < 0.01; ***p < 0.001 (Mann-Whitney U
test).
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Because of the anatomical and temporal specificity of expression, the
immediate-early gene c-fos has been suggested to be a
molecular component of the photic pathway for entrainment of the
circadian clock (Rusak et al., 1990). It has been used as a functional
marker for the activation of SCN neurons by light and the distinct
induction of both mRNA and protein within the retinorecipient
(ventrolateral) subdivision of the SCN (Schwartz et al., 1994, 2000;
Kornhauser et al., 1996). In both wild-type and
PAC1/
mice, the level of c-fos and per gene expression
was uniformly low in the entire SCN at night (Figs. 4, 5). In wild-type
mice, light increased the level of c-fos mRNA in the
retinorecipient SCN, whereas in
PAC1/
mice the c-fos response was almost completely blunted (Fig.
4b). The clock gene mPer1 is light-inducible at
early night and has been associated with light-induced changes in
running wheel activity at night (Albrecht et al., 1997; Shigeyoshi et
al., 1997; Zylka et al., 1998; Akiyama et al., 1999; Field et al.,
2000). Consequently, we examined the gene expression of
mPer1 in the SCN of wild-type and PAC1
receptor-deficient mice. As previously reported, mPer1 gene
expression was induced in the retinorecipient zone of wild-type animals
within 1 hr after exposure to a 30 min light pulse (Albrecht et al.,
1997; Shigeyoshi et al., 1997; Zylka et al., 1998) (Fig. 4c). In contrast, the light-induced increase in
mPer1 gene expression was abolished in
PAC1/
mice, as found for c-fos (Fig. 4b,c). As
previously reported, light stimulation in the wild-type mice (Albrecht
et al., 1997) at ZT16 also induces mPer2 expression within 1 hr. As seen for mPer1, light-induced mPer2
expression was significantly blunted in
PAC1/
mice compared with wild-type animals at this time point (Fig. 4d).
Photic stimulation at late night
Thirty minute light pulse at late subjective night provoked a
marked difference in behavior between the wild-type and the PAC1/
mice. Light induced a 36.9 ± 4.3 min phase advance in the
wild-type mice well in accord with previous reports on mice (Daan and
Pittendrigh, 1976a). In contrast, a 60.0 ± 28.2 min phase delay
in locomotor activity was observed in
PAC1/
mice (Figs. 3b, 5a,
6). A full PRC performed in three
wild-type and three
PAC1/
animals excluded that the phase response to light in
PAC1/
mice was attributable to a shift of the PRC to the right (Fig. 6). At
late subjective night, a 30 min light pulse induced c-fos and mPer1 gene expression in the ventrolateral part of the
SCN to the same level in wild-type and
PAC1/
mice within 1 hr (Fig. 5b,c), indicating a PAC1
receptor-independent mechanism is involved in the light-induced
expression of these two genes at late night. Light stimulation at late
night did not induce mPer2 expression in the wild-type
animals in agreement with previous observation (Albrecht et al., 1997).
A slight but statistically significant decrease in mPer2
expression was, however, demonstrated in
PAC1/
mice compared with the wild-type animals (Fig. 5d).
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Figure 6.
Phase-dependent effects on running wheel activity
(PRC) to a 30 min light pulse (>300 lux) given to
PAC1+/+ and
PAC1/ kept in
constant darkness. CT12 represents the time point for activity onset.
Values (mean ± SEM, n = 3 for all CTs except
CT16 and CT23 where n = 7-9) are given in minutes.
Positive values represent a phase advance, and negative values
represent a phase delay of activity rhythm. *p < 0.05, **p < 0.01, Mann-Whitney U
test.
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DISCUSSION |
Light entrainment of the clock to the environmental light/dark
cycle is believed to involve induction of c-fos expression (Kornhauser et al., 1996) and the recently identified clock genes Per1 and Per2 (Albrecht et al., 1997; Shigeyoshi
et al., 1997; Zylka et al., 1998; Akiyama et al., 1999; Yan et al.,
1999; Field et al., 2000). These genes are rapidly induced by light
stimulation at early and late subjective night in the retinorecipient
(ventrolateral) zone of the SCN followed by a phase delay and/or phase
advance in the behavioral rhythm, respectively (Meijer and Rietveld,
1989). In the present study we have shown that mice lacking the
PAC1 receptor respond to light stimulation at early night by
larger phase delays in behavioral rhythm compared with wild-type mice, in addition to a marked reduction in light-induced mPer1,
mPer2, and c-fos gene expression in the retinorecipient
zone of the SCN. This dissociation between light-induced phase shift of
running wheel activity and induction of c-fos,
Per1, and Per2 gene expression in the SCN
indicates that light-induced c-fos and clock gene expression and phase shift are not always interdependent phenomena. The role of
c-fos gene expression in the SCN in circadian rhythm
regulation is not fully clarified. c-fos gene expression in
the SCN has been correlated with circadian function because of the
anatomical specificity of light-induced gene expression within the
retinorecipient zone of the SCN, circadian phase dependence of both
c-fos induction and phase-shifting behavior, and because
photic thresholds of the two processes are quantitatively correlated
(Kornhauser et al., 1990). There is, however, no perfect relation
between light-induced expression of c-fos and phase shift of
circadian rhythms, and examples of phase shift of the behavioral rhythm
without induction of c-fos exist. The lack of correlation is
particularly apparent at the transition between the delay and the
advanced periods, time spans during which light induces
c-fos gene expression to a maximum but does not alter
circadian activity rhythms (Sutin and Kilduff, 1992). In transgenic
hypertensive TGR (mRen2)27 rats, light phase shifts the activity rhythm
without induction of c-fos (Lemmer et al., 2000).
