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The Journal of Neuroscience, February 1, 1999, 19(3):1115-1121
Inhibition of Light- or Glutamate-Induced mPer1
Expression Represses the Phase Shifts into the Mouse Circadian
Locomotor and Suprachiasmatic Firing Rhythms
Masashi
Akiyama1,
Yasuko
Kouzu1,
Satomi
Takahashi1,
Hisanori
Wakamatsu1,
Takahiro
Moriya2,
Miyuki
Maetani3,
Shigenori
Watanabe3,
Hajime
Tei4,
Yoshiyuki
Sakaki4, and
Shigenobu
Shibata1, 2
1 Department of Pharmacology and Brain Science and
2 ARCHS, School of Human Sciences, Waseda
University, Tokorozawa, Saitama 359-1192, Japan,
3 Department of Pharmacology, Faculty of Pharmaceutical
Sciences, Kyushu University, Fukuoka 812-82, Japan, and
4 Human Genome Center, Institute of Medical Sciences,
University of Tokyo, Tokyo, Japan
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ABSTRACT |
mPer1, a mouse gene, is a homolog of the
Drosophila clock gene period and has been
shown to be closely associated with the light-induced resetting of a
mammalian circadian clock. To investigate whether the rapid induction
of mPer1 after light exposure is necessary for
light-induced phase shifting, we injected an antisense phosphotioate oligonucleotide (ODN) to mPer1 mRNA into the cerebral
ventricle. Light-induced phase delay of locomotor activity at CT16 was
significantly inhibited when the mice were pretreated with
mPer1 antisense ODN 1 hr before light exposure.
mPer1 sense ODN or random ODN treatment had little
effect on phase delay induced by light pulses. In addition, glutamate-induced phase delay of suprachiasmatic nucleus (SCN) firing
rhythm was attenuated by pretreatment with mPer1
antisense ODN, but not by random ODN. The present results demonstrate
that induction of mPer1 mRNA is required for light- or
glutamate-induced phase shifting, suggesting that the acute induction
of mPer1 mRNA in the SCN after light exposure is
involved in light-induced phase shifting of the overt rhythm.
Key words:
antisense oligonucleotide; circadian rhythm; firing
rhythm; mPer1; phase shift; suprachiasmatic nucleus
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INTRODUCTION |
Circadian rhythms, which persist in
the absence of environmental cues, are observed in a wide variety of
organisms (Edmunds, 1988 ). To maintain synchrony with the daily
environmental cycle, organisms responded to environmental cues,
especially light, to reset or entrain their circadian rhythms. In
mammals, the suprachiasmatic nucleus (SCN) of hypothalamus has been
shown to be a primary circadian pacemaker of locomotor activity and
various physiological phenomena (Hastings, 1997). The genetic and
molecular mechanisms that control circadian rhythms were initiated by
studies of Drosophila rhythms (Konopka and Benzer, 1971).
The circadian rhythms evident in the locomotor activity of adult flies
and in gating of eclosion were altered by mutations in two
genes, period (dPer) and timeless (tim) (Hall, 1998; Young, 1998). Protein levels and mRNA
levels of these genes undergo robust circadian oscillation, and both proteins co-regulate their own regulation by negative feedback loops.
In mammals, previous studies have demonstrated that mRNAs of
immediately early genes (IEGs), including c-fos and junB,
are markedly induced by light in the SCN (Rusak et al., 1990;
Morris et al., 1998). However, the molecular component of the
circadian clock and the mechanism by which the light entrains the
circadian clock have only been recently elucidated. The recent
isolation of dPer homologous genes, Per1 (Sun
et al., 1997; Tei et al., 1997), Per2 (Albrecht et al.,
1997; Shearman et al., 1997; Takumi et al., 1998b), and Per3
(Zylka et al., 1998; Takumi et al., 1998a) from human and mouse have
significantly clarified the molecular mechanisms of the circadian clock
in mammals. These genes are rhythmically expressed in the SCN. We
showed that brief exposure to light during subjective night results in
a large and rapid induction of mPer1 expression (Shigeyoshi
et al., 1997). The induction of mPer1 (<20 min) by light is
more rapid than the accumulation of c-fos protein (Shigeyoshi et al.,
1997). This suggests that c-fos protein is not directly involved in the
rapid induction of the mPer1 gene.
