Peripheral clock gene expression in CS mice with

bimodal locomotor rhythms

Tsuyoshi Watanabe, Mayumi Kojima, Shigeru Tomida, Takahiro J. Nakamura,

Takashi Yamamura, Nobuhiro Nakao, Shinobu Yasuo,

Takashi Yoshimura, Shizufumi Ebihara

*

Division of Biomodeling, Graduate school of Bioagricultural Sciences, Nagoya University, Furo-cho,

Chikusa-ku, Nagoya-shi 464-8601, Japan

Received 8 September 2005; accepted 16 December 2005

Available online 25 January 2006

Abstract

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting,

but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1,
mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust
oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these
mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate
that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.
# 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Keywords: CS mice; Circadian rhythm; Suprachiasmatic nucleus; Peripheral clocks; Clock genes; Restricted feeding

1. Introduction

In mammals, behavioral and physiological circadian

rhythms are driven by the suprachiasmatic nucleus (SCN) in
the hypothalamus, which receives photic input from the retina
and conveys the timing information to the peripheral organs by
way of a variety of signaling pathways (

Panda and Hogenesch,

2004; Reppert and Weaver, 2001

). Although it is relatively well

understood how the SCN receives the environmental light–dark
(LD) cycle, the output pathways from the SCN to coordinate
temporal physiology and behavior are not well understood
because multiple and complex pathways are involved in this
process.

Recent progress of molecular dissection of the circadian

clock has revealed that the self-sustained oscillation is
generated by feedback loops based on transcription and
translation of multiple clock genes. The discovery of these
clock genes led to a new finding that peripheral tissues contain

many of the same clock molecules as the SCN and widespread
peripheral tissues sustain the oscillation in vitro even after
lesions of the SCN (

Yoo et al., 2004

). Thus the current notion of

the mammalian circadian hierarchical structure is that the SCN
works to coordinate peripheral circadian rhythms of self-
sustained rather than damped oscillators (

Yamazaki et al., 2000;

Yoo et al., 2004

). Among several time cues to entrain peripheral

rhythms, feeding has been known as a dominant synchronizer,
but this does not affect the phase of the SCN (

Damiola et al.,

2000; Hara et al., 2001; Stokkan et al., 2001

). Because the SCN

controls feeding time, it is likely that the SCN synchronizes
peripheral oscillators via feeding which directly affects the
oscillators, although other time cues also seem to be involved in
peripheral entrainment (

Brown et al., 2002

).

CS mice used in the present study are reported to show

unique properties of circadian rhythms such as unstable free-
running periods (usually longer than 24 h) and distinct bimodal
rhythms under constant darkness (DD) (

Abe et al., 1999

). This

behavioral pattern is similar to rhythm splitting observed in
hamsters, but we will use the term ‘‘bimodal rhythms’’ in CS
mice. In contrast to other reports (

de la Iglesia et al., 2000;

Edelstein et al., 2003; Ohta et al., 2005

), circadian rhythms of

www.elsevier.com/locate/neures

Neuroscience Research 54 (2006) 295–301

* Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (S. Ebihara).

0168-0102/$ – see front matter # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
doi:10.1016/j.neures.2005.12.009

clock gene expression of the SCN under distinct bimodal
rhythmicity are essentially the same as normal rhythmic mice
(

Abe et al., 2001; Watanabe et al., 2003

), indicating that the

coupling between the SCN and behavioral rhythms is not robust
in CS mice. These data lead to the proposal that in CS mice, the
SCN cannot properly coordinate peripheral oscillators. To
consider this hypothesis, we examined several behavioral
rhythms and peripheral rhythms, comparing CS and normal
C57BL/6J mice.

2. Materials and methods

2.1. Animal and housing

Male CS mice (10–15 weeks old) bred in our colony and male C57BL/6J mice

(10–15 weeks old, Clea, Japan) purchased from a breeder were used. CS strain was
established from hybrids between NBC and SII strains in 1956 at the Department
of Animal Genetics, Nagoya University (

Festing, 1996

). Before the experiment,

mice were maintained under 12 h-light 12 h-dark (LD12:12) cycles at 23

1 8C.

Food and water were available ad libitum. In all of the experiments, animals were
treated in accordance with the guideline of Nagoya University.

2.2. Measurement of behavioral rhythms

Mice were housed individually in cages equipped with running wheels.

