European Journal of Neuroscience, Vol. 25, pp. 2413–2424, 2007
doi:10.1111/j.1460-9568.2007.05490.x
Constant light housing during nursing causes human DSPS
(delayed sleep phase syndrome) behaviour in Clock-mutant
mice
Yukako Wakatsuki, Takashi Kudo and Shigenobu Shibata
Department of Physiology and Pharmacology, School of Science and Engineering, Waseda University, Higashifushimi 2-7-5,
Nishitokyo, 202–0021 Japan
Keywords: circadian rhythm, clock gene, development, lighting condition, melatonin, suprachiasmatic nucleus
Abstract
Delayed sleep phase syndrome (DSPS) is very often seen among patients with sleep-wake rhythm disorders. Humans with the
3111C allele of the human Clock gene tend to demonstrate a higher evening preference on the morningness–eveningness (ME)
preference test. DSPS is thought to be an extreme form of this evening preference. Clock-mutant mice have been proposed as an
animal model of evening preference. In this study, we looked at whether constant light (LL) housing of Clock-mutant mice during
lactation would result in evening preference and ⁄ or DSPS. Housed under light–dark (LD) or constant dark (DD) conditions during the
lactation period, both wild-type and Clock-mutant mice did not show a phase-delay in the locomotor activity measured under light–
dark conditions, whereas constant light housing during lactation significantly caused a delayed onset. The magnitude of the delay
during the light–dark cycle was positively associated with free-running period measured during constant darkness. Among wild,
heterozygote, and homozygote pups born from heterozygous dams, only homozygote pups showed a delayed onset. Constant light–
housed Clock-mutant mice exhibited a lower number and delayed peak of phospho-MAPK-immunoreactive cells in core regions of
the suprachiasmatic nucleus (SCN) compared to light–dark housed wild-type or Clock-mutant mice. Activity onset returned to normal
with daily melatonin injection at the lights-off time for 5 days. The present results demonstrate that Clock-mutant mice exposed to
constant light during lactation can function as an animal model of DSPS and can be used to gain an understanding of the ethological
aspects of DSPS as well as to find medication for its treatment.
Introduction
In mammals, the master circadian pacemaker (or circadian clock)
located in the suprachiasmatic nuclei (SCN) of the hypothalamus
coordinates circadian physiological and behavioural rhythms
(reviewed in Hastings, 1997; King & Takahashi, 2000). In humans,
sleep-wakefulness, cognitive function, body temperature, and hormonal secretion cycles are regulated by intrinsic circadian clock function
(Klerman, 2005).
Clock was the first molecule to be identified as a mammalian clock
gene in a mutant mouse (Vitaterna et al., 1994; Antoch et al., 1997;
King et al., 1997). Clock-mutant mice have a lengthened locomotor
activity rhythm in heterozygotes and homozygotes and finally
abrogation of the rhythmicity in homozygotes under constant dark
(DD) conditions. A polymorphism in the 3¢-flanking region of the
human Clock homolog (3111T ⁄ C, hClock) was examined in reference
to morningness–eveningness (ME) preferences in humans. Two
research groups suggested there is a significant association between
the 3111C ⁄ C allele of hClock and evening preference (Mishima et al.,
2005; Katzenberg et al., 1998). A disorder called delayed sleep phase
syndrome (DSPS) in which patients fail to adapt their sleep–wake
cycle to the environmental time cues (reviewed in Regestein & Monk,
Correspondence: Dr Shigenobu Shibata, as above.
E-mail: [email protected]
Received 18 July 2006, revised 13 February 2007, accepted 20 February 2007
1995) is seen as an example of extreme evening preference as these
patients score very low on the ME scale (Weitzman et al., 1981).
Homozygous Clock-mutant mice carrying the ICR background
strain have previously been proposed as a mouse model of evening
preference similar to that in humans (Sei et al., 2001). Previous
research shows that constant light (LL) housing during lactation alters
the functioning of the circadian system in adult rats and mice.
Spoelstra et al. (2002) reported that the circadian rhythmicity of wheel
running activity could be restored in Clock-mutant mice placed under
LL conditions. This adaptation to light may lead to a stronger
circadian pacemaker that is not easily affected by environmental light
(Cambras et al., 1997; Canal-Corretger et al., 2000; Canal-Corretger
et al., 2001a). We set out to examine whether LL housing during
lactation could lead to evening preference and ⁄ or DSPS in Clockmutant (Jcl:ICR) mice.
The first purpose of this experiment was to examine whether a
delayed onset of nocturnal activity could be observed under the
light–dark (LD) cycle in Clock-mutant mice pre-exposed to LL
conditions for several weeks during lactation or adult periods.
Circadian and photic regulation of mitogen-activated protein kinase
(MAPK) phosphorylation has been shown to be closely related to
clock function in the mouse SCN (Obrietan et al., 1998; Nakaya
et al., 2003). In order to elucidate the mechanism underlying
delayed activity onset in Clock-mutant mice, our second purpose
was to examine the daily pattern of phospho-MAPK-immunoreactive
cell numbers in the SCN. In the clinical setting, melatonin is
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
2414 Y. Wakatsuki et al.
recommended to treat patients with DSPS (reviewed in Zisapel,
2001; Wyatt, 2004). Therefore, the final purpose was to find whether
daily melatonin given to Clock-mutant mice with a 2–3 h delay of
locomotor activity onset could help them to normalize and readjust
to the environmental LD cycle.
Materials and methods
Animals and housing
Clock-mutant mice were purchased from Jackson Laboratory (Stock
no. 002923; Bar Harbor, ME). We backcrossed Clock mutants
carrying the C57black background strain (C57Bl ⁄ 6J) to the ICR
strain (Jcl:ICR) more than eight times. In this experiment, Clockmutant mice carrying the ICR but not the C57black background
were used for purposes of comparing our data to that of Sei et al.
(2001), and in doing so, we found that a high percentage of these
mice (up to 90%) sustained circadian locomotor activity in constant
darkness (DD). Animals were maintained on an LD cycle (12h light : 12-h dark) with lights on at 08:00 h (room temperature at
23 ± 2 !C) and supplied with water and commercial chow (Oriental
Yeast Co, Ltd, Japan) ad libitum. During the light period, light
intensity was 150 lux at cage level. Zeitgeber time (ZT) 0 was
defined as the lights-on time and ZT12 as the lights-off time. Wildtype and Clock-mutant female mice were mated with wild-type and
Clock-mutant male mice, respectively. In order to examine whether
a delay of activity onset was related to Clock mutation in the dam
or her pups, Clock-mutant dams, heterozygote females, and males
were mated. Thus, wild, heterozygote, and homozygote pups were
bred by heterozygote dams. After the behavioural experiments, pup
genotype was determined using a method described in one of our
previous reports (Hoshino et al., 2006). In the present experiment,
the lactation period could have affected the activity onset; therefore,
to rule this out, the weaning day was timed to 3 weeks after birth.
