Two oscillatory components detected by forced splitting of the sleep-wake cycle in humans.

The sleep-wake cycle of human subjects was artificially split into two episodes by imposing an 8-h light and 4-h dark cycle (LD 8:4) twice a day for 7 days, which was followed by a 3-day free-running session. Sleep was permitted only in the dark period. The subjects in the ordinary group were exposed to ordinary light (ca. 500 lx) in the 8-h light period, and those in the bright light group to bright (ca. 5,000 lx) and ordinary light alternatively with bright light after the first dark period (2400-400). Split sleeps persisted in the free-running session with the major episode around the first dark period and the minor episode around the second dark period. By contrast, circadian melatonin rhythm in the free-running session significantly phase delayed in the ordinary light group, but phase advanced in the bright light group, keeping the melatonin rhythm unsplit. The length of nocturnal melatonin secretion (NMS) was significantly shortened in the bright light group. Interestingly, the falling phase of NMS advanced significantly further than the rising phase. Such a difference was not detected in the ordinary light group. Similar differences were observed in the body temperature rhythm. These findings indicated oscillatory mechanisms underlying split sleeps distinct from the circadian pacemaker and suggested an involvement of different circadian oscillators in the rising and falling phases of NMS, which is consistent with the dual oscillator model proposed for the circadian system of nocturnal rodents.NEW & NOTEWORTHY The present study demonstrated that human sleep was separated into two essentially identical components, which persisted under constant conditions, suggesting circadian oscillator underlying split-sleep episodes. The study also indicated differential light sensitivities in the rising and falling phases of circadian melatonin rhythm, indicating the involvement of two different oscillators. These results consisted of the evening and morning dual-oscillator hypothesis for the circadian pacemaker and the hierarchical model for the pacemaker and sleep-wake cycle.

Differential responses to artificial photoperiods of the rising and falling phases of human melatonin rhythm are consistent with a dual oscillator hypothesis.

Circadian rhythms and sleep-wake cycles were measured in volunteers staying singly in temporal isolation unit where they were exposed to artificial short and long light-dark (LD) cycles for 7 days. The long day consisted of 16-h light and 8-h dark (LD 16:8) and the short day consisted of 8-h light and 16-h dark (LD 8:16). During the light period, bright light of approximately 5,000 lux was given from the ceiling and during the dark period there was no illumination. Sleep was monitored by bed sensors, wrist actiwatch, and polysomnography (PSG) on the first and last nights of the schedule. Sleep length was significantly longer under LD 8:16 than under LD 16:8 and the sleep quality estimated by PSG was worse under LD 8:16 than under LD 16:8, which were comparable to natural seasonality in sleep. The circadian rhythm in plasma melatonin was measured in dim light (10 lux) before and after the LD exposures. The nocturnal melatonin secretion (NMS) was significantly longer after LD 8:16 than after LD 16:8 due to differential phase shifts of the rising and falling phases of NMS. After LD 8:16, the falling phase was much advanced than the rising phase, whereas after LD 16:8 the rising phase was much delayed than the falling phase, resulting in the NMS compression. These results indicate that the light sensitivity in terms of phase shifting is different in the two circadian phases, supporting a dual oscillator hypothesis with different phase-response curves for light in the human circadian system.NEW & NOTEWORTHY The present study demonstrated differential light responsiveness of the rising and falling phases of nocturnal melatonin secretion in human subjects exposed to artificial long (LD 16:8) and short days (LD 8:16) and suggested the involvement of different oscillators under these phases. The findings well mimicked the seasonality of the circadian rhythms in nature and consisted with the evening/morning dual oscillator hypothesis proposed originally for nocturnal rodents, providing a new concept for the human circadian system.

[Impacts of mastication frequency on circadian rhythm of glucose metabolism].