Activity rhythms were measured by a computer system (The Chronobiology
Kit, Standord Software System, CA). The activity time (a) was measured by an
eye-fitted method connecting the activity onsets and offsets. The light intensity
was 300–500 and 0 lx in L and D phase of LD cycles, respectively. Water and
food intake rhythms were also measured with the Chronobiology Kit.

2.3. Genes expression in LD and DD condition

After entrainment to LD condition or keeping under constant darkness (DD)

condition for 2 weeks, mice were humanely decapitated after cervical transloca-
tion according to the law (no. 105) and notification (no. 6) of the Japanese
Government and the tissues (SCN, liver, adrenal gland and heart) were collected at
either zeitgeber time (ZT) or circadian time (CT) 0, 3, 6, 9, 12, 15, 18, 21. We
selected these tissues, because (1) the tissues show robust oscillation of clock gene
expression that is necessary to compare the pattern between CS mice and C57BL/
6J mice and (2) clock gene expression in these tissues is affected by a change of
environmental cues including feeding and photoperiod (

Carr et al., 2003; Stokkan

et al., 2001

). In addition, we selected the adrenal gland, a pituitary-dependent

endocrine organ, which secretes glucocorticoids affecting the mouse peripheral
clock (

Balsalobre et al., 2000

). In the experiment under DD condition, because not

all CS mice showed clear bimodality, we selected only distinctive ones for the
experiment. Activity onsets of evening components were defined as CT12 in the
bimodal mice (

Abe et al., 2001; Watanabe et al., 2003

).

2.4. Restricted feeding schedule

To examine the effects of restricted feeding (RF) on peripheral clock (liver),

mice were given a RF schedule for 6 days. During the RF schedule, mice were
allowed to access food for 4 h starting at ZT5. On seventh day, mice for RF
group were given no food. Free feeding (FF) group were allowed to feed
throughout the experiment. This RF schedule has been proved to be effective for
phase-shifting circadian rhythms of mPer1, mPer2 and Dbp expression in the
liver (

Hara et al., 2001

). Liver tissues were collected at ZT0, ZT3, ZT6 and ZT9

in both RF and FF group on seventh day.

2.5. Real-time quantitative PCR

Total RNA in peripheral tissues (liver, adrenal gland and heart) of the mice

was isolated using Trizol Regent. Genomic DNA was degraded using DNaseI.

First strand cDNA was synthesized using ReverTra Ace (TOYOBO, Japan). To
analyze the expression level of each mRNAs in peripheral tissues, real-time PCR
was performed using ABI PRISM 7000 detection system (Applied Biosystems,
USA). Gene specific primers were designed using Primer Express software
(Applied Biosystems, USA). Primer sequence were 5

0

-CGACTCCGGCAA-

GATCGAA-3

0

, 5

0

-GGTCCCCCAGGCTCTCTACT-3

0

for mouse cyclophilin

(GenBank accession number,

X58990

), 5

0

-AGAAGAAAACAGCACCAGCT-

3

0

, 5

0

-TCTTGAGTTATAAGAACCCCAACATG-3

0

for mouse Per1 (

AF022992

),

5

0

-TGGCCGCTGTAGACACTACATT-3

0

,

5

0

-CTCTATCCAGTAAGCTTCA-

CAGACTGTAA-3

0

for mouse Bmal1 (

AB014494

) and 5

0

-CGCGCAGGCTT-

GACATCTA-3

0

, 5

0

-GGATCAGGTTCAAAGGTCATTAGC-3

0

for mouse Dbp

(

AK140243

). Real-time PCR amplification was performed after 30 s of denature

at 95 8C, and then run for 45 cycles at 95 8C for 5 s, 60 8C for 31 s. We confirmed
that single amplicon was detected in each real-time PCR reaction using the
dissociation curve of SDS 1.0 software (Applied Biosystems, USA). Quantifica-
tion of cDNAs was determined by comparing the threshold cycles for amplifica-
tion of the unknowns with those of six concentrations of each standards using SDS
1.0 software (Applied Biosystems, USA). After the calculation of concentration of
cDNAs, mPer1, mBmal1 and Dbp mRNA levels were normalized using cyclo-
philin mRNA levels. Each relative value was obtained by normalizing using the
maximum value that was set to 100.

