Mammalian Circadian System Research Articles

Photoneuroendocrine, circadian and seasonal systems: from photoneuroendocrinology to circadian biology and medicine.

This contribution highlights the scientific development of two intertwined disciplines, photoneuroendocrinology and circadian biology. Photoneuroendocrinology has focused on nonvisual photoreceptors that translate light stimuli into neuroendocrine signals and serve rhythm entrainment. Nonvisual photoreceptors first described in the pineal complex and brain of nonmammalian species are luminance detectors. In the pineal, they control the formation of melatonin, the highly conserved hormone of darkness which is synthesized night by night. Pinealocytes endowed with both photoreceptive and neuroendocrine capacities function as "photoneuroendocrine cells." In adult mammals, nonvisual photoreceptors controlling pineal melatonin biosynthesis and pupillary reflexes are absent from the pineal and brain and occur only in the inner layer of the retina. Encephalic photoreceptors regulate seasonal rhythms, such as the reproductive cycle. They are concentrated in circumventricular organs, the lateral septal organ and the paraventricular organ, and represent cerebrospinal fluid contacting neurons. Nonvisual photoreceptors employ different photopigments such as melanopsin, pinopsin, parapinopsin, neuropsin, and vertebrate ancient opsin. After identification of clock genes and molecular clockwork, circadian biology became cutting-edge research with a focus on rhythm generation. Molecular clockworks tick in every nucleated cell and, as shown in mammals, they drive the expression of more than 3000 genes and are of overall importance for regulation of cell proliferation and metabolism. The mammalian circadian system is hierarchically organized; the central rhythm generator is located in the suprachiasmatic nuclei which entrain peripheral circadian oscillators via multiple neuronal and neuroendocrine pathways. Disrupted molecular clockworks may cause various diseases, and investigations of this interplay will establish a new discipline: circadian medicine.

Mouse Models in Circadian Rhythm and Melatonin Research.

This contribution reviews the role of inbred and transgenic mouse strains for deciphering the mammalian melatoninergic and circadian system. It focusses on the pineal organ as melatonin factory and two major targets of the melatoninergic system, the suprachiasmatic nuclei (SCN) and the hypophysial pars tuberalis (PT). Mammalian pinealocytes sharing molecular characteristics with true pineal and retinal photoreceptors synthesize and secrete melatonin into the blood and cerebrospinal fluid night by night. Notably, neuron-like connections exist between the deep pinealocytes and the habenular/pretectal region suggesting direct pineal-brain communication. Control of melatonin biosynthesis in rodents involves transcriptional regulation including phosphorylation of CREB and upregulation of mPer1. In the SCN, melatonin acts upon MT1 and MT2 receptors. Melatonin is not necessary to maintain the rhythm of the SCN molecular clockwork, but it has distinct effects on the synchronization of the circadian rhythm by light, facilitates re-entrainment of the circadian system to phase advances in the level of the SCN molecular clockwork by acting upon MT2 receptors and plays a stabilizing role in the circadian system as evidenced from locomotor activity recordings. While the effects in the SCN are subtle, melatonin is essential for PT functions. Via the MT1 receptor it drives the PT-intrinsic molecular clockwork and the retrograde and anterograde output pathways controlling seasonal rhythmicity. Although inbred and transgenic mice do not show seasonal reproduction, the pathways from the PT are fully intact if the animals are melatonin proficient. Thus, only melatonin-proficient strains are suited to investigate the circadian and melatoninergic systems.

Steven A. Brown and the synchronization of circadian rhythms by body temperature cycles.

The mammalian circadian timing system has a hierarchical architecture, with a central pacemaker located in the brain's suprachiasmatic nucleus orchestrating rhythms in behaviour and physiology. In cooperation with environmental cycles, it synchronizes the phases of peripheral oscillators operating in most cells of the body. Even cells kept in tissue culture harbour self-sustained and cell-autonomous circadian clocks that keep ticking throughout their lifespan. The master pacemaker in the suprachiasmatic nucleus is synchronized primarily by light-dark cycles, whereas peripheral oscillators are phase entrained by a multitude of systemic signalling pathways. These include pathways depending on feeding-fasting cycles, cellular actin polymerization dynamics, endocrine rhythms and, surprisingly, body temperature oscillations. Using tissue culture and murine models, Steve Brown was the first one to demonstrate that shallow rhythms of mammalian body temperature are timing cues (zeitgebers) for peripheral circadian clocks.