Postprandial glucose concentration is dependent on the time of day and its concentration in the morning is lower than in the evening. The circadian rhythm of glucose metabolism is regulated by the central circadian pacemaker in the suprachiasmatic nucleus (SCN). Both the SCN circadian clock and the pancreatic clock play important role in generating and maintaining the circadian rhythm of glucose metabolism. Also, short sleep duration and circadian misalignment are closely associated with a decrease in insulin sensitivity and an increase in type2 diabetes. Increased frequency of mastication and/or thorough chewing has been reported to alter the secretion of hormones related to appetite and energy metabolism. Furthermore, we have reported that the effect of mastication on postprandial glucose metabolism is dependent on the time of day and frequency of mastication. Morning mastication but not evening decreases postprandial blood glucose concentrations and increases insulin secretion at 30 min and so-called the insulinogenic index as a marker of early-phase ß-cell function. This novel finding may aid in reducing the incidence of obesity and type 2 diabetes mellitus. This review covers the basic concept of the mammalian human circadian system, the underlying mechanism causing phase adjustment of the circadian rhythms in the SCN and peripheral organs, and the effect of eating behavior (e.g., chewing frequency) on the circadian rhythm of glucose metabolism.

Nonphotic entrainment of central and peripheral circadian clocks in mice by scheduled voluntary exercise under constant darkness.

In mammals, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) entrains to an environmental light-dark (LD) cycle and organizes the temporal order of circadian rhythms in physiology and behavior. Previously, some studies have demonstrated that scheduled exercise could entrain the free-running behavior rhythm in nocturnal rodents. However, it remains unknown whether entrainment by scheduled exercise alters the internal temporal order of the behavioral circadian rhythms or clock gene expression in the SCN, extra-SCN brain regions, and peripheral organs when mice are entrained to the scheduled exercise under constant darkness (DD). In the present study, we examined circadian rhythms in locomotor activity and clock gene Per1 expression by bioluminescence reporter (Per1-luc) in the SCN, arcuate nucleus (ARC), liver, and skeletal muscle of mice entrained to an LD cycle, mice free-running under DD, and mice entrained to daily exposure to a new cage with a running wheel (NCRW) under DD. All mice showed a steady-state entrainment of behavioral circadian rhythms to NCRW exposure under DD in parallel with shortening of the α when compared with that under DD. The temporal order of behavioral circadian rhythms and the Per1-luc rhythms in the SCN and peripheral tissues but not in the ARC were maintained in the mice entrained to the NCRW and LD cycles; in contrast, the temporal order was altered in the mice under DD. The present findings reveal that the SCN entrains to daily exercise, and daily exercise reorganizes the internal temporal order of behavioral circadian rhythms and clock gene expression in the SCN and peripheral tissues.

Time of Day of Vaccination Does Not Associate With SARS-CoV-2 Antibody Titer Following First Dose of mRNA COVID-19 Vaccine.

The immune system exhibits circadian rhythms, and its response to viral infection is influenced by the circadian clock system. Previous studies have reported associations between the time of day of vaccination against COVID-19 and production of anti-SARS-CoV-2 antibody titer. We examined the effect of vaccination time of day on anti-SARS-CoV-2 antibody titer after the first dose of vaccination with the mRNA-1273 (Moderna) COVID-19 vaccine in an adult population. A total of 332 Japanese adults participated in the present study. All participants were not infected with SARS-CoV-2 and had already received the first dose of mRNA-1273 2 to 4 weeks prior to participating in the study. The participants were asked to provide basic demographic characteristics (age, sex, medical history, allergy, medication, and mean sleep duration), the number of days after the first dose of vaccination, and the time of day of vaccination. Blood was collected from the participants, and SARS-CoV-2 antibody titers were measured. Ordinary least square regression was used for assessing the relationship between basic demographic characteristics, number of days after vaccination, time of day of vaccination, and the log10-transformed normalized antibody titer. The least square mean of antibody titers was not associated with the vaccination time and sleep durations. The least square means of antibody titers was associated with age; the antibody titers decreased in people aged 50 to 59 years and 60 to 64 years. The present findings demonstrate that the vaccination time with mRNA-1273 was not associated with the SARS-CoV-2 antibody titer in an adult population, suggesting that these results do not support restricting vaccination to a particular time of day. The present findings may be useful in optimizing SARS-CoV-2 vaccination strategies.