Under the present experimental conditions, as the occurrence of
delayed activity onset was the same for males and females, we
combined the data. Some wild and Clock-mutant mice without a
delay of activity onset under LD and some Clock-mutant mice with
a delay of activity onset under LL were killed in order to examine
phospho-MAPK immunohistochemistry in the SCN. Animal experiments and animal care were conducted under the permission of the
‘Experimental Animal Welfare Committee in the School of Science
and Engineering at Waseda University’ (Permission #05G09).
Definition of DSPLS in the animal models
Although there is a diagnostic criterion for DSPS in the clinic, there is
no definition for delayed sleep phase like syndrome (DSPLS) in
animal experiments. In the present experiment, we identified DSPLS
by the negative phase-angle (phase delays) at the onset of locomotor
activity in comparison to the onset with lights-off. Patients with DSPS
show a 2–3 h phase delay in sleep onset compared to their desired
sleep onset. Therefore, in the present experiment, we identified mice
with a > 2-h delay in activity onset as DSPLS-positive mice; however,
we generally measured the magnitudes of the phase angles for
statistical comparison purposes.
Locomotor activity measurement
After randomly selecting 4–8 mice from each dam, the representative
mice were housed individually in transparent plastic cages
(35 · 20 · 20 cm) for the measurement of locomotor activity.
Locomotor activity rhythms under the LD cycle (150 lux, top of
individual cage) or DD were measured by area sensors (FA-05 F5B
Omron, Japan) with a thermal radiation detector system (Akiyama
et al., 1999). Locomotor activity was continuously recorded in 6-min
epochs by a PC-9801 computer. The onset of activity was automatically plotted by ClockLab software (Actimetrics, Wilmette, IL, USA),
and the daily or circadian rhythmicity of activity was assessed for
approximately one to two weeks using a chi-square periodogram
(Sokolove & Bushell, 1978) in the range of 20–28 h. If the Qp value
did not reach the statistically significant level (P < 0.05), the activity
rhythm was considered arrhythmic.
Selection of mice for locomotor activity measurement
Due to the limited apparatus available for locomotor measurement, a
representative 2–5 pups were randomly (male, female) selected from
the litter for the measurement of locomotor activity. The selected
number of pups (20–40%) was dependent on total pup numbers from
each dam. Experiments in which we measured the locomotor activity
of all pups from each dam are specified.
Melatonin injection
Melatonin (Nakarai-tesk, Tokyo, Japan) was dissolved into 100%
ethanol and then diluted with water to a final concentration of 1%
ethanol. Clock-mutant mice with DSPLS received, on five consecutive
days, either subcutaneous injections of melatonin (0.5 mg ⁄ kg, n ¼ 5)
or (as a control) vehicle solution (1% ethanol, n ¼ 5) at ZT12. The
phase angle of activity onset was compared before and after melatonin
injection.
Sample preparation
Clock-mutant mice were divided into DSPLS or non-DSPLS groups
based on a behavioural assessment. Wild-type and Clock-mutant mice
were then killed at ZT0, 3, 6, 9,12, 15, 18, or 21 under the LD
conditions in which DSPLS was observed. Each mouse was deeply
anaesthetized with ether and intracardially perfused with ice-cold
saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer.
For immunohistochemistry, after perfusion, the brains were quickly
removed, postfixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for at least 24 h at 4 !C, and cryoprotected in 20% sucrose
in phosphate buffer saline overnight at 4 !C. Brain sections 40-lm
thick were made using a cryostat (HM505E; Microm, Walldorf,
Germany) and then placed in 2 · SSC (16.7 mm NaCl, 16.7 mm
C6H5O7Na3, pH 7.0) or PBS until processing for immunohistochemical staining.
Immunohistochemistry
Free-floating sections were sequentially treated with 1% H2O2 for
15 min, 1% goat serum in 0.03% Triton X-100 containing phosphate
buffer saline for 1 h, and incubated with rabbit phospho-MAPK
antibody (1 : 100 dilution, New England Biolabs, Hitchin, UK) for
48 h at 4 !C. A biotinylated goat anti-rabbit IgG secondary antibody
(Vector Laboratories, CA, USA) was used at a 1 : 200 dilution.
Labelling was visualized using a biotin–avidin–peroxidase kit (ABC
Elite Kit, Vector Laboratories, CA, USA) with 0.01% diaminobenzidine and 0.035% H2O2.
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
Clock-mutant mice and DSPS 2415
Cell count
Microphotographs of immunohistochemical sections were taken using
a BX-51 microscope (Olympus, Tokyo, Japan) connected to a DP50
CCD camera (Olympus). Immunolabelled neurons in the SCN were
counted in subregions (core and shell; ventromedial and dorsolateral)
according to the method of Nakaya et al. (2003). The number of cells
expressing phospho-MAPK immunoreactivity was counted by a
researcher blind to mouse genotype and housing conditions. The
averaged cell numbers of six sections taken from rostral to caudal
areas of the SCN were calculated. The six sections per animal are
treated as n ¼ 1 in the data analysis. Three to four animals per
experimental condition comprise each ZT point.
Statistical analysis
The values are expressed as means ± SEM. For statistical analysis,
one-way or two-way anova was applied, followed by the use of
Scheffe’s test or Student’s t-test. In some experiments, we used the
Fisher exact-probability test for incidence. StatView for Windows
version 5.0 (SAS Institute, NC, USA) statistical analysis software
was used.
Results
Delayed phase angle of locomotor activity onset in Clock-mutant
mice housed in LL during early developmental periods
In Fig. 1A–F, we show the representative actograms of wild-type
mice under LD or LL conditions, or Clock-mutant mice under LD
or LL conditions. We obtained a total of 18, 24, and 17 activity
records of a group of representative pups from four wild-type dams
housed in LD, LL, and DD, respectively. We also obtained a total
of 28, 32 (two mice were arrhythmic), and 31 activity records of a
group of representative pups from 5, 6, and 6 Clock-mutant dams
housed in LD, LL, and DD, respectively (Fig. 1G). Neither LL nor
DD housing affected pup growth and survival rates both in wildtype and Clock-mutant pups (data not shown). A negative phaseangle (delay; minus value) similar to DSPS was highly observed in
Clock-mutant mice, and a significant difference was observed in LL
groups (**P < 0.01, Scheffe’s test; Fig. 1G). If DSPLS was
designated as a > 2-h phase-delay angle, LL-housed mice showed a
significantly high incidence of DSPLS (P < 0.01, Fisher exact
probability test) compared to mice in LD or DD conditions
(Fig. 1H). However, the magnitude of the phase-delay angle was
almost similar among LD ()3.18 ± 0.60 h, n ¼ 4), LL
()3.20 ± 0.30 h, n ¼ 15), and DD ()3.02 ± 0.29 h, n ¼ 6) mice,
if comparing only mice with DSPLS. Wild-type mice did not
display any arrhythmicity. In contrast, we did find arrhythmic mice
in two out of 30 of the Clock-mutant mice housed under LL
conditions (Fig. 1H). We did not see any gender differences in the
incidence of DSPLS in Clock-mutant mice (male, )2.2 ± 0.38 h;
female, )2.1 ± 0.34 h).