2.6. In situ hybridization

To analyze mPer1 and mBmal1 mRNA levels in the SCN of CS and C57BL/

6J mice, in situ hybridization was carried according to a previous report
(

Yoshimura et al., 2000

). Antisense 45 mer oligonucleotide probes (5

0

-

GTCCCTGGTGCTTTACCAGATGCACATCCTTACAGATCTGCTGGA-3

0

for mPer1, GenBank accession number,

AF022992

; 5

0

-GCCATTGCTGCCT-

CATCGTTACTGGGACTACTTGATCCTTGGTCG-3

0

for Bmal1,

AB014494

)

were labeled with [

33

P]dATP (NEM Life Science Products, Boston, MA).

Coronal 20 mm sections of the SCN were prepared using a cryostat. Hybridiza-
tion was carried out overnight at 42 8C. After glass slides were washed, they
were air-dried and appose to Biomax-MR film (Kodak, Rochester, NY) for 2
weeks with

14

C-labeled standards (America Radiolabeled Chemicals). The

relative optical density was measured by using a computed image-analyzing
system (MCID Imaging Research) and converted into the radioactive value
(nCi) using the

14

C-labeled standard measurement. Subtracting background

values obtained from the corpus callosum normalized the data.

2.7. Statistics

Results were expressed as mean

S.E.M. Two-way ANOVA and un-paired

Student’s t-test were used for comparison among multiple groups and between
two groups, respectively.

3. Results

3.1. Behavioral rhythms

Activity profile analysis revealed that a is longer in CS than

C57BL/6J mice under LD condition (

Fig. 1

A). Mean value of a

was 16.08

0.17 h for CS (n = 28) and 12.47
0.11 h for

C57BL/6J (n = 25) mice (Student’s t-test, P < 0.0001). The
level of an evening peak was clearly lower in CS mice than that
C57BL/6J. In addition, total counts of wheel running per day
were significantly lower in CS than C57BL/6J (P < 0.001). In
CS mice, a morning component remarkably increased and its
duration extended to the middle of daytime. Typical bimodal
rhythms in CS mice, thus, are in the state that two components
with the same amplitude diverge with a long interval. This
typical rhythm was more frequently observed under DD
condition than LD condition, although some mice showed the
typical bimodal pattern in LD condition as DD condition

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

296

(

Fig. 5

). To see other behavioral rhythms in CS mice, water and

food intake rhythms were measured in LD condition. As shown
in

Fig. 1

B and C, both water and food intake exhibited evening

and morning peaks, which corresponded to those observed in
wheel running activity. The ratio of night-time access to water
and food was 53.8

2.2% (Student’s t-test, P < 0.01, n = 5)

and 67.3

2.5% (P < 0.01, n = 7), respectively in CS mice,

and 74.2

2.5% (P < 0.01, n = 6) and 78.9
1.8% (P < 0.01,

n = 7) in C57BL/6J mice.

3.2. Gene expression rhythms in the SCN

mPer1 and mBmal1 expression rhythms in the SCN showed

the peak at ZT6 and ZT15, respectively in both CS and C57BL/
6J mice and the patterns were essentially similar with no
significant differences between the two strains (

Fig. 2

).

3.3. Gene expression rhythms in peripheral tissues

In the liver, there were significant differences in the pattern

of mPer1 and mBmal1 expression between CS and C57BL/6J
mice (two-way ANOVA, F(1, 36) = 21.30, P < 0.0001 for
mPer1 and F(1, 36) = 16.18, P < 0.001 for mBmal1) (

Fig. 3

).

In CS mice, mBmal1 expression exhibited two peaks and the
amplitude of mPer1 rhythms was significantly reduced. In the
adrenal gland, mPer1 and Dbp expression patterns differed
significantly between the two strains (F(1, 36) = 7.71, P < 0.01
for mPer1, F(1, 36) = 18.89, P < 0.0001 for Dbp) and the
amplitude of these genes decreased in CS mice. In the heart,
expression patterns of all genes examined were different
between the two strains (F(1, 36) = 23.20, P < 0.0001 for
mPer1, F(1, 36) = 5.09, P < 0.05 for mBmal1 and F(1,
36) = 18.13, P = 0.0001 for Dbp). Under DD condition, the

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

297

Fig. 1. Behavioral rhythms of CS mice and C57BL/6J mice under LD condition. (A) Locomotor activity rhythms of CS mice and C57BL/6J mice. Representative
locomotor activity rhythms are shown in an upper column. Wheel running counts per 20 min were represented as mean

S.E.M. for 10 days (n = 28 for CS mice,

n = 25 for C57BL/6J mice) in a lower column. Water intake rhythms (B) and food intake rhythms (C) are shown. Representative water and food intake rhythms of CS
(left) and C57BL/6J (center) mice are shown. Number of access to water or food was represented every 20 min as mean counts for 6 days. The ratio of access during
the dark (ZT12-0) to a whole day is compared between CS and C57BL/6J mice (right).