Behavior and physiology in female Cricetulus barabensis are associated with the expression of circadian genes.

The circadian clock regulates the behavior, physiology, and metabolism of mammals, and these characteristics, such as sleep-wake cycles, exercise capacity, and hormone levels, exhibit circadian rhythms. Light signaling is the main stimulator of the mammalian circadian system. The photoperiod regulates the reproductive cycle of seasonal breeding animals, and the circadian clock plays a pivotal role in this process. However, the role of the clock in coordinating animal behavior and physiology in response to photoperiodic changes needs further investigation. The present study investigated the changes and correlation of behavioral activities, physiological indicators, and gene expression in female striped hamsters (Cricetulus barabensis) within 24 h under a 12L:12D photoperiod. We found that the daily rhythms of sleep-wake and open field were significant in hamsters. The expression of clock genes, melatonin receptor genes, and genes involved in general metabolism oscillated significantly in central and peripheral tissues (brain, hypothalamus, liver, ovary, and thymus) and was significantly associated with behavior and physiology. Our results revealed that the neuroendocrine system regulated the rhythmicity of behavior and physiology, and central and peripheral clock genes (Bmal1, Clock, Per1, Per2, Cry1, and Cry2), melatonin receptor genes (MT1, MT2, and GPR50), and metabolizing genes (SIRT1, FGF21, and PPARα) played important roles. Our results suggest that central and peripheral circadian clocks, melatonin receptors, and genes involved in general metabolism may play key roles in maintaining circadian behavior and metabolic homeostasis in striped hamsters. Our results may have important implication for rodent pest control.

A minimal model of peripheral clocks reveals differential circadian re-entrainment in aging.

The mammalian circadian system comprises a network of endogenous oscillators, spanning from the central clock in the brain to peripheral clocks in other organs. These clocks are tightly coordinated to orchestrate rhythmic physiological and behavioral functions. Dysregulation of these rhythms is a hallmark of aging, yet it remains unclear how age-related changes lead to more easily disrupted circadian rhythms. Using a two-population model of coupled oscillators that integrates the central clock and the peripheral clocks, we derive simple mean-field equations that can capture many aspects of the rich behavior found in the mammalian circadian system. We focus on three age-associated effects that have been posited to contribute to circadian misalignment: attenuated input from the sympathetic pathway, reduced responsiveness to light, and a decline in the expression of neurotransmitters. We find that the first two factors can significantly impede re-entrainment of the clocks following perturbation, while a weaker coupling within the central clock does not affect the recovery rate. Moreover, using our minimal model, we demonstrate the potential of using the feed-fast cycle as an effective intervention to accelerate circadian re-entrainment. These results highlight the importance of peripheral clocks in regulating the circadian rhythm and provide fresh insights into the complex interplay between aging and the resilience of the circadian system.

Effects of the neonatal intensive care environment on circadian health and development of preterm infants.

The circadian system in mammals ensures adaptation to the light-dark cycle on Earth and imposes 24-h rhythmicity on metabolic, physiological and behavioral processes. The central circadian pacemaker is located in the brain and is entrained by environmental signals called Zeitgebers. From here, neural, humoral and systemic signals drive rhythms in peripheral clocks in nearly every mammalian tissue. During pregnancy, disruption of the complex interplay between the mother's rhythmic signals and the fetal developing circadian system can lead to long-term health consequences in the offspring. When an infant is born very preterm, it loses the temporal signals received from the mother prematurely and becomes totally dependent on 24/7 care in the Neonatal Intensive Care Unit (NICU), where day/night rhythmicity is usually blurred. In this literature review, we provide an overview of the fetal and neonatal development of the circadian system, and short-term consequences of disruption of this process as occurs in the NICU environment. Moreover, we provide a theoretical and molecular framework of how this disruption could lead to later-life disease. Finally, we discuss studies that aim to improve health outcomes after preterm birth by studying the effects of enhancing rhythmicity in light and noise exposure.