To investigate whether induction of mPer1 transcripts by
light exposure is necessary for light-induced phase shifts, we injected an antisense phosphotioate oligonucleotide (ODN) to mPer1
mRNA intracerebroventricularly 1 hr before light exposure. We
found that inhibition of light-induced mPer1 expression by
antisense oligonucleotide in vivo significantly represses
light-induced phase shifts of the mouse circadian locomotor rhythm. We
have reported that treatments with glutamate, NMDA, or substance
P, or stimulation of the optic chiasm produce changes in the phase of
the firing rhythm of SCN neurons in vitro with a
phase-response curve similar to that induced by light exposure
in vivo (Shibata et al., 1992, 1994; Shibata and Moore,
1993). Direct application of mPer1 antisense ODN to
the SCN in hypothalamic slices in vitro produced an
attenuation of the glutamate-induced phase shift in a manner similar to
the reduction of the light-induced phase shifts observed in
vivo. These results suggest that acute induction of mPer1 mRNA in the SCN after light exposure is involved in
light-induced phase shifts of overt rhythms.
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MATERIALS AND METHODS |
Phosphotioate ODNs. The published sequence of
mPer1 was used to design an antisense ODN targeted to the
region of the mRNA containing the initiation ATG. The sequences of the
mPer1 antisense and sense ODNs (18-mer) were 5'-TAG GGG ACC
ACT CAT GTC T-3' and 5'-A GAC ATG AGT GGT CCC CTA-3', respectively. The
sequences of random ODN (18-mer) and vasopressin precursor gene (AVP)
antisense ODN (18-mer) were 5'-CCG TTA GTA CTG AGC TGA C-3' and 5'-CAT
CCT GGC GAG CAT AGG T-3', respectively. The random ODN contained an equivalent GC content as the antisense and sense ODNs of
mPer1. All ODNs were purified by HPLC to reduce the possible
toxicity of phosphotioate ODNs.
Animals and surgery. Male Balb/c mice (Takasugi
Saitama, Japan) purchased 6 weeks postpartum were maintained on a 12 hr
light/dark cycle with light on at 8:30 A.M. Animals were given food and
water ad libitum. Stainless steel guide cannulas (6.0 mm, 23 gauge) were implanted bilaterally intracerebroventricularly (4.5 mm
anterior and 1.1 mm lateral to lambda and depth of 2.1 mm below the
skull) using a stereotaxic frame (Narishige, Tokyo, Japan). After
2 d recovery, animals were moved to continuous darkness for at
least 2 weeks before ODN administration. For assessment of the
locomotor activity, mice were housed individually, and their locomotor
activity rhythm was measured by area sensors (model FA-05 F5B; Omron,
Tokyo, Japan) with a thermal radiation detector system, and data were stored on a personal computer.
After free-running for 14-20 d in constant darkness, mice were
randomly assigned to mPer1 antisense ODN, mPer1
sense ODN, mPer1 random ODN, AVP antisense ODN, or vehicle
(sterilized saline). A 5 µl aliquot of each ODN (2-6 nmol) was
unilaterally injected into the lateral ventricle via an injection
cannula (external diameter, 0.35 mm) extending 0.5 mm below the tip of
the guide cannula at a rate of 1 µl min 1 using a
10 µl Hamilton syringe. Injection was performed at circadian time 1 (CT1; CT12, onset time of locomotor activity), CT4, CT8, CT15, or CT21,
then animals were returned to their individual cages. For light
exposure experiments, implanted mice were again randomly assigned an
ODN, and 60 min after the injection, each animal was exposed to a light
pulse lasting 15 min at CT16. Light (20 lux) was administered while the
mice were in a Plexiglas cylinder. After treatment, animals were
returned to constant darkness. Some mice were first exposed to the
light, and then ODNs were administered 0 or 120 min after light
exposure. Each group received a repeated intracerebroventricular
injection (3 or 4 times for each animal) after at least 14 d.