A fixed single meal in the subjective day prevents free-running of the human sleep-wake cycle but not of the circadian pacemaker under temporal isolation.

Effects of a fixed single meal per day were examined on the circadian pacemaker and sleep-wake cycle in subjects under temporal isolation. When the time of single meal was allowed to take at any time of day (ad-lib meal), the sleep-wake cycle as well as the circadian rhythms in plasma melatonin, cortisol, and core body temperature were significantly phase-delayed in 8 days. On the other hand, when the time of meal was fixed at 1800 h in local time (RF meal), the phase-shift of sleep-wake cycle was not significant while those of the circadian rhythms were significant. The differential effects of a fixed single meal schedule were confirmed in most individual subjects. There was no evidence for the prefeeding increase in plasma cortisol and leptin levels under the fixed single meal schedule. The plasma ghrelin level was apparently high before meal in both ad-lib and RF meal groups, which was, however, likely sculptured by a nonspecific prandial drop and gradual increase after meal intake. Single meal augmented the prandial increase of plasma insulin levels by four to five times. These findings indicate that a single meal at a fixed time of the day during the subjective day failed to prevent the human circadian pacemaker but prevented the sleep-wake cycle from free running for at least 8 days under temporal isolation, suggesting that mealtime was a potent nonphotic time cue for the human sleep-wake cycle.

Effects of Lactococcus lactis subsp. cremoris YRC3780 daily intake on the HPA axis response to acute psychological stress in healthy Japanese men.

BACKGROUND: Lactococcus lactis subsp. cremoris (YRC3780), which is isolated from kefir, has been associated with anti-allergic effects in humans. However, it remains unknown whether daily intake of YRC3780 attenuates the response to psychological stress in humans in parallel with changes to the gut microbiome. We examined the fundamental role of YRC3780 in the gut microbiome, stress response, sleep, and mental health in humans. METHODS: Effects of daily intake of YRC3780 on the hypothalamic-pituitary-adrenal (HPA) axis response to acute psychological stress were investigated in a double-blind, placebo-controlled clinical trial involving 27 healthy young men (mean age and body mass index: 23.5 years and 21.5 kg/m2) who were randomly assigned to placebo (n = 13) or YRC3780 (n = 14) groups. The HPA axis response to acute psychological stress, the diurnal rhythm of HPA axis activity, and gut microbiome were assessed and compared between the two groups. RESULTS: The results showed that daily intake of YRC3780 significantly lowered morning salivary cortisol levels compared with placebo. In addition, salivary cortisol levels following a social stress test significantly decreased +40 min after beginning the TSST in the YRC3780-treated group compared to placebo. There were no significant differences between the two groups in terms of actigraphy-based sleep quality, but the subjective sleep quality and mental health were significantly improved in the YRC3780-treated group compared to placebo. CONCLUSIONS: Our study suggests that daily intake of YRC3780 improves the HPA axis response to acute psychological stress, which might be associated with a decrease in morning cortisol levels.

Effects of different light incident angles via a head-mounted device on the magnitude of nocturnal melatonin suppression in healthy young subjects.

Bright light is a primary zeitgeber (synchronizer) for the central circadian pacemaker in humans. Recently, head-mounted devices for light therapy have been developed to treat patients suffering from circadian rhythm sleep disorders. In this study, to evaluate the influence of the light incident angle of head-mounted devices on the human circadian pacemaker, we examined the effects of bright light (ca.10000 lx) from two different angles (55° vs. 28°) on the suppression of melatonin secretion at night. Twenty-nine subjects (25.1 ± 6.3 SD years) participated in the present study. The subjects were kept under dim light conditions (< 5 lx) from 4 h before their habitual bedtime, followed by exposure to 1 h of bright light at two different angles during their habitual bedtime. Saliva samples were collected every hour under dim light conditions and then collected every 30 min during the bright light exposure. To assess the effect of the light incident angle on ipRGCs mediating light-evoked pupillary constriction, pupil size was measured in before and after exposure to bright light. Melatonin suppression in the group exposed to light at 28° was significantly higher than that in the group with light at 55° (p < 0.001). The pupillary constriction was significantly greater in the group exposed to light at 28° than that in the group with light at 55° (p < 0.001). The present findings suggest that the light incident angle is an important factor for bright light therapy and should be considered to effectively use head-mounted devices in home and clinical settings.