*

P

< 0.01, un-paired Student’s t-test. In all experiments, the animals were kept

in LD 12:12 shown as the bar above each column.

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

298

Fig. 2. Expression profiles of clock genes in the SCN of CS and C57BL/6J mice under LD condition. (A) Representative autoradiographs of mPer1 and mBmal1 are
shown for CS and C57BL/6J mice. (B) Temporal changes of mPer1 and mBmal1 values are indicated in CS (open circle) and C57BL/6J mice (closed circle). The bar
above each graph means LD condition. Each value is the mean

S.E.M. (n = 3–4).

Fig. 3. Expression profiles of clock genes in peripheral tissues of CS and C57BL/6J mice under LD condition. Bars above each graph are LD condition. Large
asterisks show significant differences between CS (open circle) and C57BL/6J (closed circle) mice analyzed by two-way ANOVA (P < 0.05). Small asterisks
represent significant differences at each time point between two strains by un-paired Student’s t-test (P < 0.05). Each value is indicated as mean

S.E.M. (n = 3–4).

amplitude of mPer1 rhythms in the liver was significantly
reduced in CS mice (F(1, 50) = 20.81, P < 0.0001) and this
tendency was also observed in Dbp (

Fig. 4

). A bimodal pattern

of mBmal1 expression in CS mice was similar to that observed
in LD condition.

3.4. Effects of RF on gene expression rhythms in the liver

In C57BL/6J mice, RF schedule shifted the peak time of

mPer1 and Dbp from ZT9 to ZT3 in the liver (

Figs. 3 and 5

).

This schedule also shifted the peak from ZT15 to ZT6 for
mPer1 and from ZT9 to ZT3 for Dbp in CS mice. In addition,
RF significantly increased expression levels of both genes
during the period of observation (ZT0-9) in CS mice and as a
result, no essential differences in RF-induced gene expression
between two strains could be observed.

4. Discussion

In the present study, we demonstrated that CS mice exhibit

unusual circadian rhythms of behaviors and gene expression in
the peripheral tissues in spite of normal robust oscillation in the
SCN.

It has been reported that the anti-phase oscillation of clock

genes in the left and right SCN is reflected in behavioral rhythm
splitting in hamster and mice exposed to LL condition (

de la

Iglesia et al., 2000; Ohta et al., 2005

). In addition, hamsters

showing rhythm splitting induced by introducing daily novel
running wheel exhibited the bimodal expression of mPer1 and
mPer2 in the SCN (

Gorman and Lee, 2001; Edelstein et al.,

2003

). In these cases, behavioral circadian rhythms clearly

correlated with oscillatory pattern of clock genes in the SCN.
However, this is not the case of CS mice, in which even in the

situation of typical bimodal rhythms, circadian profiles of clock
genes in the SCN were essentially similar to those in C57BL/6J
mice and no asymmetric clock gene expression between the left
and right SCN occurred (

Abe et al., 2001; Watanabe et al.,

2003

). In addition, we preliminarily observed that mPer1

promotor activity showed unimodal oscillation in the coronal
and horizontal SCN slices in mPer1<luc transgenic mice
exhibiting bimodal rhythms which were produced by mating
with CS mice (unpublished observation). Although more
precise cellular and regional examination within the SCN might
be required, the results so far suggest that circadian oscillation
in the SCN is not properly reflected in behavior rhythms in CS
mice. Thus CS mice seem to be a unique model to study the
mechanism of the output pathway from the SCN controlling
behavioral rhythms.