Injections were randomly administered into the right or left ventricle.
To verify that ODNs were administered into the cerebral ventricle, mice
were injected with 5 µl of saturated fast green, and their brains
were examined macroscopically after sectioning. Anisomycin (50 µg in
5 µl of saline) and MK-801 (10 µg in 5 µl of saline) were also
intracerebroventricularly injected in the same manner. After treatment,
animals were returned to constant darkness. The phase of the rhythm was
assessed visually by applying a straight edge to the onset of activity
on successive days before the light pulse and again beginning ~3 d
after a light treatment and determining the difference in phases on the
day of the light exposure (Daan and Pittendrigh, 1976). At least four independent experiments using different mice were done at each group.
Slice culture and measurement of neural activity rhythm. On
the first day, coronal hypothalamic slices (400 µm thickness) including SCN were obtained between zeitgeber time 9-11
(ZT9-11) from male Balb/c mice (10-14 weeks). Then, slices
were preincubated and treated with vehicle or mPer1
antisense, sense, or random ODNs (each 20 µM) in Krebs'
Ringer's solution (in mM: NaCl 129, KCl 4.2, MgSO4 1.3, KH2PO4 1.2, CaCl2 1.5, glucose 25, NaHCO3 22.4, and HEPES
25, with gentamycin 0.5 mg/ml, pH 7.3-7.4) for 4 hr (ZT12-16). At
ZT16, the slices were removed into the buffer containing glutamate (10 µM) for 15 min. After drug treatment, perfusion with
normal medium was reinstated. The spontaneous action potentials of
single SCN cells were recorded extracellularly through glass electrodes
filled with 3 M NaCl during the second day in vitro. Stable single unit activity was recorded over 5 min
intervals. The activities of all cells recorded during a single
experiment were averaged into 2 hr intervals using 1 hr lags. Previous
studies have shown that this procedure yields a pattern of electrical activity for the population of SCN neurons that varies little between
animals, and that the time of peak electrical activity is a reliable
marker of the phase of the SCN pacemaker (Shibata and Moore, 1993;
Shibata et al., 1994).
Biochemical analysis. To detect injected biotinylated ODN,
anesthetized mice were perfused intracardially with ice-cold saline and
4% paraformamide in 0.1 M phosphate buffer (PB), pH 7.4, and then their brains were removed, post-fixed for 24 hr at 4°C and placed in 0.1 M PB with 20% sucrose for 24 hr. The
distributions of ODN in the serial cryostat sections (30 µm)
containing SCN were determined using a Vecstatain ABC Elite kit (Vector
Laboratories, Burlingame, CA).
RT-PCR analysis. The effect of mPer1 antisense
ODN on mPer1 expression in the SCN was examined by RT-PCR.