Basic concepts and unique features of human circadian rhythms: implications for human health.

Most physiological functions and behaviors exhibit a robust approximately 24-hour rhythmicity (circadian rhythm) in the real world. These rhythms persist under constant conditions, but the period is slightly longer than 24 hours, suggesting that circadian rhythms are endogenously driven by an internal, self-sustained oscillator. In mammals, including humans, the central circadian pacemaker is located in the hypothalamic suprachiasmatic nucleus. The primary zeitgeber for this pacemaker is bright sunlight, but nonphotic time cues also affect circadian rhythms. The human circadian system uniquely exhibits spontaneous internal desynchronization between the sleep-wake cycle and core body temperature rhythm under constant conditions and partial entrainment of the sleep-wake cycle in response to nonphotic time cues. Experimental and clinical studies of human circadian rhythms must take into account these unique features. This review covers the basic concepts and unique features of the human circadian system, the mechanisms underlying phase adjustment of the circadian rhythms by light and nonphotic time cues (eg, physical exercise), and the effects of eating behavior (eg, chewing frequency) on the circadian rhythm of glucose metabolism.

Morning Mastication Enhances Postprandial Glucose Metabolism in Healthy Young Subjects.

Postprandial glucose concentration is dependent on the time of day and its concentration in the morning is lower than in the evening. However, whether it is dependent on mastication at different times of the day has not been studied before. We hypothesized that mastication affects insulin-mediated glucose metabolism differently in the morning and evening in healthy individuals. Firstly, nine healthy male volunteers (22.0 ± 0.7 SEM years, body mass index 22.0 ± 1.0 kg/m2) performed a 75-g oral glucose tolerance test (OGTT). One week after the OGTT, they participated in a high-carbohydrate food (rice) consumption test with 10 or 40 chews per mouthful. Each experiment was conducted in the morning (0800 h) and evening (2000 h) on the same day. Blood samples were collected before and at 30-min intervals for 120 min after glucose or rice consumption. The incremental area under the curve (iAUC) for glucose in the OGTT was significantly lower in the morning than in the evening, whereas the iAUC for insulin was similar at both times. In participants who chewed 40 times, the iAUC for glucose after rice consumption was significantly lower in the morning than in the evening but was similar at both times in individuals who chewed 10 times. Chewing 40 times in the morning (but not the evening) significantly increased insulin secretion at 30 min. This suggests that morning mastication improves early-phase insulin secretion after rice consumption. This novel finding may aid in reducing the incidence of obesity and type 2 diabetes mellitus.

Hypothalamic-pituitary-adrenal axis differentially responses to morning and evening psychological stress in healthy subjects.

AIM: The hypothalamic-pituitary-adrenal (HPA) axis responds to changing environmental demands including psychological stressors. The aim of the present study was to assess whether the time of day effects on the acute response of HPA axis activity to acute psychological stress. METHOD: We studied 27 healthy young subjects. The subjects were participated two experiments as follows. In the first experiment, subjects were instructed to keep their regular sleep schedule for 2 weeks which were measured by using a wrist-worn activity monitor. Afterward, to evaluate a diurnal rhythm of salivary cortisol, eight saliva samples were collected during waking period every 2 hours from when the subjects woke up. In the second experiment, the subjects were randomly assigned to two groups. The Trier Social Stress Test (TSST) was performed either in the morning (n = 14) or in the evening (n = 13). We measured diurnal rhythm of salivary cortisol and stress response of salivary cortisol and heart rate by the TSST. Morning and evening tests were started at 2 hours and 10 hours after woke up, respectively. RESULTS: All subjects showed a normal diurnal rhythm of salivary cortisol concentration, with a peak in the morning immediately after awaking and a minimum in the evening. The salivary cortisol response after the TSST was significantly increased from the prestress level in the morning but not in the evening. CONCLUSION: The HPA response to acute psychological stress was more pronounced in the morning than in the evening, correlating with the circadian regulation of cortisol synthesis.