Furthermore, c-fos knock-out mice were still able to respond
to light pulses, although they showed a damped phase-response curve
(Honrado et al., 1996). Together with the present data showing that
light-induced c-fos expression in the SCN of
PAC1/
mice was completely blunted at early night, light-induced
c-fos expression may be involved but is not necessary for
light-induced phase shift of the behavioral rhythms at this time point.
Moreover, PAC1 receptor activation seems necessary for
light-induced c-fos expression at early night.
Interestingly, the recently identified clock genes mPer1 and
mPer2, which have been implicated in photic responses at
night (Albrecht et al., 1997; Shigeyoshi et al., 1997; Zylka et al., 1998; Akiyama et al., 1999; Field et al., 2000) did not, like c-fos, increase after light stimulation at early night in
PAC1/
mice despite the larger phase delay. At late night when photic stimulation provoked a phase delay in
PAC1/
mice in contrast to a phase advance in wild-type, mPer1, and c-fos gene expression of
PAC1/
mice was similar to the wild type. This indicates that PAC1
receptor signaling plays a minor if any role in light-induced gene
expression of mPer1 and c-fos gene at this time
point. Per1 gene expression has been linked to light-induced
phase shifts because of (1) a rapid (30-60 min) and robust induction
of Per1 mRNA in the ventrolateral zone of the SCN
(Shigeyoshi et al., 1997), (2) a correlation between light-induced
Per1 expression and light-induced phase shift (Shigeyoshi et
al., 1997), and (3) blocking of light-induced phase shift with antisense oligonucleotides to Per1 mRNA (Akiyama et al.,
1999). A possible role of Per2 in photic phase shifting at
early night is suggested because of the light-induced stimulation of
Per2 mRNA in the same part of the SCN as seen for
Per1 at this time (Albrecht et al., 1997; Takumi et al.,
1998). The present study clearly demonstrates that
PAC1/
mice are able to phase shift without induction of Per1 and
with a blunted Per2 mRNA expression in the ventrolateral
part of the SCN at early night. PAC1 receptor signaling
seems important for light-induced Per gene regulation, and
the results suggest that other factors are important for light-induced
phase shift of the behavioral rhythm.
The lack of PAC1 receptor signaling seems to affect the
rhythmic expression of the Per1 genes. During LD condition
mPer1gene expression showed a slight but statistically
significant increase at dawn, whereas mPer2 was similar in
both wild-type and
PAC1/
mice, suggesting that in
PAC1/
mice the molecular machinery regulating mPer1 entrainment
could be disturbed. During constant darkness,
PAC1/
mice had a significant shortened compared with wild-type animals, supporting the notion that PAC1 receptor signaling
participates in the regulation of the core clock elements controlling
the . As previously reported in mice (Shigeyoshi et al., 1997) and
rats (Yan et al., 1999), the expression level of the mRNA of
mPer at midsubjective day was uniformly high
throughout the ventrolateral and dorsomedial SCN in wild-type and
PAC1/animals
(data not shown), indicating that circadian expression of the
per genes takes place in the same subregions of the SCN of
both wild-type and
PAC1/animals.
It remains to be investigated whether other components of the molecular
"clock" are changed in
PAC1/
mice. The first identified mammalian clock gene,
clock (Antoch et al., 1997; King et al., 1997) has
been shown to influence the regulation of light sensitivity of the
clock. Clock mutants had a blunted expression of both c-fos,
Per1, and Per2 in the SCN after light stimulation
at night (Shearman and Weaver, 1999), although these mice were able to
entrain to light (Antoch et al., 1997; King et al., 1997). Also the
cryptochromes CRY1 and CRY2, which are parts of the molecular clock
(van der Horst et al., 1999; Vitaterna et al., 1999), showed changed
light-induced Per expression at night (Okamura et al.,
1999). In CRY2-deficient mice, light-induced Per1 expression
is reduced 50-60%, whereas the light-induced phase shift (6 hr light
stimulation) was increased compared with wild-type animals (Thresher et
al., 1998). In CRY1-lacking mice, light-induced Per1
expression is blunted, but Per2 expression is normal
(Vitaterna et al., 1999). Future studies will clarify whether the CRY
and/or the clock genes could be target for
PAC1 receptor signaling.