Mice were entrained to light/dark cycle for 2 weeks. Mice were
transferred to constant darkness for one extra daily cycle, and at
ZT15, mice were administered antisense ODN (2, 4, and 6 nmol) and
vehicle. Half of both groups received light treatment (20 lux, 15 min)
at ZT16. At ZT17.5, brains (n = 4 for each group) were
removed and placed in ice-cold saline. Slices (0.5-mm-thick) of mice
brain that contained SCN were frozen on dry ice, and the SCNs were
punched out with a 26 gauge needle. Total RNA from the SCN
(n = 4) was extracted in each group by Trizol solution
(BRL, Bethesda, MD). A one-step RT-PCR system (BRL) was used for
reverse transcription of ~100 ng of RNA, and mPer1,
mPer2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA were amplified by PCR. RT-PCR reactions were performed for 21 cycles with mPer1, mPer2, and GAPDH primers in a
single tube. The primer pairs used for the amplification of each
product are as follows: 5'-CCA GGC CCG GAG AAC CTT TTT-3' and 5'-CGA
AGT TTG AGC TCC CGA AGT G-3' (mPer1); 5'-ACA CCA CCC CTT ACA
AGC TTC-3' and CGC TGG ATG ATG TCT GGC TC-3' (mPer2); and
5'-GAC CTC AAC TAC ATG GTC TAC A-3' and TGG CCG TGA TGG CAT GGA CT-3'
(GAPDH). The sizes of the PCR products of mPer1,
mPer2, and GAPDH were 402, 779, and 436 bp, respectively.
PCR products were run on 3% agarose gels, and DNA in the appropriate
bands were detected with an EDAS-120 system (Eastman Kodak, Rochester, NY).
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RESULTS |
Phase shift effects of mPer1 antisense ODN on
various CTs
Administration of 6 nmol of mPer1 antisense ODN at
various CTs (CT1, 8, 15, 21) were compared with vehicle administration (Fig. 1B). Significant
phase delays were observed when mPer1 antisense ODN was
administrated at CT1. There were no significant differences between
mPer1 antisense ODN administrated at other CTs. To examine whether this ODN shifting effect is specific to mPer1
antisense ODN, we examined the effects of intracerebroventricular
administration of four different ODNs and anisomycin on behavioral
rhythms (Fig. 1A,C). Administration
of anisomycin, which inhibits protein synthesis, has been shown to
induce phase shifts when it was injected into the SCN region (Inouye et
al., 1988). Two ODNs, mPer1 antisense ODN and AVP antisense
ODN, had specific mRNA targets, whereas the other two ODNs, sense and
random ODNs, lacked specific mRNA targets. We found that phase delays
were observed when anisomycin (50 µg) or mPer1 antisense
(6 nmol) ODN was administered (p < 0.01;
Student's t test). No significant phase shifts were
observed after injection of the other ODNs or vehicle (Fig.
1C). The magnitude of the phase shifts by mPer1
antisense ODN were dose-related, with injection of 4 nmol of the
mPer1 antisense ODN producing a phase shift approximately
half the size of the one at a 6 nmol dose. No phase delays were
observed at 2 nmol doses.
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Figure 1.
Effect of ODN administration at CT1 on the mouse
circadian locomotor rhythm. A, Locomotor activity
records of vehicle (a), mPer1
antisense ODN (b), random ODN
(c), and anisomycin
(d)-injected mice. Each animal was injected at
CT1 ( in the figure) intracerebroventricularly (5 µl; 1 µl
min1) and returned to constant darkness.
B, Phase-response curve for mPer1
antisense ODN administration at CT1, CT8, CT15, and CT21.
Numbers in parentheses indicate the number of
experiments. Injection of mPer1 antisense ODN at CT1
induced a significant phase delay (**p < 0.01;
Student's t test). C, Phase shifts of
mouse locomotor rhythm by various ODNs or anisomycin injection at CT1.
0, Vehicle; A, mPer1
antisense ODN; S, sense ODN; R, random
ODN; C, AVP antisense ODN. The number in
the figure indicates the amount (in nanomoles) of injected ODN.
Numbers in parentheses indicate the number of
experiments. Injection of mPer1 antisense ODN and
anisomycin significantly phase delayed locomotor rhythm
(**p < 0.01; Student's t
test).
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Effect of ODN on light-induced phase shifts
We previously demonstrated that mPer1 induction by
light is strongly correlated with phase shifts in behavioral rhythms
(Shigeyoshi et al., 1997). Thus, we examined the effect of
mPer1 antisense ODN on light-induced phase shifts (Fig.
2A,B).
