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The ovarian steroid hormones, estrogen and progesterone, the levels of which fluctuate dynamically with the estrous cycle, alter circadian behavioral rhythms in mammals. However, it remains unclear whether the sleep-wake rhythm fluctuates with the menstrual cycle in humans. To ascertain the relationship between the menstrual cycle and sleep-wake rhythms, we evaluated the objective and long-term sleep-wake rhythms of ten healthy women using a recently developed wearable device. The results showed a strong negative correlation between the sleep midpoint and the quasi-peak value (an indicator of rhythm robustness), and a positive correlation between the length of the menstrual cycle (days) and social jetlag (hours). These results suggest that healthy women with late sleeping habits have a disturbed sleep-wake rhythm and that irregular habits prolong the menstrual cycle. The sleep midpoint and quasi-peak values showed variations during the menstrual cycle. The quasi-peak values in the follicular phase were significantly higher than those in the menstrual and luteal phases. In rodents, the phase of locomotor activity rhythm advances, and activity increases at night during proestrus. The increase in quasi-peak values during the follicular phase, when estrogen is relatively high, may be due to the increased activity caused by estrogen. These results suggest that ovarian steroid hormones influence sleep-wake rhythms in women. Verifying the results of this study under various conditions is necessary; however, accurately predicting the day of ovulation using only the acquisition of sleep-wake rhythms with wearable devices will be possible., Competing Interests: Conflict of interestThe authors declare that they have no conflicts of interest concerning this article., (© The Author(s) 2024.)

The suprachiasmatic nucleus (SCN) is the central clock for circadian rhythms. Animal studies have revealed daily rhythms in the neuronal activity in the SCN. However, the circadian activity of the human SCN has remained elusive. In this study, to reveal the diurnal variation of the SCN activity in humans, we localized the SCN by employing an areal boundary mapping technique to resting-state functional images and investigated the SCN activity using perfusion imaging. In the first experiment ( n  = 27, including both sexes), we scanned each participant four times a day, every 6 h. Higher activity was observed at noon, while lower activity was recorded in the early morning. In the second experiment ( n  = 20, including both sexes), the SCN activity was measured every 30 min for 6 h from midnight to dawn. The results showed that the SCN activity gradually decreased and was not associated with the electroencephalography. Furthermore, the SCN activity was compatible with the rodent SCN activity after switching off the lights. These results suggest that the diurnal variation of the human SCN follows the zeitgeber cycles of nocturnal and diurnal mammals and is modulated by physical lights rather than the local time., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Green tea (GT) catechins exhibit antiviral effects in experimental studies. However, we lack clinical evidence on the preventive effects of catechin concentrations in gargling against acute upper respiratory tract infections (URTIs). Therefore, we aimed to investigate the concentration-dependence of GT catechins in gargling on the incidence of URTIs. We conducted an open-label randomized study. The target population consisted of 209 students from the University of Shizuoka and Meiji University, who were randomly assigned to high-catechin (approximate catechin concentration: 76.4 mg/dL), low-catechin (approximate catechin concentration: 30.8 mg/dL), and a control water gargling (catechin concentration: 0 mg/dL) group. All participants gargled water or GT daily for 12 weeks. The symptoms of URTIs were recorded on a daily survey form by participants. The incidences of URTIs occurred in 6 (9.1%), 7 (10.8%), and 11 (15.7%) participants in the high-catechin, low-catechin, and water groups, respectively. Cox proportional hazards analysis, using background factors and prevention status as covariates, revealed a hazard ratio of 0.57 (95% Confidence Interval (CI): 0.21-1.55, p = 0.261) for the high-catechin vs. water group and 0.54 (95% CI: 0.20-1.50, p = 0.341) for the low-catechin vs. water group. Our findings showed the incidence of URTIs in a concentration-dependent GT gargling was not significantly different, partly owing to the low event rates caused by intense precautions against the coronavirus disease 2019 pandemic. Our study would serve as a foundation for the development of an advanced protocol with optimal concentrations and a larger number of participants.

The central circadian clock of the suprachiasmatic nucleus (SCN) is a network consisting of various types of neurons and glial cells. Individual cells have the autonomous molecular machinery of a cellular clock, but their intrinsic periods vary considerably. Here, we show that arginine vasopressin (AVP) neurons set the ensemble period of the SCN network in vivo to control the circadian behavior rhythm. Artificial lengthening of cellular periods by deleting casein kinase 1 delta (CK1δ) in the whole SCN lengthened the free-running period of behavior rhythm to an extent similar to CK1δ deletion specific to AVP neurons. However, in SCN slices, PER2::LUC reporter rhythms of these mice only partially and transiently recapitulated the period lengthening, showing a dissociation between the SCN shell and core with a period instability in the shell. In contrast, in vivo calcium rhythms of both AVP and vasoactive intestinal peptide (VIP) neurons in the SCN of freely moving mice demonstrated stably lengthened periods similar to the behavioral rhythm upon AVP neuron-specific CK1δ deletion, without changing the phase relationships between each other. Furthermore, optogenetic activation of AVP neurons acutely induced calcium increase in VIP neurons in vivo. These results indicate that AVP neurons regulate other SCN neurons, such as VIP neurons, in vivo and thus act as a primary determinant of the SCN ensemble period., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tsuno et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many female mammals have recurring cycles of ovulation and sexual behaviors that are regulated by reproductive hormones and confer reproductive success. In addition to sexual behaviors, circadian behavioral rhythms of locomotor activity also fluctuate across the estrous cycle in rodents. Moreover, there is a bidirectional relationship between circadian rhythms and estrous cyclicity since mice with disrupted circadian rhythms also have compromised estrous cycles resulting in fewer pregnancies. In the present study, we assessed whether extending day length, which alters circadian rhythms, normalizes estrous cyclicity in mice. We found that Period ( Per ) 1 / 2 / 3 triple knockout (KO) mice, that have disabled canonical molecular circadian clocks, have markedly disrupted estrous cycles. Surprisingly, extending the day length by only 2 h per day restored regular 4- or 5-day estrous cycles to Per1 / 2 / 3 KO mice. Longer days also induced consistent 4-day, rather than 5-day, estrous cycles in wild-type C57BL/6J mice. These data demonstrate that extending daytime light exposure could be used for enhancing reproductive success., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (© 2023 The Authors.)