In order to determine whether DSPLS was related to shortening of
the activity period (so-called alpha) or shifting of the activity period,
we examined the offset time of locomotor activity. Activity offset was
strongly (Fig. 1F), moderately (Fig. 1D), or slightly (Fig. 1E) inhibited
by lights-on stimuli. The majority of Clock-mutant mice housed under
LL exhibited slight to moderate inhibition, and only a few mice (three
out of 30) showed a strong inhibition to lights-on stimuli. The phasedelay angle and length of the activity period (offset time minus onset
time) was )0.8 ± 0.3 h and 13.9 ± 0.5 h, respectively, in Clock-
mutant mice under LD conditions and )2.2 ± 0.3 h (P < 0.01 vs. LD,
Student’s t-test) and 13.1 ± 0.4 h (P > 0.05 vs. LD, Student’s t-test),
respectively, in Clock-mutant mice under LL conditions.
To explore whether LL housing during lactation caused DSPLS
reversibly or irreversibly, a representative eight mice from three
different Clock-mutant dams were examined to determine how long
DSPLS could be maintained. Data showed clear and stable DSPLS
that lasted for a long period (50 days, n ¼ 8; Fig. 2A). We also
measured the locomotor activity of four DSPLS mice for 100 days and
one mouse for 200 days, and found that stable DSPLS lasted through
most of the observed periods (Fig. 2B).
Critical period of LL housing to foster pup DSPLS in Clockmutant mice in the developmental stage
Days 10–20 have been identified as the critical postnatal days for
sensitivity to LL in rats (Canal-Corretger et al., 2001b). We
examined whether there was a critical period for LL housing for
DSPLS pups born from Clock-mutant mice. Figure 3A depicts the
experimental design. In this experiment, we measured the locomotor
activity of a group of representative pups from each dam. When
adult, 10-week-old, Clock-mutant mice were exposed to LL for
5 weeks (Fig. 3H), DSPLS was not observed in comparison with
LD-housed Clock-mutant mice (Fig. 3B). When comparing mice
exposed to LL for 5 weeks from birth (Fig. 3C) to those exposed
from the weaning day 3 weeks after birth (Fig. 3G), there were no
similar amplitudes of negative phase-angles between the two groups.
This data suggests to us two possibilities; one is that LL housing
before weaning does not cause DSPLS, and the second is that
2 weeks of LL housing from the weaning day is enough to produce
DSPLS. To explore the first possibility further, Clock-mutant pups
were exposed to LL housing until weaning (Fig. 3D), and then we
examined the phase-angle under the LD cycle. LL housing until
weaning was enough to produce DSPLS. To investigate the second
possibility, Clock-mutant infants were exposed to LL housing for
2 weeks after weaning (Fig. 3E). Two weeks of LL housing after
weaning was also enough to cause DSPLS. As LL housing for
2 weeks after pups turned 5-weeks old did not cause DSPLS
(Fig. 3F), at least 2 weeks of exposure to LL until 5 weeks of age
seems to be the critical time window for DSPLS.
DSPLS-related Clock mutation of the dam or her pups
To elucidate whether the DSPLS occurrence in LL conditions
referred to the dam or her pups, we mated heterozygote males and
females and produced wild-type, heterozygote, and homozygote
pups. As heterozygote dams (n ¼ 7) equally bred all pups of any
genotype until weaning in both LD or LL, the ratio of wild-type,
heterozygote-type, and homozygote-type pups was approximately 1
(n ¼ 11) : 2 (n ¼ 26) : 1 (n ¼ 13). In this experiment, the activity
rhythm of all pups from seven different dams was recorded. Only
homozygote mice bred under LL exhibited DSPLS in all genotypes,
while homozygote mice bred under LD conditions did not
(Fig. 4A).
In the next experiment, we simultaneously recorded the locomotor
activity rhythm of all Clock-mutant pups and their dams housed under
LD or LL conditions during nursing periods. In this experiment, we
studied seven dam ⁄ pup pairings (three for LD, four for LL). Although
Clock-mutant dams and pups were housed under LD conditions, one
dam showed DSPLS (3-h phase delay, Fig. 4B, dam#3), while her
pups showed only a small phase delay of activity onset (1.5-h phase
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
2416 Y. Wakatsuki et al.
Fig. 1. Delayed onset of locomotor activity onset in Clock-mutant mice housed in constant light (LL) conditions during early developmental periods. (A–F)
Representative double-plotted actogram in LD cycle of the different experimental conditions. (D–F) Actogram from Clock-mutant pups housed under LL conditions.
(E) shows an example in which lights-on does not inhibit the activity, whereas (F) shows an example in which lights-on clearly inhibits the activity. The white and
black bars above each graph show the light and dark periods of the LD cycle (ZT0, lights-on; ZT12, lights-off). (G) Mean values of the phase angle under different
experimental conditions. Vertical values represent phase angle (h), and positive and negative values indicate an advanced or delayed phase angle, respectively. The
numbers in parentheses represent the number of dams and their pups, respectively, meaning 2–5 pups per dam were used to determine locomotor activity rhythm.
**P < 0.01 vs. Clock-mutant LD housing (Scheffe’s test). ##P < 0.01 vs. wild-type LL housing (Student’s t-test). (H) Number of pups showing non-DSPLS (white
column), DSPLS (> 2-h delayed phase angle, hatched column), or arrhythmicity (no significant rhythmicity assessed by chi-square periodogram, black column). In
LL housing, two out of 27 Clock-mutant pups showed arrhythmicity. **P < 0.01 vs. Clock-mutant LD or DD housing (Fisher exact probability test).
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
Clock-mutant mice and DSPS 2417
Fig. 2. Persistence of DSPLS in Clock-mutant mice when recorded under LD cycle. (A) Mean values of the phase angle of eight representative Clock-mutant pups
from three different dams. Vertical values represent phase angle (h) and negative values indicate a delayed phase angle. The numbers in parentheses represent the
number of dams and pups, respectively. (B) Representative double-plotted actogram of locomotor activity in Clock-mutant mice maintained on an LD cycle. A total
of four mice were recorded for 100 days and one mouse for 200 days. These mice were exposed to LL during early development. The white and black bars above
each graph show the light and dark periods of the LD cycle (ZT0, lights-on; ZT12, lights-off).
