Nana N. Takasu

Differential Response of Period 1 Expression within the Suprachiasmatic Nucleus

The suprachiasmatic nuclei (SCNs) of the hypothalamus contain a circadian clock that exerts profound control over rhythmic physiology and behavior. The clock consists of multiple autonomous cellular pacemakers distributed throughout the rat SCN. In response to a shift in the light schedule, the SCN rapidly changes phase to achieve the appropriate phase relationship with the shifted light schedule. Through use of a transgenic rat in which rhythmicity in transcription of the Period 1 gene was measured with a luciferase reporter (Per1-luc), we have been successful in tracking the time course of molecular rhythm phase readjustments in different regions of the SCN that occur in response to a shift in the light schedule. We find that different regions of the SCN phase adjust at different rates, leading to transient internal desynchrony in Per1-luc expression among SCN regions. This desynchrony among regions is most pronounced and prolonged when the light schedule is advanced compared with light schedule delays. A similar asymmetry in the speed of phase resetting is observed with locomotor behavior, suggesting that phase shifting kinetics within the SCN may underlay the differences observed in behavioral resetting to advances or delays in the light schedule.

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 −/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.

The suprachiasmatic nucleus: age-related decline in biological rhythms.

Aging is associated with changes in sleep duration and quality, as well as increased rates of pathologic/disordered sleep. While several factors contribute to these changes, emerging research suggests that age-related changes in the mammalian central circadian clock within the suprachiasmatic nucleus (SCN) may be a key factor. Prior work from our group suggests that circadian output from the SCN declines because of aging. Furthermore, we have previously observed age-related infertility in female mice, caused by a mismatch between environmental light–dark cycles and the intrinsic, internal biological clocks. In this review, we address regulatory mechanisms underlying circadian rhythms in mammals and summarize recent literature describing the effects of aging on the circadian system.

Recovery from Age-Related Infertility under Environmental Light-Dark Cycles Adjusted to the Intrinsic Circadian Period

Female reproductive function changes during aging with the estrous cycle becoming more irregular during the transition to menopause. We found that intermittent shifts of the light-dark cycle disrupted regularity of estrous cycles in middle-aged female mice, whose estrous cycles were regular under unperturbed 24-hr light-dark cycles. Although female mice deficient in Cry1 or Cry2, the core components of the molecular circadian clock, exhibited regular estrous cycles during youth, they showed accelerated senescence characterized by irregular and unstable estrous cycles and resultant infertility in middle age. Notably, tuning the period length of the environmental light-dark cycles closely to the endogenous one inherent in the Cry-deficient females restored the regularity of the estrous cycles and, consequently, improved fertility in middle age. These results suggest that reproductive potential can be strongly influenced by age-related changes in the circadian system and normal reproductive functioning can be rescued by the manipulation of environmental timing signals.

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.

Cryptochrome-dependent circadian periods in the arcuate nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus is responsible for controlling behavioral activity rhythms, such as a free running rhythm in constant darkness. Rodents have several circadian oscillators in other brain regions including the arcuate nucleus (ARC). In specific conditions such as food anticipatory activity rhythms in the context of timed restricted feeding, an alternative circadian pace-making system has been assumed by means of circadian oscillators like the SCN. Despite extensive lesion studies, the anatomic locations of extra-SCN circadian pacemakers responsible for regulating behavioral rhythms have not been found. In the present study, we investigated circadian rhythms in the SCN and extra-SCN region of the arcuate nucleus (ARC) by analyzing PER2::LUCIFERASE expression in specific regions from wild-type C57BL/6, Cry1(-/-), and Cry2(-/-) mice. Compared to wild-type animals, we observed period shortening in both the SCN and ARC of Cry1(-/-) mice and period lengthening in Cry2(-/-) mice. Interestingly, the periods in the ARC of both genotypes were identical to those in the SCN. Moreover, the amplitudes of PER2::LUC rhythms in the ARC of all animals were decreased compared to those in the SCN. These data suggest that the ARC is a candidate circadian pacemaker outside the SCN.

In Vivo Monitoring of Multi-Unit Neural Activity in the Suprachiasmatic Nucleus Reveals Robust Circadian Rhythms in Period1−/− Mice

The master pacemaker in the suprachiasmatic nucleus (SCN) controls daily rhythms of behavior in mammals. C57BL/6J mice lacking Period1 (Per1−/−) are an anomaly because their SCN molecular rhythm is weak or absent in vitro even though their locomotor activity rhythm is robust. To resolve the contradiction between the in vitro and in vivo circadian phenotypes of Per1−/− mice, we measured the multi-unit activity (MUA) rhythm of the SCN neuronal population in freely-behaving mice. We found that in vivo Per1−/− SCN have high-amplitude MUA rhythms, demonstrating that the ensemble of neurons is driving robust locomotor activity in Per1−/− mice. Since the Per1−/− SCN electrical activity rhythm is indistinguishable from wild-types, in vivo physiological factors or coupling of the SCN to a known or unidentified circadian clock(s) may compensate for weak endogenous molecular rhythms in Per1−/− SCN. Consistent with the behavioral light responsiveness of Per1 −/− mice, in vivo MUA rhythms in Per1 −/− SCN exhibited large phase shifts in response to light. Since the acute response of the MUA rhythm to light in Per1−/− SCN is equivalent to wild-types, an unknown mechanism mediates enhanced light responsiveness of Per1−/− mice. Thus, Per1−/− mice are a unique model for investigating the component(s) of the in vivo environment that confers robust rhythmicity to the SCN as well as a novel mechanism of enhanced light responsiveness.

The central clock controls the daily rhythm of Aqp5 expression in salivary glands

Salivary secretion displays day–night variations that are controlled by the circadian clock. The central clock in the suprachiasmatic nucleus (SCN) regulates daily physiological rhythms by prompting peripheral oscillators to adjust to changing environments. Aquaporin 5 (Aqp5) is known to play a key role in salivary secretion, but the association between Aqp5 and the circadian rhythm is poorly understood. The aim of our study was to evaluate whether Aqp5 expression in submandibular glands (SMGs) is driven by the central clock in the SCN or by autonomous oscillations. We observed circadian oscillations in the activity of period circadian protein homolog 2 and luciferase fusion protein (PER2::LUC) in cultured SMGs with periodicity depending on core clock genes. A daily rhythm was detected in the expression profiles of Aqp5 in SMGs in vivo. In cultured SMGs ex vivo, clock genes showed distinct circadian rhythms, whereas Aqp5 expression did not. These data indicate that daily Aqp5 expression in the mouse SMG is driven by the central clock in the SCN.