Introduction: The trigeminal nerve conveys delicate sensations such as warmth, pain, and tactile pressure in the oral and facial regions, and most trigeminal afferent cell bodies are located in the trigeminal ganglion. Our previous study has shown that sensations in trigeminal nerve innervated areas, specifically in the maxillofacial region, exhibit diurnal variation and that sensitivity changes time-dependently. In this study, we aimed to clarify the rhythm of expression of clock gene in the trigeminal ganglion of mice to elucidate the mechanism of circadian regulation in the same area., Methods: Immunohistochemistry examined the expression of the PER2 protein in the suprachiasmatic nucleus and trigeminal ganglion of wild-type mice. To measure gene expression as bioluminescence, PERIOD2::LUCIFERASE knock-in (PER2::LUC) mice were used. Unilateral trigeminal ganglion and brain sections including the suprachiasmatic nucleus were incubated ex vivo . Bioluminescence levels were then measured using a highly sensitive photodetector. The same experiments were then conducted with Cry1 gene-deficient ( Cry1 -/- ) or Cry2 gene-deficient ( Cry2 -/- ) mice., Results: In the trigeminal ganglion, immunohistochemistry localized PER2 protein expression within the neuronal cell body. Mouse trigeminal ganglion ex vivo tissues showed distinct circadian oscillations in PER2::LUC levels in all genotypes, wild-type, Cry1 -/- , and Cry2 -/- . The period was shorter in the trigeminal ganglion than in the suprachiasmatic nucleus; it was shorter in Cry1 -/- and longer in Cry2 -/- mice than in the wild-type mice., Conclusion: The expression of Per2 in neurons of the trigeminal ganglion in ex vivo culture and the oscillation in a distinct circadian rhythm suggests that the trigeminal ganglion is responsible for the relay of sensory inputs and temporal gating through autonomous circadian oscillations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shirakawa, Ohno, Yamagata, Kuramoto, Oda, Nakamura, Nakamura and Sugimura.)

In mammals, the suprachiasmatic nucleus (SCN) is a principal circadian pacemaker that optimizes the timing of behavioral rhythms and physiological events. Normally, circadian behavioral rhythms are entrained by the environmental light-dark (LD) cycle via the SCN. However, daily rhythms of other synchronizing signals, such as food availability, also emerge. When food availability is restricted to a single recurring daytime meal in nocturnal rodents, they exhibit increased activity during the hours immediately preceding feeding time; this is called food anticipatory activity (FAA). Many reports suggest that FAA is mediated by the food-entrainable oscillator (FEO) with circadian properties, but not the SCN. However, the neural locus and timekeeping mechanisms of the FEO, including its relationship with gastrointestinal hormone signaling, remain unclear. Herein, to examine whether secretin receptor signaling is necessary for the FEO, the effect of daily food restriction was studied in secretin receptor-deficient (Sctr -/- ) mice. Adult wild-type (WT) and Sctr -/- mice were housed in separate cages containing a running wheel, with ad libitum food access and in a LD cycle (12 hours:12 hours) for at least 2 weeks. After acclimation to the condition, food access times were gradually restricted and 4-hour restricted feeding lasted over 10 days. Subsequently, mice had ad libitum food access for 2 days and then fasted for 2 days. Thereafter, robust FAAs were observed in both WT and Sctr -/- mice during restricted feeding and subsequent fasting. These results indicate that secretin receptor signaling is not essential for the timekeeping mechanism of FEO., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The circadian rhythms of physiology and behavior are based on molecular systems at the cellular level, which are regulated by clock genes, including cryptochrome genes, Cry1 and Cry2. In mammals, the circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus maintains the circadian rhythms throughout the body. Cry1 and Cry2 play distinct roles in regulating the circadian rhythm. However, the different effects of manipulating clock genes in heterozygous and homozygous alleles, Cry1 and Cry2, remain unclear. Therefore, this study aimed to understand the haplosufficiency of cryptochrome genes in regulating the circadian system. We examined wheel-running activity rhythms and PER2::LUC expression rhythms in SCN slices and pituitary explants in mice. Compared with wild-type mice, Cry1 - / - or Cry2 - / - mice had shortened or lengthened periods in free-running behavioral rhythms and PER2::LUC expression in the SCN and pituitary gland. Cry1 +/ - mice had similar circadian rhythms as wild-type mice, although Cry2 +/ - mice had lengthened periods. The amplitude of PER2::LUC expression exhibited faster damping in Cry1 - / - mice. Therefore, Cry1 deficiency affects the circadian period length and stability of the circadian system. A single allele of Cry2 deficiency affects the circadian rhythm, whereas that of Cry1 deficit is compensated., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which coordinates the circadian rhythm and controls locomotor activity rhythms. In addition to SCN cells, the peripheral tissues and embryonic fibroblasts also have clock genes, such as Per1/2 and Bmal1 , which generate the transcriptional-translational feedback loop to produce an approximately 24-h cycle. Aging adversely affects the circadian clock system and locomotor functions. Oak extract has been reported to improve age-related physiological changes. However, no study has examined the effect of oak extract on the circadian clock system., Methods: We examined the effects of oak extract and its metabolites (urolithin A [ULT] and ellagic acid [EA]) on clock gene expression rhythms in mouse embryonic fibroblasts (MEFs) and SCN. Furthermore, locomotor activity rhythm was assessed in young and aged mice., Results: Chronic treatment with EA and ULT delayed the phase of PER2::LUC rhythms in SCN explants, and ULT prolonged the period of PER2::LUC rhythms in MEFs in a dose-dependent manner and increased the amplitude of PER2::LUC rhythms in MEFs, though only at low concentrations. Acute treatment with ULT affected the phase of PER2::LUC rhythms in MEFs depending on the concentration and timing of the treatment. In addition, oak extract prolonged the activity time of behavioral rhythms in old mice and tended to increase daily wheel-running revolutions in both young and old mice., Conclusions: These results suggest that oak extract is a novel modulator of the circadian clock in vitro and in vivo., Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-021-00365-2., Competing Interests: Conflict of interestThe authors declare that they have no conflicts of interest concerning this article., (© The Author(s), under exclusive licence to Japanese Society of Sleep Research 2021.)