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
2418 Y. Wakatsuki et al.
Fig. 3. Initiation and duration of the effect of LL housing during the
developmental stage in Clock-mutant mice. (A) Schematic schedule of
LL ⁄ LD housing in Clock-mutant mice. (a–h) The different experimental
schedules, alphabetized to correspond with the results section. White and
hatched columns represent LL housing and LD housing, respectively. ‘R’
indicates when mice were removed from specified housing conditions.
(B) Mean values of phase angle for the different experimental conditions.
Vertical values represent phase angle (h), and negative values indicate a delayed
phase angle. The numbers in the parentheses represent the number of dams and
pups, respectively, meaning 2–5 pups per dam were used to determine
locomotor activity rhythm. **P < 0.01 vs. schedule (a) (Scheffe’s test).
##
P < 0.01, NS > 0.05 vs. schedule (b) (Scheffe’s test).
delay). In contrast, in LL-housed mice, one dam (Fig. 4B, dam#5)
showed only a small phase delay of activity onset (0.1-h phase delay),
while her pups exhibited clear DSPLS ()3.5 ± 0.8).
Free-running period of DSPLS mice under DD conditions
There is a general rule that mice with a long free-running period have a
negative phase angle (phase delay from lights-off), and those with a
short free-running period have a positive phase angle (phase advance
from lights-off) (Piitendrigh & Daan, 1976). From this aspect, we
examined the free-running period of Clock-mutant mice. Figure 5A
shows DSPLS and non-DSPLS actograms taken from Clock-mutant
mouse recordings under DD conditions, as well as the lengthened freerunning period in DSPLS mutant mice. Clock-mutant mice housed in
LL showed a long free-running period (26.0 ± 0.2 h, P < 0.05 vs. LD
housing, Student’s t-test) compared to those mice housed in LD
(24.9 ± 0.1 h). There was a negative correlation between the amplitude
of the phase delay and free-running tau in both LD-housed (r ¼ 0.60,
P < 0.01) and LL-housed (r ¼ 0.62, P < 0.01) Clock-mutant mice
(Fig. 5A and B), but not in LL-housed wild-type mice (r ¼ 0.47,
P > 0.05). As there was no difference in the slope of the correlation
curve between LD and LL groups, the LL-housed group may have had
an extended free-running period. Wild-type mice showed a similar freerunning tau (23.8 ± 0.2 h) when LL-housed (Fig. 5B) in comparison
with wild-type, LD-housed mice (24.0 ± 0.2 h). As activity intensity
itself is known to affect the free-running period, we measured the
activity counts. Daily total activity counts were 8870 ± 930 in Clockmutant mice under LD conditions, and 9180 ± 1182 (P > 0.05 vs. LD,
Student’s t-test) in LL conditions.
Fig. 4. DSPLS related to Clock mutation of dam or her pups. (A) Three and
four heterozygous dams and all their pups were housed under LD or LL
conditions, respectively. These dams bred pups that were wild-type (four for
LD, seven for LL), heterozygous (11 for LD, 15 for LL), or homozygous (five
for LD, eight for LL). Each column represents the mean values of the phase
angle under different experimental conditions. Vertical values represent phase
angle (h), and positive and negative values indicate an advanced or delayed
phase angle, respectively. The numbers in parentheses represent the number of
pups. Among pup genotypes, only homozygous pups housed under LL
conditions showed a significant delayed phase angle. **P < 0.01 vs. wild-type
LL housing (Scheffe’s test). ##P < 0.01 vs. Clock-mutant LD housing
(Student’s t-test). (B) Locomotor activities were recorded for the Clockmutant dam and all her pups simultaneously. White columns show the phase
angle of the dam and black columns that of her pups. Vertical values represent
phase angle (h), and positive and negative values indicate an advanced or
delayed phase angle, respectively. Numbers in the parentheses reflect the
number of pups recorded. Dam #1–7 identifies the separate dam ⁄ pup pairings.
For example, dam #3 showed signs of DSPLS (3-h phase delay), but her pups
showed only a small phase delay in activity onset (1.5-h phase delay). In
contrast, the pups of dam #5 exhibited clear DSPLS, while their dam showed
only a small phase delay (0.2-h phase delay).
Daily rhythms of phospho-MAPK-immunoreactive cell numbers
in the SCN of wild-type and Clock-mutant mice
As circadian and photic regulation of MAPK phosphorylation has
been shown to be related to clock function in the SCN of mice
(Obrietan et al., 1998; Nakaya et al., 2003), we examined the
expression pattern of phospho-MAPK-immunoreactive cells in the
SCN. We prepared wild-type mice housed in LD conditions, Clockmutant mice without DSPLS housed in LD conditions, and Clockmutant mice with DSPLS housed in LL conditions. In the SCN of
wild-type mice, a daily rhythm of positive cell numbers was observed
in the shell area, with a peak around ZT0 and ZT3, whereas a similar
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
Clock-mutant mice and DSPS 2419
Fig. 5. Free-running periods of locomotor activity in Clock-mutant mice. (A) Representative double-plotted actograms of Clock-mutant non-DSPLS (Upper) or
DSPLS (Lower) mice recorded under LD then DD conditions. The white and black bars above each graph show the light and dark periods of the LD cycle (ZT0,
lights-on; ZT12, lights-off). (B) Relationship between phase angle and free-running periods (tau, h) for the different experimental conditions. Vertical values
represent phase angle (h), and positive and negative values indicate an advanced or delayed phase angle, respectively. Solid and broken lines in the figure identify the
correlation lines for Clock-mutant mice housed in LL or LD, respectively. In this experiment, we show not all pups rather only pups with a free-running locomotor
activity rhythm in DD conditions (eight out of eight for LD wild-type, 24 out of 24 for LD Clock-mutant, and 20 out of 22 for LL Clock-mutant mice).
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
2420 Y. Wakatsuki et al.
daily rhythm was observed in the core area, with a peak around ZT18
and ZT21 (Fig. 6A and B). The daily rhythm of phospho-MAPKimmunoreactive cell numbers was dampened in the SCN shell area of
Clock-mutant, non-DSPLS (F1,7 ¼ 17.2, one-way anova, P < 0.01)
and Clock-mutant, DSPLS (F1,7 ¼ 31, one-way anova, P < 0.01)
mice (Fig. 6A and B). In the SCN core area, Clock-mutant mice with
DSPLS displayed a low amplitude of rhythm (F7,40 ¼ 22.7, P < 0.01
vs. wild-type; F7,40 ¼ 10.9, P < 0.01 vs. non-DSPLS, two-way
anova). There were no significant differences between wild-type
and Clock-mutants with non-DSPLS (F7,32 ¼ 0.15, P > 0.05 vs. wildtype, two-way anova). The peak time in Clock-mutant mice with
DSPLS was shifted to ZT0 compared to wild-type and Clock-mutant
non-DSPLS groups (Fig. 6B).