Although the amplitude of mPer1 and Dbp rhythm was

significantly decreased in the tissues of CS mice, mBmal1 rhythm
was not clearly attenuated. Instead, mBmal1 particularly in the
liver showed bimodal pattern without attenuation. It is
noteworthy that CS mice exhibited different expression pattern
of clock genes in peripheral tissues when compared with other
mouse strains (

Damiola et al., 2000; Hara et al., 2001

). Because

behavioral changes are reported to affect peripheral oscillation
(

Damiola et al., 2000; Stokkan et al., 2001

), it is possible that

unusual peripheral oscillation of CS mice is due to altered
behavioral rhythms. To test this possibility, we examine the effect
of RF on peripheral gene expression in CS mice. As a result,
mPer1 and Dbp expression in the liver of CS mice normally
responded to RF and showed the similar expression pattern to that
of C57BL/6J mice. These results indicate that the peripheral
clock of CS mice has indistinguishable properties from that of
C57BL/6J, which suggests that unusual behavioral rhythms lead
to unusual peripheral gene expression in CS mice, although other

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

299

Fig. 4. Locomotor activity rhythms and expression profiles of clock genes in peripheral tissues of CS and C57BL/6 mice under DD condition. (A) Representative
actograms of CS (left) and C57BL/6J (center) mice in DD condition. E and M means evening component and morning component, respectively. Right column
indicates a of CS and C57BL/6J mice with shaded and white bars representing mean activity time and rest time

S.E.M. (n = 35 for CS mice; n = 25 for C57BL/6J

mice), respectively. The bar at the above of each graph means light–dark condition. (B) Expression profiles of mPer1 (left), mBmal1 (center) and Dbp (right) in the
liver of CS (open circle) and C57BL/6 (closed circle) mice under DD condition. The black bar at the above of each graph means constant dark condition. A large
asterisk represents significant differences between two strains by two-way ANOVA (P < 0.05). Small asterisks represent differences at each time point between two
strains by un-paired Student’s t-test (P < 0.05). Each value is indicated as mean

S.E.M. (n = 3–5).

factors such as body temperature cannot be ruled out (

Brown

et al., 2002

).

If the above-mentioned hypothesis is correct, fundamental

cause of unusual circadian rhythms in CS mice seems to be
abnormal downstream pathways from the SCN. Although the
mechanisms by which the SCN controls behavioral rhythms are
still poorly understood, both neural and humoral pathways
seem to be involved in the downstream from the SCN (

Ralph

et al., 1990; Silver et al., 1996

). We do not know if CS mice

have defects in these output pathways, however future work on
these pathways is required to better understand abnormal
rhythms of CS mice.

In summary, the present results indicated that a primary

cause of unusual circadian rhythms of behavior and peripheral
clock gene expression in CS mice seems to be the downstream
pathway from the SCN. Because it is rare that animals exhibit
abnormal circadian behavior in spite of normal oscillation in
the SCN, CS mice might provide an important insight to
understand the hierarchical structure of the mammalian
circadian system.

Acknowledgements

We thank Nagoya University Radioisotope Center for its

facilities. This research was supported in part by a Grant-in

Aid from a Ministry of Education, Science and Culture to
S.E.

References

Abe, H., Honma, S., Honma, K., Suzuki, T., Ebihara, S., 1999. Functional

diversities of two activity components of circadian rhythm in genetical
splitting mice (CS strain). J. Comp. Physiol. A 184, 243–251.

Abe, H., Honma, S., Namihira, M., Masubuchi, S., Ikeda, M., Ebihara, S.,

Honma, K., 2001. Clock gene expressions in the suprachiasmatic nucleus
and other areas of the brain during rhythm splitting in CS mice. Mol. Brain
Res. 87, 92–99.

Balsalobre, A., Brown, S.A., Marcacci, L., Tronche, F., Kellendonk, C., Reich-

ardt, H.M., Schutz, G., Schibler, U., 2000. Resetting of circadian time in
peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347.

Brown, S.A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U., 2002.

Rhythms of mammalian body temperature can sustain peripheral circadian
clocks. Curr. Biol. 12, 1574–1583.

Carr, A.J., Johnston, J.D., Semikhodskii, A.G., Nolan, T., Felino, R.A., Cagm-

pang, F.R., Stirland, J.A., Loudon, A.S., 2003. Photoperiod differentially
regulates circadian oscillators in central and peripheral tissues of the Syrian
hamster. Curr. Biol. 13, 1543–1548.