Mice injected with vehicle at CT15 followed by exposure to a light
pulse for 15 min at CT16 had a marked phase delay in the circadian
rhythm of locomotor activity of ~2 hr. MK-801, an NMDA receptor
antagonist, injected intracerebroventricularly at CT15 markedly
depressed the light-induced phase delay at CT16, as previously reported
(p < 0.01; Student's t test)
(Colwell et al., 1990; Shibata et al., 1994). Injection of
mPer1 antisense ODN at CT15 attenuated the light-induced
phase delay at CT16 in a dose-dependent manner [phase shift,
 0.480 ± 0.194 hr (6 nmol injection of antisense ODN) vs
2.204 ± 0.141 hr (vehicle injection); p = 0.0001]. However, injection of mPer1 antisense alone at
CT15 did not alter locomotor activity (Fig. 2B).
mPer1 antisense administration immediately after light
exposure (CT16.3) also inhibited the light-induced phase shift, but
less efficiently, and administration of it 2 hr after light exposure (CT18) did not inhibit the phase delay (Fig. 2B). The
other ODNs injected at CT15 did not affect the light-induced phase
delay at CT16.
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Figure 2.
Effect of ODN injection on light-induced phase
delay of locomotor activity rhythm. Mice were injected with ODNs at
CT15 under the safety light, 1 hr after injection, mice were exposed to
light (20 lux) for 15 min and returned to constant darkness.
A, Locomotor activity records of vehicle
(a), mPer1 antisense
(b), random ODN (c), and
AVP antisense ODN (d)-injected mouse.
B, Light-induced phase shifts in mPer1
antisense ODN (A), sense ODN
(S), random ODN (R), AVP
antisense ODN (C), and MKC-801 (MK)-injected
mouse. * indicates that antisense ODN was administered 2 hr after the
light pulse. The number in the figure indicates the
amount (nanomoles) of injected ODN. 0 indicates the
vehicle administration. Numbers in parentheses indicate
the number of experiments. Preinjection of mPer1
antisense ODN (4 and 6 nmol) and MK-801 significantly reduced
light-induced phase shift (**p < 0.01; Student's
t test). Injection of mPer1 antisense ODN
2 hr after light exposure did not have any effects.
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Effect of antisense ODN on in vitro SCN neural
activity rhythm
We and other researchers have reported that treatments with
glutamate produce changes in the phase of the firing rhythm of SCN
neurons in vitro with a phase-response curve similar to
that induced by light exposure in vivo (Shibata et al.,
1994; Shirakawa and Moore, 1994). Thus, we examined the effects of
mPer1 antisense ODN in vitro. In control
experiments, coronal hypothalamic slices containing whole SCN were
treated in vitro for 4 hr on day 1 between ZT12 and ZT16
with drug-free perfusion medium. In these experiments, the mean peak of
electrical activity on the subsequent day occurred at ZT6-7
(ZT6.0 ± 0.5; n = 4) (Fig.
3A,B).
For slices treated with glutamate in vitro at ZT16 on day 1, the peak was around ZT9 on day 2 (Fig. 3). Glutamate-induced phase
delay at ZT16 was significantly blocked by 4 hr pretreatment with
mPer1 antisense ODN (ZT12-16) but not by pretreatment with
random ODN. However, mPer1 antisense ODN did not produce
phase changes when applied alone for 4 hr (ZT12-16).
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Figure 3.
Effect of mPer1 antisense ODN on
glutamate-induced phase delay of SCN firing rhythm in
vitro. A, The average neuronal activity rhythms
in the SCN recorded from mice slice on day 2. Each point
indicates the 2 hr means ± SEM of firing rate of single SCN cells
from ZT2-14. B, Average phase shifts induced by
glutamate and glutamate plus mPer1 antisense ODN. Each
bar indicates the peak of firing rate (mean ± SEM). Numbers in parentheses indicate the number of
slices. Preincubation of mPer1 antisense ODN
significantly reduced glutamate-induced phase shift
(*p < 0.05 vs glutamate alone; Student's
t test). Glu, Glutamate;
A, mPer1 antisense ODN; R,
mPer1 random ODN.