Several studies have suggested a strong interaction between the circadian clock and lipid metabolism in mammals. The circadian clock is driven by endogenous cyclic gene expression patterns, commonly referred to as clock genes, and transcription-translation negative feedback loops. Clock genes regulate the transcription of some lipid metabolism-related genes; however, the relationship between the circadian clock and triglyceride (TG) accumulation at the cellular level remains unclear. Here, we evaluated rhythms of intracellular TG accumulation levels as well as the expression of clock genes and lipid metabolism-related genes for 54 h in mouse and bovine adipose-derived cell cultures. To the best of our knowledge, this study represents the first report demonstrating that TG accumulation exhibits diurnal variations, with the pattern differing among cell types. Furthermore, we found that expression of clock genes and corresponding lipid metabolism-related genes exhibited circadian rhythms. Our results suggest that the cellular clock regulates lipid metabolism-related genes to relate circadian rhythms of TG accumulation in each cell type. We anticipate that the amount of fat stored depends on the timing of the supply of glucose-the precursor of fat. The findings of this study will contribute to the advancement of chrono-nutrition., (© 2022 Japanese Society of Animal Science.)

Management of time and circadian disruption is an extremely important factor in basic research on pain and analgesia. Although pain is known to vary throughout the day, the mechanism underlying this circadian variation remains largely unknown. In this study, we hypothesized that the process of pain transmission to the central nervous system (after receiving nociceptive stimuli from outside the body) would show day-night differences. Ten-week-old male mice were kept under a strict 12/12-h light/dark cycle for at least 10 days. Formalin was then injected into the second branch region of the trigeminal nerve and the duration of pain-related behaviors (PRBs) was assessed. Immunohistochemical staining was then performed, and the c-Fos-immunopositive cells in the trigeminal spinal tract subnucleus caudalis (Sp5C) were counted. The results showed that the duration of PRBs was longer and the number of c-Fos immunopositive cells in the Sp5C was higher at nighttime than during the day. In addition, the trigeminal ganglia (TG) were extracted from the mice and examined by quantitative real-time PCR to evaluate the daytime and nighttime expression of nociceptive receptors. The results showed that the mRNA expression of transient receptor potential ankyrin 1 in the TG was significantly higher at night than during the day. These results suggest that pain in the trigeminal nerve region is more intense at nighttime, when rodents are active, than during the daytime, partly due to differences in nociceptor expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niiro, Ohno, Yamagata, Yamagata, Tomita, Kuramoto, Oda, Nakamura, Nakamura and Sugimura.)

The dorsal striatum forms part of the basal ganglia circuit that is a major regulator of voluntary motor behavior. Dysfunction in this circuit is a critical factor in the pathology of neurological (Parkinson's and Huntington's disease) as well as psychiatric disorders. In this study, we employed in vivo real-time monitoring of multiple unit neural activity (MUA) in the dorsal striatum of freely moving mice. We demonstrate that the striatum exhibits robust diurnal and circadian rhythms in MUA that peak in the night. These rhythms are dependent upon the central circadian clock located in the suprachiasmatic nucleus (SCN) as lesions of this structure caused the loss of rhythmicity measured in the striatum. Nonetheless, chronic treatment of methamphetamine (METH) makes circadian rhythms appear in MUA recorded from the striatum of SCN-lesioned mice. These data demonstrate that the physiological properties of neurons in the dorsal striatum are regulated by the circadian system and that METH drives circadian rhythms in striatal physiology in the absence of the SCN. The finding of SCN-driven circadian rhythms in striatal physiology has important implications for an understanding of the temporal regulation of motor control as well as revealing how disease processes may disrupt this regulation., Competing Interests: None., (© 2021 The Authors.)

