In vivo recording of suprachiasmatic nucleus dynamics reveals a dominant role of arginine vasopressin neurons in circadian pacesetting.

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.

Diurnal Variation of Brain Activity in the Human Suprachiasmatic Nucleus.

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.

GABA from vasopressin neurons regulates the time at which suprachiasmatic nucleus molecular clocks enable circadian behavior.

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 GABAA 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 Ca2+ 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.

Circadian clock regulates tear secretion in the lacrimal gland.

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.

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation.

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.

Photic phase-response curves for cycling female mice.

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.

TLR-mediated secretion of endoplasmic reticulum aminopeptidase 1 from macrophages.

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-ß 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-ß and TNF-α, which in turn lead to enhanced cytosolic Ca(2+) levels and calmodulin activation.

Long days restore regular estrous cyclicity in mice lacking circadian rhythms.

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.

Circadian rhythm of PERIOD2::LUCIFERASE expression in the trigeminal ganglion of mice.

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.

Age-related decline in circadian output.

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.

Secretin receptor-deficient mice exhibit robust food anticipatory activity.

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.

Diurnal variations of triglyceride accumulation in mouse and bovine adipocyte-derived cell lines.

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.

Oak extracts modulate circadian rhythms of clock gene expression in vitro and wheel-running activity in mice.

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.

Role of heterozygous and homozygous alleles in cryptochrome-deficient mice.

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.

In vivo monitoring of circadian timing in freely moving mice.

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.

Electrophysiological Approaches to Studying the Suprachiasmatic Nucleus.

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.

Chronic methamphetamine uncovers a circadian rhythm in multiple-unit neural activity in the dorsal striatum which is independent of the suprachiasmatic nucleus.

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.

Diurnal Variation in Trigeminal Pain Sensitivity in Mice.

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.

The role of the neuropeptides PACAP and VIP in the photic regulation of gene expression in the suprachiasmatic nucleus.

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.

Modeling circadian regulation of ovulation timing: age-related disruption of estrous cyclicity.

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.