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Distribution of ODN in the brain and effect of antisense ODN
on mRNA
Distribution of antisense ODN was examined by injection and
staining of biotinylated ODN in the fixed slice section of the brain.
The ODN were most extensively distributed around the third ventricle,
including the SCN (Fig.
4A). Inhibition of
mPer1 induction by mPer1 antisense ODN in the SCN
1.5 hr after light exposure at CT16 was examined by RT-PCR. Before
amplification, RNA were preliminarily tested for possible genomic DNA
contamination. Gel analysis showed bands of expected lengths. Light
exposure at CT16 induced expression of mPer1 mRNA (180 ± 24% of nonlight group; n = 4) and mPer2
mRNA (160 ± 25%; n = 4) 90 min after light
pulse. Light induction of mPer1 mRNA was considerably
inhibited when 4 or 6 nmol of mPer1 antisense ODN was
administrated (Fig. 4B,C). Administration of 6 nmol of mPer1 antisense significantly
reduced the expression of mPer1 mRNA (68 ± 8.7% of
random ODN treatment; n = 4; p < 0.05;
Student's t test) but not that of mPer2 mRNA (99 ± 11% of random ODN treatment; n = 4;
p > 0.05; Student's t test). This result
suggests that phenotypic effects of mPer1 antisense ODN
treatment on light-induced phase delay are mediated by the specific
inhibition of mPer1 expression in the SCN.
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Figure 4.
Effects of mPer1
antisense ODN on the mPer1 expression in the SCN.
A, Distribution of biotinylated ODN 2 hr after injection
into the brain. mPer1 antisense ODN (5'-biotnylated; 6 nmol in 5 ul) was microinjected intracerebroventricularly. Mice were
killed 2 hr later, followed by detection of biotinylated ODN. An
arrow on the top slice shows antisense
ODN injection site. An arrow on the
bottom slice shows the position of SCN. The ODNs were
most extensively distributed around the third ventricle including the
SCN. B, Inhibition of light induction of
mPer1 transcript in the SCN by in vivo
mPer1 antisense ODN treatment. Total RNA was isolated 1.5 hr
after light exposure from mPer1 antisense ODN-pretreated
mice, and mPer1, mPer2, and GAPDH RNA
were amplified by an RT-PCR method. Lane 1, Treated with
vehicle; lane 2, treated with 2 nmol of antisense ODN;
lane 3, treated with 4 nmol of ODN; lane
4, treated with 6 nmol of antisense ODN. The PCR products of
mPer1, mPer2, and GAPDH gene are
indicated by arrows. C, Semiquantitative
analysis of RT-PCR products shown in B. The band
intensity of RT-PCR products of mPer1 and
mPer2 mRNA was measured by one-dimensional
analysis software (Eastman Kodak), and their amounts were normalized
against GAPDH.
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DISCUSSION |
Administration of mPer1 antisense ODN at CT1, but not
at other CTs, significantly delayed the locomotor rhythm of mice.
Injection of vehicle, mPer1 sense ODN, or mPer1
random ODN at CT1 had little effect. It is currently believed that
circadian oscillators, including those in mammals, are comprised of
transcription/translation-based negative feedback loops controlled by
clock genes (Hall, 1998; Reppert, 1998; Young, 1998). Peripheral or
intra-SCN injections of translation inhibitors such as anisomycin or
cycloheximide at early subjective days (CT1-4) have been reported to
produce a phase delay in wheel-running rhythms (Takahashi and Turek,
1987; Inouye et al., 1988). Transcript of mPer1 is
endogenously rhythmic with a consistent peak of expression in the
subjective day at CT4 (Tei et al., 1997). We observed that the
injection of mPer1 antisense ODN 4 hr before the light pulse
did not block the light-induced phase delay of locomotor rhythm (data
not shown). Therefore, the largest reduction of mPer1
expression by antisense ODN might occur when antisense ODN is injected
2-3 hr before the mPer1 peak and may be the reason why
antisense ODN delayed the circadian rhythm only at CT1.