Although diurnal variations have been observed in tear film parameters in various species, the molecular mechanisms that control circadian tear secretion remain unclear. The aim of our study was to evaluate the role of clock genes in the lacrimal gland (LG) in regulation of tear secretion. Tear volume was measured by cotton thread test in core clock genes deficient (Cry1 -/- Cry2 -/-- ) mice which are behaviorally arrhythmic. Real-time quantitative RT-PCR was used to examine expression profiles of core clock genes in the LG including Per1, Per2, Per3, Clock, Bmal1. All experiments were performed under a 12 h of light and 12 h of darkness (LD) and constant dark (DD) conditions. Under both LD and DD conditions, diurnal and circadian rhythms were observed in tear secretion of wild-type mice with tear volume increased in the objective and subjective night while disruption in diurnal and circadian variations of tear secretion were found in Cry1 -/- Cry2 -/-- mice. In wild-type mice, the expression level of major clock genes in the LG showed oscillatory patterns under both LD and DD conditions. In contrast, expression clock genes in the lacrimal gland of Cry1 -/- Cry2 -/-- mice showed complete loss of oscillation regardless of environmental light conditions. These findings confirmed the presence of diurnal and circadian rhythms of tear secretion and provided evidences supporting a critical role for the clock in the control of tear secretion., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABA A receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca 2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks., Competing Interests: The authors declare no competing interest.

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a bilaterally paired structure in the hypothalamus known as the suprachiasmatic nucleus (SCN). Understanding the mammalian circadian system will require a detailed multilevel analysis of neural SCN circuits ex vivo and in vivo. Many of the techniques and approaches that are used for the analysis of the circuitry driving circadian oscillations in the SCN are similar to those employed in other brain regions. There is, however, one fundamental difference that needs to be taken into consideration, that is, the physiological, cell, and molecular properties of SCN neurons vary with the time of day. In this chapter, we will consider the preparations and electrophysiological techniques that we have used to analyze the SCN circuit focusing on the acute brain slice and intact, freely moving animal.

The circadian clocks within the hypothalamic-pituitary-gonadal axis control estrous cycles in female rodents. The suprachiasmatic nucleus (SCN), where the central clock is located, generates daily signals to trigger surge release of luteinizing hormone (LH), which in turn induces ovulation. It has been observed in aged rodents that output from the SCN such as neuronal firing activity is declined, and estrous cycles become irregular and finally stop. Circadian clock mutants display accelerated reproductive aging, suggesting the complicated interplay between the circadian system and the endocrine system. To investigate such circadian regulation of estrous cycles, we construct a mathematical model that describes dynamics of key hormones such as LH and of circadian clocks in the SCN and in the ovary, and simulate estrous cycles for various parameter values. Our simulation results demonstrate that reduction of the amplitude of the SCN signal, which is a symptom of aging, makes estrous cycles irregular. We also show that variation in the phase of the SCN signal and changes in the period of ovarian circadian clocks exacerbates the aging effect on estrous cyclicity. Our study suggests that misalignment between the SCN and ovarian circadian oscillations is one of the primary causes of the irregular estrous cycles.

Placental leucine aminopeptidase/insulin-regulated aminopeptidase (P-LAP/IRAP) regulates vasopressin and oxytocin levels in the brain and peripheral tissues by controlled degradation of these peptides. In this study, we determined the relationship between P-LAP/IRAP and vasopressin levels in subregions of the murine brain. P-LAP/IRAP expression was observed in almost all brain regions. The expression patterns of P-LAP/IRAP and vasopressin indicated that cells expressing one of these protein/peptide were distinct from those expressing the other, although there was significant overlap between the expression regions. In addition, we found reciprocal diurnal rhythm patterns in P-LAP/IRAP and arginine vasopressin (AVP) expression in the hippocampus and pituitary gland. Further, synchronously cultured PC12 cells on treatment with nerve growth factor (NGF) showed circadian expression patterns of P-LAP/IRAP and enzymatic activity during 24 h of incubation. Considering that vasopressin is one of the most efficient peptide substrates of P-LAP/IRAP, these results suggest a possible feedback loop between P-LAP/IRAP and vasopressin expression, that regulates the function of these substrate peptides of the enzyme via translocation of P-LAP/IRAP from intracellular vesicles to the plasma membrane in brain cells. These findings provide novel insights into the functions of P-LAP/IRAP in the brain and suggest the involvement of these peptides in modulation of brain AVP functions in hyperosmolality, memory, learning, and circadian rhythm., (Copyright © 2020 Goto, Nakamura, Ogawa, Hattori and Tsujimoto.)

Salt is an essential nutrient; however, excessive salt intake is a prominent public health concern worldwide. Various physiological functions are associated with circadian rhythms, and disruption of circadian rhythms is a prominent risk factor for cardiovascular diseases, cancer, and immune disease. Certain nutrients are vital regulators of peripheral circadian clocks. However, the role of a high-fat and high-salt (HFS) diet in the regulation of circadian gene expression is unclear. This study aimed to investigate the effect of an HFS diet on rhythms of locomotor activity, caecum glucocorticoid secretion, and clock gene expression in mice. Mice administered an HFS diet displayed reduced locomotor activity under normal light/dark and constant dark conditions in comparison with those administered a normal diet. The diurnal rhythm of caecum glucocorticoid secretion and the expression levels of glucocorticoid-related genes and clock genes in the adrenal gland were disrupted with an HFS diet. These results suggest that an HFS diet alters locomotor activity, disrupts circadian rhythms of glucocorticoid secretion, and downregulates peripheral adrenal gland circadian clock genes., Competing Interests: The authors have declared that no competing interests exist.