Daily injection of melatonin advanced activity onset in DSPLS
Clock-mutant mice
Because of the reported usefulness of melatonin for treating DSPS
patients (Mundey et al., 2005), we injected Clock-mutant DSPLS mice
with 0.5 mg ⁄ kg of melatonin at ZT12 once a day for 5 days to
observe the changes. Daily melatonin injection clearly and significantly (P < 0.05, vs. vehicle treatment) advanced the onset time of
activity (Fig. 7B and C), whereas daily vehicle treatment did not
(Fig. 7A). Melatonin treatment significantly advanced activity onset
during the actual treatment vs. pretreatment periods (day 0 vs. day )3,
P < 0.05, Student’s paired t-test), and activity onset slowly returned to
the pretreatment level after melatonin withdrawal (day +6 vs. day 0,
P < 0.05, Student’s paired t-test) (Fig. 7C).
Discussion
We examined whether the observed evening preference, based on the
human ME scale, in Clock-mutant mice (Sei et al., 2001) could be
restored when these mice were reared under LL conditions. Our results
support the notion that these mice can act as a good animal model for
the study of human DSPS. Clock-mutant mice exhibited a clear
negative phase-angle (phase delay) in locomotor activity onset and
offset during the LD cycle, only when reared under LL but not DD
conditions during nursing periods. In contrast, a phase-delay was
never observed in wild-type mice under either LL or DD conditions
during nursing or adult periods. In the present experiment, we did not
examine sleep-onset or sleep-offset, and therefore we conservatively
conclude that Clock-mutant mice can function as an adequate animal
model for DSPS.
We have yet to understand how LL during the developmental period
affects the circadian system, especially in Clock-mutant animals.
Previous research shows that LL housing during lactation alters the
functioning of the circadian system in adult rats and mice and prevents
arrhythmicity in adults under LL conditions (Cambras et al., 1997;
Canal-Corretger et al., 2000; Canal-Corretger et al., 2001a). When
light is present from the day of birth, the animal adapts accordingly,
and manifestation of the circadian rhythm is permitted even under LL.
This adaptation may lead to a stronger circadian pacemaker that is not
easily affected by environmental light. Considering the present results,
however, because Clock-mutant mice exposed to LL during lactation
showed a longer free-running period recorded in DD conditions, there
may actually be a weaker circadian pacemaker in these mice. It may be
that an adaptation mechanism(s) to LL during lactation is impaired in
Clock-mutant mice.
Spoelstra et al. (2002) demonstrated an abnormal response to LL
housing in Clock-mutant mice; in LL conditions, restoration of the
circadian rhythmicity of wheel-running activity appeared in Clockmutant mice with previous arrhythmicity in DD conditions. The
degree of self-sustainment of the central pacemaker partly depends on
feedback from activity. In Clock-mutant mice, overall activity is
suppressed by LL to a lesser extent than in wild-type mice. Thus,
Spoelstra et al. (2002) believed the mechanism that maintained
circadian rhythmicity in Clock-mutant mice under LL may be
attributable to the weak suppression of behavioural activity in LL.
At present, the reason for the discrepancy between our results and that
of Spoelstra et al. (2002) observation remains elusive. Spoelstra et al.
(2002) used adults and not infants, Clock-mutant mice carrying the
C57Bl ⁄ 6J strain vs. the ICR strain, and they assessed motor activity
through wheel-running rather than a sensor system. The coupling of
oscillators may be tighter in Clock-mutant mice with the ICR strain vs.
C57BL ⁄ 6J, because only 10% of the colony with the former strain
showed arrhythmicity in DD (present result), while approximately
70% of the latter strain of mice showed arrhythmicity (Spoelstra et al.,
2002). Thus, different experimental conditions may have led to
different results.
Critical postnatal days for sensitivity to LL have been identified and
persistence of the circadian locomotor rhythm under LL occurs when
rats are exposed to LL around postnatal days 10–20 for at least
2 weeks (Canal-Corretger et al., 2001b). We suggested that LL
housing around the weaning day (day 21 after birth) may provide a
critical window of opportunity during development to cause DSPLS in
Clock-mutant pups. When applied at the critical time point, 2 weeks of
LL from the time mice are offspring until they reach 5 weeks of age
may be the ideal length of time to cause DSPLS. LL housing for
5 weeks in adult mice did not produce DSPLS in Clock mutants.
Functional change in the SCN may be responsible for the
appearance of DSPLS in Clock-mutant mice housed in LL during
lactation. To test this idea, we examined the daily rhythm of phosphoMAPK-immunoreactive cells in the SCN of Clock-mutant mice. When
reared under LL (DSPLS), Clock mutants showed a more dampened
and delayed daily rhythm of phospho-MAPK-immunoreactive cell
numbers in the core region of the SCN compared to Clock-mutant
mice reared under LD conditions (non-DSPLS). This outcome may be
related to lengthening of the oscillation and low sensitivity to lights-on
stimuli in Clock-mutant mice with DSPLS. In the shell region of the
SCN, Clock-mutant mice with or without DSPLS displayed a damping
rhythm of phospho-MAPK-immunoreactive cell numbers, which may
be related to the effect of the Clock mutation itself. Several findings
have suggested that photic and gastrin-releasing peptide (GRP) –
induced phase shifts are associated primarily with a distinct subset of
SCN core cells (Silver et al., 1996; Antle et al., 2005). Other than with
light (Obrietan et al., 1998), MAPKs are modulated in the SCN by a
variety of extracellular signals such as NGF, PACAP, GRP or EGF
(Antle et al., 2005; Butcher et al., 2005; Pizzio et al., 2005; Hao &
Schwaber, 2006). Thus, Clock-mutant mice with DSPLS have
impaired photic and ⁄ or signalling cascades such as VIP ⁄ PACAP
and GRP in the SCN. During the past couple of years, SCN cellular
coupling mechanisms concerning VIP and GRP have been intensively
studied and clarified in chronobiology (Maywood et al., 2006; Brown
et al., 2005; Karatsoreos et al., 2006). However, we still do not know
whether the change of phospho-MAPK-immunoreactive cell numbers
in the SCN is the cause or result of DSPLS.