In this study, we demonstrated that blockade of acute mPer1
induction after light exposure by antisense ODN prevents the
light-induced phase shifts of the circadian activity rhythm. This block
in light-induced phase shift was caused by selective inhibition of
mPer1 induction, because mPer1 antisense ODN
alone did not interfere with the free-running rhythm at CT16. Moreover,
it is interesting that both mPer1 mRNA expression and phase
delay of locomotor activity induced by light at CT16 were reduced by 4 nmol of mPer1 antisense ODN but not by 2 nmol. Thus, we
observed the parallel reduction of mPer1 expression and
phase delay. In the present experiment, 4 and 6 nmol of
mPer1 antisense ODN reduced to 60-70% of mPer1
RNA expression induced by light exposure. Although we do not detect the
protein production of mPer1 after light exposure, we can estimate
30-40% reduction of mPer1 mRNA may affect the
light-induced phase changes in mouse behavior. Present results suggest
that the reduction of light-induced phase delay by antisense ODN
in vivo is a result of the inhibition of light-induced acute
induction of mPer1 gene in the SCN. Further experiments are
needed to locate the specific region of antisense ODN action (for
example, direct antisense ODN injection into the SCN or immunostaining
of mPer1 antibody there).
Transcript of mPer1 is rapidly induced by light in a
time-of-day-dependent manner (Shigeyoshi et al., 1997). The
responsiveness of mPer1 mRNA to light is gated so that
little or no increase was seen during the subjective day, whereas
robust induction was seen during the subjective night. Gating is also
present in light-induced phase shifts of behavioral rhythm. Their dose
and threshold is closely correlated with mPer1 inducibility
in the SCN. These results with our present results suggest that
mPer1 plays a central role in the circadian clock.
mPer2 gene was also shown to be induced by light but in a
delayed manner compared with mPer1 (Takumi et al., 1998b),
possibly reflecting a different regulatory mechanism. Recently,
mPer3 has been isolated and shown not to be light inducible (Takumi et al., 1998a; Zylka et al., 1998), suggesting that
mPer genes have different roles in the light-induced phase
shift. Therefore, injection of mPer2 or mPer3
antisense ODN or cocktails containing antisense ODNs of mPer
genes may be a good strategy for determining the roles of these genes.
To exclude the possibility that other regions of the brain might have
added to the effects of mPer1 antisense ODN treatment, we
examined the neural rhythm of SCN using slice culture. Administration of mPer1 antisense ODN blocked the glutamate-induced phase
delay of the SCN circadian firing rhythm. Thus, glutamate-induced phase shifts may be involved in the expression of mPer1 mRNA in
the SCN. SCN is entrained to the environmental light/dark cycle via a
retinal projection, the retinohypothalamic tract (RHT). Glutamate is a
transmitter of the RHT (de Vries et al., 1993). Glutamate and NMDA
application to rat SCN in vitro have been reported to cause
phase delays in SCN firing rhythms when applied at early subjective
night (Shibata et al., 1994; Shirakawa and Moore, 1994). Furthermore,
glutamate receptor antagonists and inhibitors of nitric oxide synthase,
calmodulin, or calcium calmodulin kinase II antagonize phase shifts in
the SCN firing rhythm induced by glutamate or NMDA in vitro
(Shibata et al., 1994; Watanabe et al., 1994; Fukushima et al., 1997).
Therefore, we cannot rule out the possibility that mPer1
antisense ODN interferes with these biochemical steps. However, the
sequence specificity of the ODNs on light- or glutamate-induced phase
delay strongly suggest this is not the case.