In mammals, the timing of behavior and physiological activity is controlled by the suprachiasmatic nucleus (SCN) in the hypothalamus. Incidentally, secretin is a peptide hormone that promotes digestive activities and regulates water reabsorption. In recent studies, exogenous administration of secretin has been reported to induce secretion of oxytocin in the supraoptic nucleus of the hypothalamus and modulate social behavior. These results indicate that secretin is involved in the neural network that controls social behavior and plays important roles in the central nervous system. In the present study, we investigated the effects of secretin on circadian rhythms, by assessing circadian rhythms during wheel-running behavior in secretin receptor-deficient (Sctr -/- ) mice. Male adult wild-type (WT) and Sctr -/- mice were housed in separate cages containing a wheel. Every minute of the wheel-running activity was monitored during the normal light-dark (LD) cycle (12:12 h) and in constant darkness (DD). Significant differences were observed in the free-running period between the WT and Sctr -/- mice. However, no significant differences were observed in the daily wheel-running revolutions between WT and Sctr -/- mice, in the LD and DD conditions. Moreover, the ratio of the daily activity phase to the rest phase (α/ρ) was significantly smaller in Sctr -/- than that in WT mice in the DD condition. Secretin receptors were expressed in the SCN cells. These findings suggest that secretin receptors are involved in the central circadian clock in the SCN and the circadian system in general., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: Light-emitting diodes that emit high-intensity blue light are associated with blue-light hazard. Here, we report that blue light disturbs circadian rhythms by interfering with the clock gene in the suprachiasmatic nucleus (SCN) and that suppression of blue light at night ameliorates metabolic abnormalities by controlling circadian rhythms., Methods: C57BL/6J mice were exposed to 10-lux light for 30 minutes at Zeitgeber time 14 for light pulse with blue light or blue-light cut light to induce phase shift of circadian rhythms. Phase shift, clock gene expression in SCN, and metabolic parameters were analyzed. In the clinical study, healthy participants wore blue-light shield eyewear for 2 to 3 hours before bed. Anthropometric data analyses, laboratory tests, and sleep quality questionnaires were performed before and after the study., Results: In mice, phase shift induced with a blue-light cut light pulse was significantly shorter than that induced with a white light pulse. The phase of Per2 expression in the SCN was also delayed after a white light pulse. Moreover, blood glucose levels 48 hours after the white light pulse were higher than those after the blue-cut light pulse. Irs2 expression in the liver was decreased with white light but significantly recovered with the blue-cut light pulse. In a clinical study, after 1 month of wearing blue-light shield eyeglasses, there were improvements in fasting plasma glucose levels, insulin resistance, and sleep quality., Conclusions: Our results suggest that suppression of blue light at night effectively maintains circadian rhythms and metabolism.

Serum testosterone concentration decreases with age in humans and rodents. Accordingly, old male mice show changes in locomotor activity rhythms: a lengthened free-running period and decreased activity levels among others. To investigate whether testosterone replacement improves the age-related decline in circadian rhythmicity, we examined the effects of testosterone on the circadian rhythms of wheel running activity in old male mice. Intact male C57BL/6J mice (18-22 months old) were subcutaneously implanted with silicone tubes packed with testosterone propionate (TP) or cholesterol. TP treatment significantly decreased the daily wheel running revolutions in a normal light/dark (LD) cycle and in constant darkness (DD), but did not affect the free-running period. The same experiment performed on young male gonadectomized mice (3-5 months old) demonstrated that TP treatment significantly increased activity levels in both LD and DD. These results suggest that testosterone replacement exacerbates the age-related decline in circadian rhythmicity.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi-functional enzyme. In this study, we analysed its role in lipopolysaccharide-induced inflammatory response in wild-type and ERAP1-knockout mice. Following lipopolysaccharide injection, ERAP1 was secreted into the blood, increasing leucine aminopeptidase activity and NO synthesis therein. Among the amino acids tested, arginine concentration was significantly increased in wild-type mice compared to ERAP1-knockout mice. These results suggest that ERAP1 behaves similar to acute-phase proteins, which are secreted into the blood in response to infectious/inflammatory stimuli and are involved in enhancing NO synthesis as a host defense mechanism.

The photic entrainment system is critical for the internal circadian clock to be synchronized by external time cues. In nocturnal rodents, exposure to light during the early subjective night causes a phase delay, whereas it causes a phase advance during the late subjective night. This is represented by a phase-response curve (PRC). The PRC of females has not been well studied due to their estrous cycles. Our aim in this study was to understand the characteristics of photic entrainment in female cycling rodents and identify differences in photic entrainment among the stages of the estrous cycle. To establish two types of PRC, immediate PRC (iPRC) and steady state PRC (ssPRC), in each stage of the estrous cycle, we recorded circadian rhythms of wheel running activity, applying a 15-min light pulse to cycling female mice in constant darkness. In the iPRC, which was evaluated on the next day of the light pulse, the amount of phase shift in the diestrus was larger than that in the metestrus stage at circadian time (CT) 2. Similarly, the amount of phase shift in metestrus was larger than that in proestrus at CT 10. In the ssPRC, which was evaluated after completion of a new steady state, no significant estrous variations in the amount of photic phase shifts were detected for any CTs. Although these results indicate that the intrinsic photic entrainment system is not influenced by the estrous cycle, it may affect photoreception and cause sudden behavioral changes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ability of the circadian clock to adapt to environmental changes is critical for maintaining homeostasis, preventing disease, and limiting the detrimental effects of aging. To date, little is known about age-related changes in the entrainment of peripheral clocks to external cues. We therefore evaluated the ability of the peripheral clocks of the kidney, liver, and submandibular gland to be entrained by external stimuli including light, food, stress, and exercise in young versus aged mice using in vivo bioluminescence monitoring. Despite a decline in locomotor activity, peripheral clocks in aged mice exhibited normal oscillation amplitudes under light-dark, constant darkness, and simulated jet lag conditions, with some abnormal phase alterations. However, age-related impairments were observed in peripheral clock entrainment to stress and exercise stimuli. Conversely, age-related enhancements were observed in peripheral clock entrainment to food stimuli and in the display of food anticipatory behaviors. Finally, we evaluated the hypothesis that deficits in sympathetic input from the central clock located in the suprachiasmatic nucleus of the hypothalamus were in part responsible for age-related differences in the entrainment. Aged animals showed an attenuated entrainment response to noradrenergic stimulation as well as decreased adrenergic receptor mRNA expression in target peripheral organs. Taken together, the present findings indicate that age-related circadian disorganization in entrainment to light, stress, and exercise is due to sympathetic dysfunctions in peripheral organs, while meal timing produces effective entrainment of aged peripheral circadian clocks., Competing Interests: The authors declare no conflict of interest.