Ohta et al. (2005) reported that LL desynchronizes adult SCN
clock neurons, but does not compromise their ability to generate a
circadian rhythm in Per1-GFP transgenic mice. In addition to the
LL housing effect on adult SCN clock function, Ohta et al. (2006)
recently demonstrated that LL has both acute and long-term
disruptive effects on developing biological clocks in the SCN in
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
Clock-mutant mice and DSPS 2421
Fig. 6. Daily profile of phospho-MAPK-immunoreactive cell numbers in the SCN of wild-type and Clock-mutant LD-housed mice, or Clock-mutant LL-housed
mice. (A) Daily profile of immunohistochemistry of phospho-MAPK-immunoreactive-positive cells in the SCN of wild-type and Clock-mutant mice killed under
the LD cycle. In this experiment, we selected Clock-mutant mice with non-DSPLS in LD conditions or DSPLS in LL conditions. (B) Average number of cells
showing phospho-MAPK immunoreactivity in the shell or core area of the SCN. Each point represents three wild-type LD-housed (open circles), three Clock-mutant
LD-housed (closed squares), and four Clock-mutant LL-housed (closed circles) mice. (C) The SCN was divided into two subregions; the shell area (many phosphoMAPK-immunoreactive cells observed in wild-type at ZT0 and ZT3) and the core area (many phospho-MAPK-immunoreactive cells observed in wild-type at ZT18
and ZT21). These immunohistochemical figures are enlargements of those seen in wild-type mice at ZT0 and ZT18 in A.
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
2422 Y. Wakatsuki et al.
Fig. 7. Recovery effect of daily melatonin injection on DSPLS in Clock-mutant mice. One representative double-plotted actogram of a Clock-mutant DSPLS mouse
is shown for vehicle injection (A) and two representative actograms for melatonin (0.5 mg ⁄ kg) injections (B and C). The white and black bars above each graph
show the light and dark periods of the LD cycle (ZT0, lights-on; ZT12, lights-off). (D) Mean values of phase angle of melatonin- (n ¼ 5) or vehicle- (n ¼ 5) treated
mice. Vertical values represent the phase angle (h), and negative values indicate a delayed phase angle. Closed circles, melatonin. Open circles, vehicle. Before and
after drug treatment, the mean phase delays of three-constitutive days were calculated. Day 0 is the average of the phase delays for injection periods lasting 5 days.
#
P < 0.05 vs. vehicle treatment (Student’s t-test). *P < 0.05 vs. pretreatment (Day )3) (Student’s paired t-test).
the same transgenic pups. Thus, LL exposure may have more
severely affected SCN clock function in pups rather than adults
when LL housing was conducted during lactation periods. Further
experiments that examine the change of clock molecules in the
SCN of Clock-mutant mice under LL exposure during lactation are
necessary. There are reports that exposure to LL causes a
behavioural arrhythmicity in locomotor activity that corresponds
to a loss of rhythmicity in Per2 mRNA and PER2 immunoreactive
cell number in the SCN (Sudo et al., 2003) or that there is a
constitutively elevated expression level of PER2 maintained (Munoz
et al., 2005).
In the present experiment, only Clock-mutant homozygote mice
exhibited DSPLS among the wild, heterozygote, and homozygote
littermates bred by heterozygote dams under LL during lactation.
Thus, it seems that the appearance of DSPLS refers not to a Clock
mutation in dams but to that in pups. Pups born to a Clock-mutant
mother with DSPLS showed no signs of DSPLS when reared under
LD conditions during lactation. In contrast, pups born to a non-DSPLS
Clock-mutant mother showed clear DSPLS in their activity rhythm
when reared under LL conditions during lactation. Consequently, we
propose that LL housing during lactation is a necessary and critical
factor in the appearance of DSPLS, as assessed during the LD cycle.
Patients with DSPS may suffer from a different pathological
phase relationship between the endogenous circadian phases and
sleep timing compared to people with a strong evening preference
(Ozaki et al., 1996; Duffy et al., 1999; Uchiyama et al., 2000;
Iwase et al., 2002). Although there is a significant association
between the 3111C ⁄ C allele of hClock and evening preference
(Mishima et al., 2005; Katzenberg et al., 1998), Takano et al.
(2004) suggested another possible mechanism related to human
DSPS – a missense variation in human CK1eipsilon. Through this
mechanism, Clock-mutant mice would show DSPLS through a
change of CK1eipsilon activity. It is important to discuss other
possible mechanisms for us to eventually come to understand the
real pathogenesis in the future.
Mongrain et al. (2004) reported that ME preference, as identified by
the ME scale, refers to two distinct mechanisms, one associated with a
difference in circadian period and phase of entrainment and the other
associated with chronobiological aspects of sleep regulation. For DSPS
patients seen as extreme evening types on the ME scale continuum, it has
been suggested that this continuum can run on two parallel paths; one
related to an extremely late circadian phase and long endogenous period
and another related to an abnormal phase angle caused by a deficiency in
sleep-wake regulation (Mongrain et al., 2004). Naylor et al. (2000)
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
Clock-mutant mice and DSPS 2423
demonstrated that Clock-mutant mice have a disruption in not only the
core circadian clock system, but also in sleep parameters during
entrainment to the LD cycle, as well as during recovery from 6-h sleep
deprivation in LD. Based on other previous and our present results, we
propose that Clock-mutant mice housed under LL during lactation can
function as a sufficient animal model of the evening preference seen in
humans.
Several types of treatment for DSPS patients have been suggested,
such as phototherapy, chronotherapy, and exogenous melatonin
administration (reviewed in Wyatt, 2004; Zisapel, 2001). Melatonin,
given several hours before its endogenous peak at night, effectively
advances sleep time in DSPS patients and facilitates sleep. Armstrong
et al. (1993) reported that melatonin (1 mg ⁄ kg) and S20098
(1.3 mg ⁄ kg), an analogue of melatonin, successfully reverse the
effects in the rat animal model of DSPLS. In the present experiments,
injection of 0.5 mg ⁄ kg melatonin at ZT12 significantly advanced the
phase-delay in Clock-mutant mice, but melatonin injection at other
times such as ZT3 and ZT6 did not change the phase-delay (data not
shown). Only after the withdrawal of melatonin treatment did we see
the phase-delay slowly return to its pretreatment level. From a
pharmacological point of view, Clock-mutant mice with DSPLS may
be a useful model to evaluate drugs that can effectively treat DSPS.
One drawback to the use of melatonin is that no multicentred research
trials have been able to verify the safety of melatonin administration
on child development, and so far the possible side-effects of melatonin
include convulsions (Sheldon, 1998) and problems with sexual
maturation (Ubuka et al., 2005; Kriegsfeld et al., 2006).
In summary, Clock-mutant mice exposed to LL during lactation
demonstrated a phase-delay in activity onset. Thus, in order to
understand the ethological aspects and drug treatment of DSPS or ME
preference, Clock-mutant mice may function as a useful animal model.
Acknowledgements
This study was partially supported by grants awarded to S.S. from the Japanese
Ministry of Education, Science, Sports, and Culture (18390071), and Waseda
University Grant for Special research Projects (2005A-069).