Light-induced phase shifts of circadian rhythms induce immediately
early genes (IEGs) such as c-fos, junB, and
NGFI-A mRNAs specifically in the SCN (Rusak et al., 1990; Morris
et al., 1998). Blockade of expression of c-fos or Jun B expression in
the SCN has been shown to inhibit light-induced phase shifts in
mammalian circadian clocks (Wollnik et al., 1995). These proteins are
believed to dimerize and bind to AP-1, which are CRE/CaRE consensus
sequences that are present in the promoters of many genes (Takeuchi et
al., 1993). The light-induced induction of IEGs is also gated as
mPer1 and mPer2. The time courses of c-fos and
mPer1 mRNA induction are similar, but it is unknown whether
c-fos protein is involved in transcription of mPer1 (or
mPer2) or the induction of the c-fos and mPer are simultaneous.
In this study, we used antisense ODN as pharmacological tools to
inhibit mPer1 expression in vivo and in
vitro. The mechanism of inhibition of physiological systems by
antisense ODNs is believed to be the result of specific hybridization
of the antisense ODN to its complementary mRNA, causing disruption of
the translation of the mRNA into protein (Talamo, 1998). We have not
determined the amounts of mPer1 protein expression, because we have not
obtained anti-mPer1 antibody. Antisense ODN is also believed to bind to the genomic DNA region of the corresponding gene and inhibit binding of
transcription factors and to bind to mRNA and accelerate degradation of
targeted mRNA by RNaseH (Kashihara et al., 1998). Both of these mechanisms should lower the level of mRNA. These effects may be sequence-specific; arising from inhibition of imperfectly matched target genes, or sequence-independent effects on gene expression. Antisense ODNs may also effect nontargeted genes or even be toxic to
physiological systems (Talamo, 1998). In the present study, we showed
that mPer1 mRNA in the SCN was reduced by treatment with
mPer1 antisense ODN, but treatment did not affect
mPer2 and GAPDH mRNA levels, demonstrating that the
antisense ODN used in this study specifically effects only
mPer1 gene expression. We used phosphotioate-substituted
ODNs, which have longer biological half-lives than unsubstituted ODNs
but may be toxic (Agrawal et al., 1991). In this study, some animals
exhibited altered locomotor activity for the first several hours after
injection. However, this effect was observed in both mPer1
antisense ODN-injected animals and control ODNs-injected animals,
suggesting this change is caused by a toxic effect of the
administration of ODNs. In all cases, locomotor activity was
restored to normal under constant darkness. In our previous experiments
(Ono et al., 1996, Watanabe et al., 1996), methamphetamine and
adenosine antagonists inhibited the light-induced phase shift, although
these chemicals increase or decrease motor activity, respectively.
Thus, the circadian oscillator may be unaffected by ODN injection. We
also demonstrated that ODNs injected intracerebroventricularly were
distributed in specific regions of the brain after 2 hr, especially
around the third ventricle including the SCN. However, other regions of
the brain might have added to the effects of mPer1 antisense ODN treatment.
In summary, the present results indicate that acute induction of
mPer1 mRNA after light exposure is necessary for
light-induced phase shifting of the mouse locomotor rhythm. Further
genetic dissection of mPer genes, possibly with knock-out
mice is useful to identify the role of these genes in detail.
| |
FOOTNOTES |
Received Aug. 25, 1998; revised Nov. 9, 1998; accepted Nov. 17, 1998.
This study was partially supported by grants to S.S. from Research
Project for Future Program (RFTF96L00310), from the Japan Society for
the Promotion of Science, and from the Japanese Ministry of Education,
Science, Sports and Culture (09470018).
Correspondence should be addressed to Shigenobu Shibata, Department of
Pharmacology and Brain Science, School of Human Sciences, Waseda
University, Tokorozawa, Saitama 359-1192, Japan. E-mail: shibata{at}human.waseda.ac.jp
| |
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Top
Abstract
Introduction