Cryptochrome deficiency enhances transcription but reduces protein levels of pineal Aanat

Cryptochrome (Cry) 1 and 2 are essential for circadian rhythm generation, not only in the suprachiasmatic nucleus, the site of the mammalian master circadian clock, but also in peripheral organs throughout the body. CRY is also known as a repressor of arylalkylamine-N-acetyltransferase (Aanat) transcription; therefore, Cry deficiency is expected to induce constantly high pineal melatonin content. Nevertheless, we previously found that the content was consistently low in melatonin-proficient Cry1 and Cry2 double-deficient mice (Cry1−/−/Cry2−/−) on C3H background. This study aims to clarify the mechanism underlying this discrepancy. In the Cry1−/−/Cry2−/− pineal, expression levels of Aanat and clock gene Per1 were consistently high with no circadian fluctuation on the first day in constant darkness, demonstrating that CRY acts in vivo as a repressor of the pineal circadian clock and AANAT. In contrast, the enzyme activity and protein levels of AANAT remained low throughout the day, supporting our previous observation of continuously low melatonin. Thus, effects of Cry deficiency on the responses of ß-adrenergic receptors were examined in cultured pineal glands. Isoproterenol, a ß-adrenergic stimulant, significantly increased melatonin content, although the increase was smaller in Cry1−/−/Cry2−/− than in WT mice, during both the day and night. However, the increase in cAMP in response to forskolin was similar in both genotypes, indicating that CRY deficiency does not affect the pathway downstream of the ß-adrenergic receptor. These results suggest that a lack of circadian adrenergic input due to CRY deficiency decreases ß-receptor activity and cAMP levels, resulting in consistently low AANAT levels despite abundant Aanat mRNA.

Two Coupled Circadian Oscillators Are Involved in Nonphotic Acceleration of Reentrainment to Shifted Light Cycles in Mice.

The onset and offset of an activity band in the circadian behavioral rhythm are known to differentially reentrain to shifted light-dark cycles (LD). Differential reentrainment could be explained by different light responsivities of circadian oscillators underlying these phase-markers. In contrast, reentrainment is accelerated by exposure to nonphotic time cues such as timed wheel-running. However, the relationship between the 2 oscillators and nonphotic acceleration of reentrainment is largely unknown. We examined phase-shifts of the mouse behavioral rhythm in response to an 8-h phase-advanced shift of LD and effects of behavioral interventions: maintained in a home cage (HC), exposed to a running wheel (RW) in HC (HC+RW), transferred to a new cage (NC), and exposed to RW in NC (NC+RW). Each intervention was given for 3h from the beginning of the shifted dark period and repeated for 4 days. Following the last dark period, the mice were released into constant darkness (DD). As a result, activity onset and offset were differentially phase-shifted. The activity onset on the first day of DD (DD1) was phase-advanced from the baseline slightly in HC and HC+RW, substantially in NC+RW, but not significantly in NC. The amount of phase-shift was significantly larger in the NC+RW than in the other groups. In contrast, the activity offset was significantly advanced in all groups by 6 to 8 h. The differential phase-shifts resulted in shortening of the activity band (α compression). The α compression was gradually relieved upon exposure to DD (α decompression), and the activity band finally became stable. Interestingly, the magnitude of phase-shifts of activity offset, but not of activity onset, in the following DD was negatively correlated with the extent of α compression in DD1. These findings indicate that the 2 circadian oscillators underlying activity onset and offset are involved in asymmetric phase-shifts and nonphotic acceleration of reentrainment.

Mistimed wheel running interferes with re-entrainment of circadian Per1 rhythms in the mouse skeletal muscle and lung.