Glutamate is considered to be the primary transmitter mediating light
information to the clock (Ding et al., 1994; Ebling, 1996). There is,
however, increasing evidence that PACAP, which is costored with
glutamate in the RHT (Hannibal et al., 2000), participates in photic
entrainment of the clock (Kopp et al., 1997, 1999; von Gall et al.,
1998; Harrington et al., 1999; Chen et al., 1999). However, the changes
in rhythms observed in the PAC1 receptor-deficient mice were
not predictable from previous studies, all performed in "acute"
models. In vitro and in vivo application of
glutamate or NMDA induce phase delay of the endogenous rhythm at early
night similar to light (Ding et al., 1994; Mintz and Albers, 1997;
Mintz et al., 1999). In vitro application of PACAP in
micromolar concentration increases the glutamate-induced phase delay
(Chen et al., 1999), whereas nanomolar concentration of PACAP alone
induces a phase shift similar to that observed for glutamate
(Harrington et al., 1999). The present data indicate that the
PAC1 receptor in the SCN is involved in the light-induced phase shift at night. The larger phase delay after light stimulation in
the behavioral rhythm of
PAC1/
mice compared with wild-type animals suggests that the clock sensitivity to light is increased when the PAC1 receptor
system is missing. The changed light responsiveness at night is most likely caused by compensatory changes in PAC1
receptor-deficient mice. We have previously shown that the cAMP-PKA
pathway is responsible for the PACAP effects during subjective day
(Hannibal et al., 1997), and there is accumulating evidence that both
cAMP-PKA and calcium-dependent pathways via
PKC/IP3 are involved in the PACAP-mediated effects at night (von Gall et al., 1998; Kopp et al., 1999; Tischkau et
al., 2000). Interestingly, application of a specific PKA inhibitor, KT5720, blocks glutamate-induced Per1 mRNA expression in the
SCN at early, but not at late, night (Tischkau et al., 2000). This finding accords well with a glutamate-PACAP interaction via the PAC1 receptor and cAMP-PKA signaling (Tischkau et al.,
2000). PAC1 receptor activation was recently shown to
release calcium from ryanodine-caffeine stores independent of inositol
trisphosphates and cAMP pathways in bovine adrenal medullary cells
(Tanaka et al., 1998). Although it remains to be shown the existence of
such pathway within the SCN, it is not unlikely because light-induced phase shift at early night is dependent on the release of intracellular calcium via ryanodine receptor activation (Ding et al., 1998). The
phase response to light stimulation is clock-controlled and dependent
of the (Daan and Pittendrigh, 1976b; Ding et al., 1994). The
mechanism that determines the direction of the phase shift is not known
but it seems to be dependent on the properties of the molecular clock
(Dunlap, 1999; King and Takahashi, 2000). This mechanism seemed to be
changed in
PAC1/
mice resulting in a light-induced phase delay in contrast to the phase
advance in wild-type mice at late night. How PAC1 receptor signaling participates in this regulation is unknown. Interestingly, application of an adenosine A2A receptor agonist
directly into the SCN at CT20 followed by a 15 min light pulse causes a
phase delay similar to that observed in
PAC1/
mice (Mintz and Albers, 2000). A fine-tuning role of adenosine receptor
activation in synaptic transmission has been shown to involve
NMDA-receptor signaling and cAMP-PKA activation (Sebastiao and
Ribeiro, 2000). Whether these signaling pathways are involved in the
altered sensitivity to photic stimulation seen in
PAC1/
mice at late night remains to be examined.
In conclusion, our data indicate that PAC1 receptor
signaling participates in the gating control of photic sensitivity of the clock and shows that light-induced phase shift and induction of the clock genes Per1 and Per 2 are not always
interdependent phenomena, suggesting that other factors are of
importance for light-induced phase shifts at night.
| |
FOOTNOTES |
Received Dec. 27, 2000; revised March 26, 2001; accepted April 12, 2001.
This work was supported by The European Commission (BIO-98-0517), The
Danish Biotechnology Centre for Cellular Communication, The Danish
Neuroscience Program, and Danish Medical Research Council Grant
9702202. The skillful technical assistance of Lea Larsen and Anita
Hansen is gratefully acknowledged.
Correspondence should be addressed to Dr. Jens Hannibal, Department of
Clinical Biochemistry, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. E-mail: J.Hannibal{at}inet.uni2.dk.
| |
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