Damiola, F., Le Minh, N., Preitner, N., Kornmann, B., Fleury-olela, F., Schibler,

U., 2000. Restricted feeding uncouples circadian oscillators in peripheral
tissues from the central pacemaker in the suprachiasmatic nucleus. Genes
Dev. 14, 2950–2961.

de la Iglesia, H.O., Meyer, J., Carpino Jr., A., Schwartz, W.J., 2000. Antiphase

oscillation of the left and right suprachiasmatic nuclei. Science 290, 799–
801.

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

300

Fig. 5. Effect of daytime restricted feeding (RF) schedule on mPer1 and Dbp expression in the liver of CS and C57BL/6J mice. Expression patterns were compared
between groups of free feeding (FF) and restricted feeding (RF) during the period from ZT0 to ZT9. Bars above each graph mean LD condition (dark and white bars)
and RF period (shade bars). Asterisks represent significant differences between FF group and RF group by un-paired Student’s t-test (

**

P

< 0.01,

*

P

< 0.05). Each

value is indicated as mean

S.E.M. (n = 3–4).

Edelstein, K., de la iglesia, H.O., Mrosovsky, N., 2003. Period gene expression

in the suprachiasmatic nucleus of behaviorally decoupled hamsters. Mol.
Brain Res. 114, 40–45.

Festing, M.F.W., 1996. Origins and Characteristics of inbred strains of mice. In:

Lyon, M.E., Rastan, S., Brown, S.D.M. (Eds.),

Genetic Variants and

Strains of the Laboratory Mouse, 3rd ed., vol. 2. Oxford University Press,
pp. 1537–1576.

Gorman, M.R., Lee, T.M., 2001. Daily novel wheel running reorganizes and

splits hamster circadian activity rhythms. J. Biol. Rhythms 16, 541–551.

Hara, R., Wan, K., Wakamatsu, H., Aida, R., Moriya, T., Akiyama, M., Shibata,

S., 2001. Restricted feeding entrains liver clock without participation of the
suprachiasmatic nucleus. Genes Cells 6, 269–278.

Ohta, H., Yamazaki, S., McMahon, D.G., 2005. Constant light desynchronizes

mammalian clock neurons. Nat. Neurosci. 8, 267–269.

Panda, S., Hogenesch, J.B., 2004. It’s all in the timing: many clocks, many

outputs. J. Biol. Rhythms 19, 374–387.

Ralph, M.R., Foster, R.G., Davis, F.C., Menaker, M., 1990. Transplanted supra-

chiasmatic nucleus determines circadian period. Science 247, 975–978.

Reppert, S.M., Weaver, D.R., 2001. Molecular analysis of mammalian circadian

rhythms. Annu. Rev. Physiol. 63, 647–676.

Silver, R., LeSauter, J., Tresco, P.A., Lehman, M.N., 1996. A diffusible coupling

signal from the transplanted suprachiasmatic nucleus controlling circadian
locomotor rhythms. Nature 382, 810–813.

Stokkan, K.A., Yamazaki, S., Tei, H., Sakaki, Y., Menaker, M., 2001. Entrain-

ment of the circadian clock in the liver by feeding. Science 290, 490–
493.

Watanabe, T., Yoshimura, T., McMahon, D.G., Ebihara, S., 2003. Unimodal

circadian rhythm in the suprachiasmatic nucleus of behaviorally splitting
mice. Neurosci. Lett. 345, 49–52.

Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block,

G.D., Sakaki, Y., Menaker, M., Tei, H., 2000. Resetting central and
peripheral circadian oscillators in transgenic rats. Science 288, 682–685.

Yoo, S.H., Yamazaki, S., Lowery, P.L., Shimomura, K., Ko, C.H., Buhr, E.D.,

Siepka, S.M., Hong, H.K., Oh, W.J., Yoo, O.J., Menaker, M., Takahashi,
J.S., 2004. PERIOD2<LUCIFERASE real-time reporting of circadian
dynamics reveals persistent circadian oscillations in mouse peripheral
tissues. Proc. Natl. Acad. Sci. U.S.A. 101, 5339–5346.

Yoshimura, T., Suzuki, Y., Makino, E., Suzuki, T., Kuroiwa, A., Matsuda, Y.,

Namikawa, T., Ebihara, S., 2000. Molecular analysis of avian circadian
clock genes. Mol. Brain Res. 78, 207–215.

T. Watanabe et al. / Neuroscience Research 54 (2006) 295–301

301