Previously, we showed the acceleration of re-entrainment to 8-h phase-advanced light/dark cycles (LD) in the circadian Per1 expression rhythms of the mouse lung and skeletal muscle by 3-h wheel running (WR) at the beginning of shifted dark phase. In the present study, the effects of WR at the end of shifted dark phase were examined on the re-entrainment in mice. LD was advanced by shortening and was delayed by lengthening the first light period in the phase-advance and phase-delay protocol, respectively. Shifted LD was continued for 4 days, which was followed by constant darkness (DD). Per1 expression was measured in the cultured tissues obtained on the first day of DD from mice carrying a bioluminescence reporter of Per1 expression. In the phase-advance protocol, re-entrainment was not influenced by WR in any circadian rhythm examined. In the phase-delay protocol, re-entrainment of the circadian locomotor rhythm was not affected by WR. However, re-entrainment of circadian Per1 rhythm was significantly decelerated in the skeletal muscle and lung. These findings indicate that the effects of WR on re-entrainment depend on the time of day and the peripheral tissues. Mistimed WR interferes with re-entrainment of circadian rhythms in the lung and skeletal muscle.

Morning and evening physical exercise differentially regulate the autonomic nervous system during nocturnal sleep in humans.

Effects of daily physical exercise in the morning or in the evening were examined on circadian rhythms in plasma melatonin and core body temperature of healthy young males who stayed in an experimental facility for 7 days under dim light conditions (<10 lux). Sleep polysomnogram (PSG) and heart rate variability (HRV) were also measured. Subjects performed 2-h intermittent physical exercise with a bicycle ergometer at ZT3 or at ZT10 for four consecutive days, where zeitgeber time 0 (ZT0) was the time of wake-up. The rising phase of plasma melatonin rhythm was delayed by 1.1 h without exercise. Phase-delay shifts of a similar extent were detected by morning and evening exercise. But the falling phase shifted only after evening exercise by 1.0 h. The sleep PSG did not change after morning exercise, while Stage 1+2 sleep significantly decreased by 13.0% without exercise, and RE sleep decreased by 10.5% after evening exercise. The nocturnal decline of rectal temperature was attenuated by evening exercise, but not by morning exercise. HRV during sleep changed differentially. Very low frequency (VLF) waves increased without exercise. VLF, low frequency (LF), and high frequency (HF) waves increased after morning exercise, whereas HR increased after evening exercise. Morning exercise eventually enhanced the parasympathetic activity, as indicated by HRV, while evening exercise activated the sympathetic activity, as indicated by increase in heart rate in the following nocturnal sleep. These findings indicated differential effects of morning and evening exercise on the circadian melatonin rhythm, PSG, and HRV.

Differential regulation of circadian melatonin rhythm and sleep-wake cycle by bright lights and nonphotic time cues in humans.

Our previous study demonstrated that physical exercise under dim lights (<10 lux) accelerated reentrainment of the sleep-wake cycle but not the circadian melatonin rhythm to an 8-h phase-advanced sleep schedule, indicating differential effects of physical exercise on the human circadian system. The present study examined the effects of bright light (>5,000 lux) on exercise-induced acceleration of reentrainment because timed bright lights are known to reset the circadian pacemaker. Fifteen male subjects spent 12 days in temporal isolation. The sleep schedule was advanced from habitual sleep times by 8 h for 4 days, which was followed by a free-run session. In the shift session, bright lights were given during the waking time. Subjects in the exercise group performed 2-h bicycle running twice a day. Subjects in the control kept quiet. As a result, the sleep-wake cycle was fully entrained by the shift schedule in both groups. Bright light may strengthen the resetting potency of the shift schedule. By contrast, the circadian melatonin rhythm was phase-advanced by 6.9 h on average in the exercise group but only by 2.0 h in the control. Thus physical exercise prevented otherwise unavoidable internal desynchronization. Polysomnographical analyses revealed that deterioration of sleep quality by shift schedule was protected by physical exercise under bright lights. These findings indicate differential regulation of sleep-wake cycle and circadian melatonin rhythm by physical exercise in humans. The melatonin rhythm is regulated primarily by bright lights, whereas the sleep-wake cycle is by nonphotic time cues, such as physical exercise and shift schedule.

Daily exposure to a running wheel entrains circadian rhythms in mice in parallel with development of an increase in spontaneous movement prior to running-wheel access.