The Mammalian Circadian Time-Keeping System.

Our physiology and behavior follow precise daily programs that adapt us to the alternating opportunities and challenges of day and night. Under experimental isolation, these rhythms persist with a period of approximately one day (circadian), demonstrating their control by an internal autonomous clock. Circadian time is created at the cellular level by a transcriptional/translational feedback loop (TTFL) in which the protein products of the Period and Cryptochrome genes inhibit their own transcription. Because the accumulation of protein is slow and delayed, the system oscillates spontaneously with a period of ∼24 hours. This cell-autonomous TTFL controls cycles of gene expression in all major tissues and these cycles underpin our daily metabolic programs. In turn, our innumerable cellular clocks are coordinated by a central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. When isolated in slice culture, the SCN TTFL and its dependent cycles of neural activity persist indefinitely, operating as "a clock in a dish". In vivo, SCN time is synchronized to solar time by direct innervation from specialized retinal photoreceptors. In turn, the precise circadian cycle of action potential firing signals SCN-generated time to hypothalamic and brain stem targets, which co-ordinate downstream autonomic, endocrine, and behavioral (feeding) cues to synchronize and sustain the distributed cellular clock network. Circadian time therefore pervades every level of biological organization, from molecules to society. Understanding its mechanisms offers important opportunities to mitigate the consequences of circadian disruption, so prevalent in modern societies, that arise from shiftwork, aging, and neurodegenerative diseases, not least Huntington's disease.

Cry1 expression during postnatal development is critical for the establishment of normal circadian period.

The mammalian circadian system generates an approximate 24-h rhythm through a complex autoregulatory feedback loop. Four genes, Period1 (Per1), Period2 (Per2), Cryptochrome1 (Cry1), and Cryptochrome2 (Cry2), regulate the negative feedback within this loop. Although these proteins have distinct roles within the core circadian mechanism, their individual functions are poorly understood. Here, we used a tetracycline trans-activator system (tTA) to examine the role of transcriptional oscillations in Cry1 and Cry2 in the persistence of circadian activity rhythms. We demonstrate that rhythmic Cry1 expression is an important regulator of circadian period. We then define a critical period from birth to postnatal day 45 (PN45) where the level of Cry1 expression is critical for setting the endogenous free running period in the adult animal. Moreover, we show that, although rhythmic Cry1 expression is important, in animals with disrupted circadian rhythms overexpression of Cry1 is sufficient to restore normal behavioral periodicity. These findings provide new insights into the roles of the Cryptochrome proteins in circadian rhythmicity and further our understanding of the mammalian circadian clock.

Mitochondrial function and E2 synthesis are impaired following alteration of CLOCK gene expression in porcine ovarian granulosa cells

Circadian locomotor output cycles kaput (CLOCK) is a critical component of the mammalian circadian clock system and regulates ovarian physiology. However, the functions and mechanisms of CLOCK in porcine granulosa cells (GCs) are poorly understood. The present study focused on CLOCK's effects on estradiol synthesis. Similarity analysis showed that CLOCK is highly conserved between pigs and other species. The phylogenetic tree analysis indicated that porcine CLOCK was most closely related to that in Arabian camels. CLOCK significantly reduced E2 synthesis in GCs. CLOCK reduced the expression of steroidogenesis-related genes at the mRNA and protein levels, including CYP19A1, CYP11A1, and StAR. CYP17A1 levels were significantly downregulated. We demonstrated that CLOCK dramatically decreased ATP content, mitochondrial copy number, and mitochondrial membrane potential (MMP) and increased reactive oxygen species levels in GCs. We observed that mitochondria were severely damaged with fuzzy and fractured cristae and swollen matrix. These findings suggest that mitochondrial function and E2 synthesis are impaired following the alteration of CLOCK gene expression in porcine ovarian GCs.

Circadian rhythms and the liver.