Abbreviations
DD, constant dark; DSPS, delayed sleep phase syndrome; DSPLS, delayed
sleep phase like syndrome; GRP, gastrin-releasing peptide; LD, light-dark; LL,
constant light; MAPK, mitogen-associated protein kinase; ME, morningnesseveningness; SCN, suprachiasmatic nucleus; ZT, zeitgeber time.
References
Akiyama, M., Kouzu, Y., Takahashi, S., Wakamatsu, H., Moriya, T., Maetani,
M., Watanabe, S., Tei, H., Sakaki, Y. & Shibata, S. (1999) Inhibition of lightor glutamate-induced mPer1 expression represses the phase shifts into the
mouse circadian locomotor and suprachiasmatic firing rhythms. J. Neurosci.,
19, 1115–1121.
Antle, M.C., Kriegsfeld, L.J. & Silver, R. (2005) Signaling within the master
clock of the brain: localized activation of mitogen-activated protein kinase by
gastrin-releasing peptide. J. Neurosci., 25, 2447–2454.
Antoch, M.P., Song, E.J., Chang, A.M., Vitaterna, M.H., Zhao, Y., Wilsbacher,
L.D., Sangoram, A.M., King, D.P., Pinto, L.H. & Takahashi, J.S. (1997)
Functional identification of the mouse circadian Clock gene by transgenic
BAC rescue. Cell, 89, 655–667.
Armstrong, S.M., McNulty, O.M., Guardiola-Lemaitre, B. & Redman, J.R.
(1993) Successful use of S20098 and melatonin in an animal model of
delayed sleep–phase syndrome (DSPS). Pharmacol. Biochem. Behav., 46,
45–49.
Brown, T.M., Hughes, A.T. & Piggins, H.D. (2005) Gastrin-releasing peptide
promotes suprachiasmatic nuclei cellular rhythmicity in the absence of
vasoactive intestinal polypeptide-VPAC2 receptor signaling. J. Neurosci.,
25, 11155–11164.
Butcher, G.Q., Lee, B., Cheng, H.Y. & Obrietan, K. (2005) Light stimulates
MSK1 activation in the suprachiasmatic nucleus via a PACAP-ERK ⁄ MAP
kinase-dependent mechanism. J. Neurosci., 25, 5305–5013.
Cambras, T., Canal, M.M., Torres, A., Vilaplana, J. & Diez-Noguera, A. (1997)
Manifestation of circadian rhythm under constant light depends on lighting
conditions during lactation. Am. J. Physiol., 272, R1039–R1046.
Canal-Corretger, M.M., Cambras, T., Vilaplana, J. & Diez-Noguera, A. (2000)
Bright light during lactation alters the functioning of the circadian system of
adult rats. Am. J. Physiol. Regul. Integr. Comp. Physiol., 278, R201–R208.
Canal-Corretger, M.M., Vilaplana, J., Cambras, T. & Diez-Noguera, A. (2001a)
Effect of light on the development of the circadian rhythm of motor activity
in the mouse. Chronobiol. Int., 18, 683–696.
Canal-Corretger, M.M., Vilaplana, J., Cambras, T. & Diez-Noguera, A. (2001b)
Functioning of the rat circadian system is modified by light applied in critical
postnatal days. Am. J. Physiol. Regul. Integr. Comp. Physiol., 280,
R1023–R1030.
Duffy, J.F., Dijk, D.J., Hall, E.F. & Czeisler, C.A. (1999) Relationship of
endogenous circadian melatonin and temperature rhythms to self-reported
preference for morning or evening activity in young and older people.
J. Invest. Med., 47, 141–150.
Hao, H. & Schwaber, J. (2006) Epidermal growth factor receptor induced
Erk phosphorylation in the suprachiasmatic nucleus. Brain Res., 1088,
45–48.
Hastings, M.H. (1997) Central clocking. TINS, 20, 459–464.
Hayes, J.M. & Balkema, G.W. (1993) Visual thresholds in mice: comparison
of retinal light damage and hypopigmentation. Vis. Neurosci., 10, 931–
938.
Hoshino, K., Wakatsuki, Y., Iigo, M. & Shibata, S. (2006) Circadian Clock
mutation in dams disrupts nursing behavior and growth of pups.
Endocrinology, 147, 1916–1923.
Iwase, T., Kajimura, N., Uchiyama, M., Ebisawa, T., Yoshimura, K., Kamei, Y.,
Shibui, K., Kim, K., Kudo, Y., Katoh, M., Watanabe, T., Nakajima, T.,
Ozeki, Y., Sugishita, M., Hori, T., Ikeda, M., Toyoshima, R., Inoue, Y.,
Yamada, N., Mishima, K., Nomura, M., Ozaki, N., Okawa, M., Takahashi,
K. & Yamauchi, T. (2002) Mutation screening of the human Clock gene in
circadian rhythm sleep disorders. Psychiatry Res., 109, 121–128.
Karatsoreos, I.N., Romeo, R.D., McEwen, B.S. & Silver, R. (2006) Diurnal
regulation of the gastrin-releasing peptide receptor in the mouse circadian
clock. Eur. J. Neurosci., 23, 1047–1053.
Katzenberg, D., Young, T., Finn, L., Lin, L., King, D.P., Takahashi, J.S. &
Mignot, E. (1998) A CLOCK polymorphism associated with human diurnal
preference. Sleep, 21, 569–576.
King, D.P. & Takahashi, J.S. (2000) Molecular genetics of circadian rhythms in
mammals. Annu. Rev. Neurosci., 23, 713–742.
King, D.P., Zhao, Y., Sangoram, A.M., Wilsbacher, L.D., Tanaka, M., Antoch,
M.P., Steeves, T.D., Vitaterna, M.H., Kornhauser, J.M., Lowrey, P.L., Turek,
F.W. & Takahashi, J.S. (1997) Positional cloning of the mouse circadian
clock gene. Cell, 89, 641–653.
Klerman, E.B. (2005) Clinical aspects of human circadian rhythms. J. Biol.
Rhythms, 20, 375–386.
Kriegsfeld, L.J., Mei, D.F., Bentley, G.E., Ubuka, T., Mason, A.O., Inoue, K.,
Ukena, K., Tsutsui, K. & Silver, R. (2006) Identification and characterization
of a gonadotropin-inhibitory system in the brains of mammals. Proc. Natl
Acad. Sci. USA, 103, 2410–2415.
Maywood, E.S., Reddy, A.B., Wong, G.K., O’Neill, J.S., O’Brien, J.A.,
McMahon, D.G., Harmar, A.J., Okamura, H. & Hastings, M.H. (2006)
Synchronization and maintenance of timekeeping in suprachiasmatic
circadian clock cells by neuropeptidergic signaling. Curr. Biol., 16, 599–
605.