Entrainment of circadian behavior rhythms by daily exposure to a running wheel was examined in mice under constant darkness. Spontaneous movement was individually monitored for more than 6 mo by a thermal sensor. After establishment of steady-state free running, mice were placed in a different cage equipped with a running-wheel for 3 h once per day at 6 AM. The daily exchange was continued for 80 days. The number of wheel revolutions during exposure to the running wheel was also measured simultaneously with spontaneous movement. In 13 out of 17 mice, circadian behavior rhythm was entrained by daily wheel exposure, showing a period indistinguishable from 24 h. The entrainment occurred in parallel with an increase in spontaneous movement immediately prior to the daily wheel exposure. A similar preexposure increase was observed in only one of four nonentrained mice. The preexposure increase appeared in 19.5 days on average after the start of daily wheel exposure and persisted for 36 days on average after the termination of the exposure schedule. The preexposure increase was detected only when daily wheel exposure came into the activity phase of the circadian behavior rhythm, which was accompanied by an increase in the number of wheel revolutions. These findings indicate that a novel oscillation with a circadian period is induced in mice by daily exposure to a running wheel at a fixed time of day and suggest that the oscillation is involved in the nonphotic entrainment of circadian rhythms in spontaneous movement.

Daily exposure to cold phase-shifts the circadian clock of neonatal rats in vivo.

Maternal rhythms entrain the prenatal and neonatal circadian clock in the suprachiasmatic nucleus (SCN) before light entrainment is established. However, the responsible time cues for maternal entrainment are not identified. To examine the role of cyclic changes of ambient temperature in maternal entrainment, blind neonatal rats carrying a clock gene (Per2) bioluminescence reporter were exposed to either of three ambient temperatures (10, 20 or 30 °C) during 6-h maternal separation in the early light phase. Cold exposure was performed from postnatal day 1 (P1) to P5. On P6, the SCN was harvested and cultured for photometric monitoring of the circadian rhythm in Per2 expression. Here we demonstrate that the daily cold exposure phase-delayed the circadian Per2 expression rhythms at P6 in a temperature-dependent manner. Exposure to 10 °C produced the largest phase-shift of 12.7 h, and exposure to 20 and 30 °C yielded moderate shifts of 4.1 and 4.5 h, respectively. There was no significant difference in the phase-shifts between the latter two temperatures, indicating that ambient temperature is not the sole factor for the phase-shift. Behavioral rhythms that developed after weaning reflected the phase-shift of clock gene expression rhythm in the SCN. These findings indicate that a daily exposure to an ambient temperature of 10 °C during the neonatal period is capable of resetting the circadian clock in the SCN, but other factors yet unidentified are also involved in maternal entrainment.

Loss of circadian rhythm and light-induced suppression of pineal melatonin levels in Cry1 and Cry2 double-deficient mice.

Cryptochrome 1 and 2 (Cry1 and Cry2) are considered essential for generating circadian rhythms in mammals. The role of Cry1 and Cry2 in circadian rhythm expression and acute light-induced suppression of pineal melatonin was assessed using Cry1 and Cry2 double-deficient mice (Cry1(-/-) /Cry2(-/-) ) developed from the C3H strain that synthesizes melatonin. We examined the circadian variation of pineal melatonin under a 12:12-h light-dark (LD) cycle and constant darkness (DD). Light suppression of pineal melatonin concentration was analyzed by subjecting a 30-min light pulse at the peak phase of melatonin concentration. Wild-type mice showed significant rhythmicity in pineal melatonin concentration with the highest level at Zeitgeber time 22 (ZT22, where time of light on was defined as ZT0) under LD or ZT18 on the first day of DD. In contrast, Cry1(-/-) /Cry2(-/-) mice did not show significant circadian rhythmicity, with only a small peak observed at ZT22 in LD. Nevertheless, a significant daily variation could be observed under DD, with a small increase at ZT6 and ZT18 h. Melatonin concentration was significantly suppressed by acute light pulse at ZT22 in wild-type mice but not in Cry1(-/-) /Cry2(-/-) mice. The present results suggest that Cry genes are required for regulating pineal melatonin synthesis via circadian and photic signals from the suprachiasmatic nucleus of the hypothalamus (SCN).