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.

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15-17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17-19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Circadian timing systems, like most physiological processes, cannot escape the effects of aging. With age, humans experience decreased duration and quality of sleep. Aged mice exhibit decreased amplitude and increased fragmentation of the activity rhythm, and lengthened circadian free-running period in both light-dark (LD) and constant dark (DD) conditions. Several studies have shown that aging impacts neural activity rhythms in the central circadian clock in the suprachiasmatic nucleus (SCN). However, evidence for age-related disruption of circadian oscillations of clock genes in the SCN has been equivocal. We hypothesized that daily exposure to LD cycles masks the full impact of aging on molecular rhythms in the SCN. We performed ex vivo bioluminescent imaging of cultured SCN slices of young and aged PER2::luciferase knock-in (PER2::LUC) mice housed under LD or prolonged DD conditions. Under LD conditions, the amplitude of PER2::LUC rhythms differed only slightly between SCN explants from young and aged animals; under DD conditions, the PER2::LUC rhythms of aged animals showed markedly lower amplitudes and longer circadian periods than those of young animals. Recordings of PER2::LUC rhythms in individual SCN cells using an electron multiplying charge-coupled device camera revealed that aged SCN cells showed longer circadian periods and that the rhythms of individual cells rapidly became desynchronized. These data suggest that aging degrades the SCN circadian ensemble, but that recurrent LD cycles mask these effects. We propose that these changes reflect a decline in pacemaker robustness that could increase vulnerability to environmental challenges, and partly explain age-related sleep and circadian disturbances.

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)-γ and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Macrophages play an important role in host defense under several immunological, inflammatory, and/or infectious conditions. In our previous work, we demonstrated that endoplasmic reticulum aminopeptidase 1 (ERAP1) was secreted from macrophages in response to LPS and IFN-γ, and it enhanced their phagocytic activity. In this study, we analyzed the mechanism of LPS/IFN-γ-induced ERAP1 secretion. LPS/IFN-γ-induced secretion of the enzyme from the murine macrophage cell line RAW264.7 was suppressed by polymyxin B. Several agonists of TLRs, such as Pam3CSK4, FSL-1, and ODN1826, induced its secretion. In contrast, neutralizing Abs to IFN-β and TNF-α receptor type 1 suppressed its secretion. Using murine peritoneal macrophages derived from TNF-α and type 1 IFNR knockout mice, we confirmed the involvement of these two cytokines in ERAP1 secretion. In addition, secretion of ERAP1 from both RAW264.7 cells and murine peritoneal macrophages was induced by A23187 and thapsigargin and inhibited by BAPTA-AM and the calmodulin inhibitor W7. These results suggest that LPS/IFN-γ-induced secretion of ERAP1 is mediated by TLRs via induction of intermediate cytokines such as IFN-β and TNF-α, which in turn lead to enhanced cytosolic Ca(2+) levels and calmodulin activation.

Elderly people often develop sleep and autonomic dysfunctions, which are regulated by circadian rhythm. Recently, we reported on the degradation of neural output from the central circadian clock in the suprachiasmatic nucleus (SCN) with aging. However, it is likely that many other factors contribute to the age-related decline in the functioning of the circadian system. In this study, we examined the effects of dopaminergic neuronal loss in the substantia nigra (SN) on circadian rhythms of mice to assess whether age-related degeneration of the dopamine system influences circadian rhythm. Young male C57BL/6J mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that selectively destroys dopaminergic neurons in the SN, and their wheel-running activities were recorded. We observed that MPTP-treated mice lost 43% of their dopaminergic neurons in the SN (on average) and demonstrated longer period of wheel-running activity rhythm in constant darkness compared with control mice. However, all the remaining circadian parameters in the MPTP-treated mice remained constant. Our findings suggest that in addition to SCN output dysfunction, age-related degeneration in the dopamine system of the brain leads to circadian rhythm irregularities., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Disruptions in sleep/wake cycles, including decreased amplitude of rhythmic behaviors and fragmentation of the sleep episodes, are commonly associated with aging in humans and other mammals. While there are undoubtedly many factors contributing to these changes, a body of literature is emerging, suggesting that an age-related decline in the central circadian clock in the suprachiasmatic nucleus (SCN) may be a key element responsible. To explore age-related changes in the SCN, we have performed in vivo multiunit neural activity (MUA) recordings from the SCN of freely moving young (3-5 months) and middle-aged (13-18 months) mice. Importantly, the amplitude of day-night difference in MUA was significantly reduced in the older mice. We also found that the neural activity rhythms are clearly degraded in the subparaventricular zone, one of the main neural outputs of the SCN. Surprisingly, parallel studies indicate that the molecular clockwork in the SCN as measured by PER2 exhibited only minor deficits at this same age. Thus, the circadian output measured at the level of neural activity rhythms in the SCN is degraded by aging, and this decline occurs before the disruption of key components of the molecular clockwork.