Mishima, K., Tozawa, T., Satoh, K., Saitoh, H. & Mishima, Y. (2005) The
3111T ⁄ C polymorphism of hClock is associated with evening preference
and delayed sleep timing in a Japanese population sample. Am. J. Med.
Genet. B Neuropsychiatr. Genet., 133, 101–104.
Mongrain, V., Lavoie, S., Selmaoui, B., Paquet, J. & Dumont, M. (2004) Phase
relationships between sleep-wake cycle and underlying circadian rhythms in
Morningness-Eveningness. J. Biol. Rhythms, 19, 248–257.
Mundey, K., Benloucif, S., Harsanyi, K., Dubocovich, M.L. & Zee, P.C. (2005)
Phase-dependent treatment of delayed sleep phase syndrome with melatonin.
Sleep, 28, 1271–1278.
Munoz, M., Peirson, S.N., Hankins, M.W. & Foster, R.G. (2005) Long-term
constant light induces constitutive elevated expression of mPER2 protein in
the murine SCN: a molecular basis for Aschoff’s rule? J. Biol. Rhythms, 20,
3–14.
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424
2424 Y. Wakatsuki et al.
Nakaya, M., Sanada, K. & Fukada, Y. (2003) Spatial and temporal regulation of
mitogen-activated protein kinase phosphorylation in the mouse suprachiasmatic nucleus. Biochem. Biophys. Res. Commun., 305, 494–501.
Naylor, E., Bergmann, B.M., Krauski, K., Zee, P.C., Takahashi, J.S., Vitaterna,
M.H. & Turek, F.W. (2000) The circadian clock mutation alters sleep
homeostasis in the mouse. J. Neurosci., 20, 8138–8143.
Obrietan, K., Impey, S. & Storm, D.R. (1998) Light and circadian rhythmicity
regulate MAP kinase activation in the suprachiasmatic nuclei. Nature
Neurosci., 1, 693–700.
Ohta, H., Mitchell, A.C. & McMahon, D.G. (2006) Constant light disrupts the
developing mouse biological clock. Pediatr Res., 60, 304–308.
Ohta, H., Yamazaki, S. & McMahon, D.G. (2005) Constant light desynchronizes mammalian clock neurons. Nature Neurosci., 8, 267–269.
Ozaki, S., Uchiyama, M., Shirakawa, S. & Okawa, M. (1996) Prolonged
interval from body temperature nadir to sleep offset in patients with delayed
sleep phase syndrome. Sleep, 19, 36–40.
Piitendrigh, C.S. & Daan, S. (1976) A functional analysis of circadian
pacemakers in nocturnal rodents. I. The stability and liability of spontaneous
frequency. J. Comp. Physiol., 106, 291–331.
Pizzio, G.A., Hainich, E.C., Plano, S.A., Ralph, M.R. & Golombek, D.A.
(2005) Nerve growth factor-induced circadian phase shifts and MAP kinase
activation in the hamster suprachiasmatic nuclei. Eur. J. Neurosci., 22,
665–671.
Regestein, Q.R. & Monk, T.H. (1995) Delayed sleep phase syndrome: a review
of its clinical aspects. Am. J. Psychiatry, 152, 602–608. Review.
Sei, H., Oishi, K., Morita, Y. & Ishida, N. (2001) Mouse model for
morningness ⁄ eveningness. Neuroreport, 12, 1461–1464.
Sheldon, S.H. (1998) Pro-convulsant effects of oral melatonin in neurologically
disabled children. Lancet, 351, 1254.
Silver, R., Romero, M.T., Besmer, H.R., Leak, R., Nunez, J.M. & LeSauter, J.
(1996) Calbindin-D28K cells in the hamster SCN express light-induced Fos.
Neuroreport, 7, 1224–1228.
Sokolove, P.G. & Bushell, W.N. (1978) The chi square periodogram: its utility
for analysis of circadian rhythms. J. Theor. Biol., 72, 131–160.
Spoelstra, K., Oklejewicz, M. & Daan, S. (2002) Restoration of self-sustained
circadian rhythmicity by the mutant clock allele in mice in constant
illumination. J. Biol. Rhythms, 17, 520–525.
Sudo, M., Sasahara, K., Moriya, T., Akiyama, M., Hamada, T. & Shibata, S.
(2003) Constant light housing attenuates circadian rhythms of mPer2 mRNA
and mPER2 protein expression in the suprachiasmatic nucleus of mice.
Neuroscience, 121, 493–499.
Takano, A., Uchiyama, M., Kajimura, N., Mishima, K., Inoue, Y., Kamei, Y.,
Kitajima, T., Shibui, K., Katoh, M., Watanabe, T., Hashimotodani, Y.,
Nakajima, T., Ozeki, Y., Hori, T., Yamada, N., Toyoshima, R., Ozaki, N.,
Okawa, M., Nagai, K., Takahashi, K., Isojima, Y., Yamauchi, T. & Ebisawa,
T. (2004) A missense variation in human casein kinase I epsilon gene that
induces functional alteration and shows an inverse association with circadian
rhythm sleep disorders. Neuropsychopharmacology, 29, 1901–1909.
Ubuka, T., Bentley, G.E., Ukena, K., Wingfield, J.C. & Tsutsui, K. (2005)
Melatonin induces the expression of gonadotropin-inhibitory hormone in the
avian brain. Proc. Natl Acad. Sci. USA, 102, 3052–3057.
Uchiyama, M., Okawa, M., Shibui, K., Kim, K., Tagaya, H., Kudo, Y., Kamei,
Y., Hayakawa, T., Urata, J. & Takahashi, K. (2000) Altered phase relation
between sleep timing and core body temperature rhythm in delayed sleep
phase syndrome and non-24-hour sleep–wake syndrome in humans.
Neurosci. Lett., 294, 101–104.
Vitaterna, M.H., King, D.P., Chang, A.M., Kornhauser, J.M., Lowrey, P.L.,
McDonald, J.D., Dove, W.F., Pinto, L.H., Turek, F.W. & Takahashi, J.S.
(1994) Mutagenesis and mapping of a mouse gene, Clock, essential for
circadian behavior. Science, 264, 719–725.
Weitzman, E.D., Czeisler, C.A., Coleman, R.M., Spielman, A.J., Zimmerman,
J.C., Dement, W., Richardson, G. & Pollak, C.P. (1981) Delayed sleep phase
syndrome. A chronobiological disorder with sleep-onset insomnia. Arch.
Gen. Psychiatry, 38, 737–746.
Wyatt, J.K. (2004) Delayed sleep phase syndrome: pathophysiology and
treatment options. Sleep, 27, 1195–1203.
Zisapel, N. (2001) Circadian rhythm sleep disorders: pathophysiology and
potential approaches to management. CNS Drugs, 15, 311–328.
ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 25, 2413–2424