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day-night difference was not observed in Cry-deficient mice. In addition, we observed day-night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals., (© 2011 Nakamura et al.)

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression., (Copyright 2010 Elsevier Inc. All rights reserved.)

Previously, we have shown that mice deficient in either vasoactive intestinal peptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit specific deficits in the behavioral response of their circadian system to light. In this study, we investigated how the photic regulation of the molecular clock within the suprachiasmatic nucleus (SCN) is altered by the loss of these closely-related peptides. During the subjective night, the magnitude of the light-induction of FOS and phosphorylated mitogen-activated protein kinase (p-MAPK) immunoreactive cells within the SCN was significantly reduced in both VIP- and PACAP-deficient mice when compared with wild-type mice. The photic induction of the clock gene Period1 (Per1) in the SCN was reduced in the VIP- but not in the PACAP-deficient mice. Baselines levels of FOS, p-MAPK or Per1 in the night were not altered by the loss of these peptides. In contrast, during the subjective day, light exposure increased the levels of FOS, p-MAPK and Per1 in the SCN of VIP-deficient mice, but not in the other genotypes. During this phase, baseline levels of these markers were reduced in the VIP-deficient mice compared with untreated controls. Finally, the loss of either neuropeptide reduced the magnitude of the light-evoked increase in Per1 levels in the adrenals in the subjective night without any change in baseline levels. In summary, our results indicate that both VIP and PACAP regulate the responsiveness of cells within the SCN to the effects of light. Furthermore, VIP, but not PACAP, is required for the appropriate temporal gating of light-induced gene expression within the SCN.

Fluctuations in circulating estrogen and progesterone levels associated with the estrous cycle alter circadian rhythms of physiology and behavior in female rodents. Endogenously applied estrogen shortens the period of the locomotor activity rhythm in rodents. We recently found that estrogen implants affect Period (Per) gene expression in the suprachiasmatic nucleus (SCN; central clock) and uterus of rats in vivo. To explore whether estrogen directly influences the circadian clock in the SCN and/or tissues of the reproductive system, we examined the effects of 17beta-estradiol (E(2)) on PER2::LUCIFERASE (PER2::LUC) expression in tissue explant cultures from ovariectomized PER2::LUC knockin mice. E(2) applied to explanted cultures shortened the period of rhythmic PER2::LUC expression in the uterus but did not change the period of PER2::LUC expression in the SCN. Raloxifene, a selective estrogen receptor modulator and known E(2) antagonist in uterine tissues, attenuated the effect of E(2) on the period of the PER2::LUC rhythm in the uterus. These data indicate that estrogen directly affects the timing of the molecular clock in the uterus via an estrogen receptor-mediated response.

In mammals, the principal circadian pacemaker driving daily physiology and behavioral rhythms is located in the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. The neural output of SCN is essential for the circadian regulation of behavioral activity. Although remarkable progress has been made in revealing the molecular basis of circadian rhythm generation within the SCN, the output pathways by which the SCN exert control over circadian rhythms are not well understood. Most SCN efferents target the subparaventricular zone (SPZ), which resides just dorsal to the SCN. This output pathway has been proposed as a major component involved in the outflow for circadian regulation. We have examined the downstream pathway of the central clock by means of multiunit neural activity (MUA) in freely moving mice. SCN neural activity is tightly coupled to environmental photic input and anticorrelated with MUA rhythm in the SPZ. In Clock mutant mice exhibiting attenuated circadian locomotor rhythmicity, MUA rhythmicity in the SCN and SPZ is similarly blunted. These results suggest that the SPZ plays a functional role in relaying circadian and photic signals to centers involved in generating behavioral activity.

The molecular basis of seasonal or nonseasonal breeding remains unknown. Although laboratory rats are generally regarded as photoperiod-insensitive species, the testicular weight of the Fischer 344 (F344) strain responds to photoperiod. Recently, it was clarified that photoperiodic regulation of type 2 iodothyronine deiodinase (Dio2) in the mediobasal hypothalamus (MBH) is critical in photoperiodic gonadal regulation. Strain-dependent differences in photoperiod sensitivity may now provide the opportunity to address the regulatory mechanism of seasonality by studying Dio2 expression. Therefore, in the present study, we examined the effect of photoperiod on Dio2 expression in photoperiod-sensitive F344 and photoperiod-insensitive Wistar rats. A statistically significant difference was observed between short and long days in terms of testicular weight and Dio2 expression in the F344 strain, while no difference was observed in the Wistar strain. These results suggest that differential responses of the Dio2 gene to photoperiod may determine the strain-dependent differences in photoperiod sensitivity in laboratory rats.

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.

p-Nonylphenol (NP) and 4-tert-octylphenol (OP) are known to mimic the action of estrogens as endocrine disruptors. However, their acute effects on the pituitary and the hypothalamus functions in vivo have been uncertain. We therefore determined their effects on the anterior pituitary, in particular, gonadotropin secretion. Two weeks after ovariectomy, the rats were given a subcutaneous injection of 10 mg NP, 10 mg OP, 10 mg bisphenol A, 1 microg 17beta-estradiol, or sesame oil alone as control. Twenty-four hours after the treatment, the expression of progesterone receptor mRNA in the anterior pituitary and the level of luteinizing hormone (LH), follicle-stimulating hormone, and prolactin were determined. The expression of progesterone receptor mRNA in the anterior pituitary was significantly increased by either NP, OP, bisphenol A, or estradiol, but bisphenol A was less effective. The level of LH was significantly decreased by either NP or OP, but not by bisphenol A and estradiol. Only estradiol significantly increased the level of prolactin. The level of follicle-stimulating hormone was unchanged by any of the treatments. To check the effects of NP and OP on pulsatile LH secretion, blood samplings were done at 6-min intervals for 3 h. Twenty-four hours after treatment in ovariectomized adult rats, we found that the injection of NP significantly decreased the amplitude of LH pulses and the mean LH concentrations, but not the frequency of LH pulses. The injection of OP significantly decreased the mean LH concentrations without affecting the frequency and amplitude of the LH pulses. Finally, the rats given an injection of NP or sesame oil were intravenously injected with 50 ng of gonadotropin-releasing hormone (GnRH) to check whether NP affected the LH secretory responsiveness of the anterior pituitary to GnRH. We found that the responsiveness to GnRH in NP-injected rats was significantly attenuated compared to the sesame oil-injected rats. The present study suggests that NP, even with a single injection, suppresses the pulsatile LH secretion in adult ovariectomized rats, probably by affecting the anterior pituitary level., (Copyright 2006 S. Karger AG, Basel.)

Although it has long been established that estrogen alters circadian rhythms in behavior, physiology, and reproductive functions in mammals, the molecular mechanism for these effects remains unknown. To explore the possibility that estrogen affects circadian rhythms by changing the expression of clock-related genes, we investigated the effects of chronic treatment with 17beta-estradiol (E2) on the expression of Per1 and Per2 genes in the brain (suprachiasmatic nucleus and cerebral cortex) and periphery (liver, kidney, and uterus) of ovariectomized rats by means of in situ hybridization and northern blotting. In the brain, E2 treatment advanced the peak of Per2 mRNA expression in the SCN; however, it failed to affect the rhythm of Per2 mRNA expression in the CX and Per1 mRNA expression in both the SCN and the CX. In nonreproductive peripheral tissues (liver and kidney), E2 delayed the phase and increased the amplitude of Per1 mRNA expression. In the reproductive tissues (uterus), biphasic rhythms in Per1 and Per2 mRNA were observed after E2 treatment. These findings suggest that the effects of estrogen are different between central and peripheral clock in the brain, and between reproductive and nonreproductive tissues in the periphery.

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin alpha/beta system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin alphas, alpha1 (Rch1), alpha3 (Qip-1), and alpha7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin alpha/beta system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.

To investigate the neural mechanisms underlying the mammalian photic entrainment of circadian rhythms, the response of neuronal extracellular firing activity to retinal light stimulation was investigated in the suprachiasmatic nucleus (SCN) of anesthetized mice during nighttime and daytime. In nighttime, most recorded SCN cells (83%) increased their firing frequency in response to retinal illumination. Some SCN cells (11%) responded by decreasing their firing rate. In daytime, the retinal illumination increased the firing rate in only 26% of the SCN cells, and no response was observed in the remaining cells. The light intensity threshold for the activation of SCN cells at zeitgeber time (ZT) 16 was approximately 3 x 10(11) photons cm(-2)s(-1) and the maximum response was observed at approximately 1 x 10(14) photons cm(-2)s(-1). Therefore, photic response in the firing of mouse SCN cells may be phase-dependent and have a higher threshold, which corresponds to properties of the photic entrainment in locomotor activity of mice.

Alkylphenols, such as p-nonylphenol (NP) and 4-tert-octylphenol (OP) and bisphenol A (BPA) are thought to mimic oestrogens in their action, and are called endocrine disrupters. We examined whether these endocrine disrupters affected progesterone receptor (PR) mRNA expression in the adult female rat neocortex. In one experiment, at 12.00 h, ovariectomized rats were given a subcutaneous injection of 10 mg of NP, 10 mg of OP or 10 mg of BPA, or sesame oil alone as control. Twenty-four hours after injection, the left side of the frontal cortex, parietal cortex and temporal cortex was collected. In a second experiment to study the time-course of the effects of BPA on PR mRNA, the ovariectomized rats were given a subcutaneous injection of 10 mg of BPA and killed 0, 6, 12 and 24 h after injection. In addition to the frontal cortex and temporal cortex, the occipital cortex was also collected. Northern blotting revealed that, in the first experiment, injection of NP, OP or BPA significantly increased PR mRNA expression in the frontal cortex but not in the parietal cortex. In the temporal cortex, BPA significantly decreased PR mRNA, but NP and OP produced no significant changes. The second experiment revealed that, in the frontal cortex, BPA induced a significant increase in PR mRNA expression at 6 h after injection, which lasted until 24 h after injection. In the temporal cortex, PR mRNA expression was significantly decreased 6 h after injection of BPA and was still significantly low 24 h after injection. No significant change was observed in the occipital cortex. These results suggest that, even in adult rats, endocrine disrupters alter the neocortical function by affecting the PR system, although the physiological significance of PR in the affected area is unknown.