This narrative review briefly describes the mammalian circadian timing system, the specific features of the liver clock, also by comparison with other peripheral clocks, the role of the liver clock in the preparation of food intake, and its relationship with energy metabolism. It then goes on to provide a chronobiological perspective of the pathophysiology and management of several types of liver disease, with a particular focus on metabolic-associated fatty liver disease (MAFLD), decompensated cirrhosis and liver transplantation. Finally, it provides some insight into the potential contribution of circadian principles and circadian hygiene practices in preventing MAFLD, improving the prognosis of advanced liver disease and modulating liver transplantation outcomes.

Strain and Age Dependent Entrainable Range of Circadian Behavior in C57BL/6 and BALB/c Mice

The mammalian circadian system has a plasticity in a certain range, rather than a strict 24-hour cycle, with considerable variations among species, strains, and ages. As the most widely used mouse strains in circadian research, C57BL/6 and BALB/c mice were well known to have different internal periods and responses to various non-24-hour light-dark cycles. However, their entrainable range of circadian behavior was not specifically studied, neither was the effect of aging. Besides, it is not well known if mice with appeared behavioral adaptation are really healthy. In the current study, we exposed C57BL/6 and BALB/c mice at 3 months and 18 months old to a series of short (T cycles < 24 h) and long (T cycles > 24 h) light-dark cycles. Wheel running activities were monitored continuously for calculation of the entrainable range and glucose homeostasis was investigated to reflect their health status. Our results showed that the range in both young and old C57BL/6 mice is between T23 and T26. By contrast, due to the strong adaptability to extreme LD cycles, the entrainable range on a circadian scale in both young and old BALB/c mice cannot be well determined. Despite the adaptation appeared at the behavioral level, glucose homeostasis revealed by glucose tolerance test and insulin tolerance test was impaired in mice upon T cycle treatment. In summary, our study explored the entrainment range in two popular mouse strains and suggested that behavioral adaptation may not well reflect their health status.

Circadian molecular clock disruption in chronic pulmonary diseases.

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future.

Circadian rhythm of lipid metabolism.

Lipids comprise a diverse group of metabolites that are indispensable as energy storage molecules, cellular membrane components and mediators of inter- and intra-cellular signaling processes. Lipid homeostasis plays a crucial role in maintaining metabolic health in mammals including human beings. A growing body of evidence suggests that the circadian clock system ensures temporal orchestration of lipid homeostasis, and that perturbation of such diurnal regulation leads to the development of metabolic disorders comprising obesity and type 2 diabetes. In view of the emerging role of circadian regulation in maintaining lipid homeostasis, in this review, we summarize the current knowledge on lipid metabolic pathways controlled by the mammalian circadian system. Furthermore, we review the emerging connection between the development of human metabolic diseases and changes in lipid metabolites that belong to major classes of lipids. Finally, we highlight the mechanisms underlying circadian organization of lipid metabolic rhythms upon the physiological situation, and the consequences of circadian clock dysfunction for dysregulation of lipid metabolism.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

The Ventral Tegmental Area and Nucleus Accumbens as Circadian Oscillators: Implications for Drug Abuse and Substance Use Disorders.

Circadian rhythms convergently evolved to allow for optimal synchronization of individuals’ physiological and behavioral processes with the Earth’s 24-h periodic cycling of environmental light and temperature. Whereas the suprachiasmatic nucleus (SCN) is considered the primary pacemaker of the mammalian circadian system, many extra-SCN oscillatory brain regions have been identified to not only exhibit sustainable rhythms in circadian molecular clock function, but also rhythms in overall region activity/function and mediated behaviors. In this review, we present the most recent evidence for the ventral tegmental area (VTA) and nucleus accumbens (NAc) to serve as extra-SCN oscillators and highlight studies that illustrate the functional significance of the VTA’s and NAc’s inherent circadian properties as they relate to reward-processing, drug abuse, and vulnerability to develop substance use disorders (SUDs).

Transcriptional Feedback Loops in the Caprine Circadian Clock System.

The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway

Context The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. Objective We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. Materials and methods We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. Results We found that Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. Discussion and conclusions Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

The Effects of Light and the Circadian System on Rhythmic Brain Function

Life on earth has evolved under the influence of regularly recurring changes in the environment, such as the 24 h light/dark cycle. Consequently, organisms have developed endogenous clocks, generating 24 h (circadian) rhythms that serve to anticipate these rhythmic changes. In addition to these circadian rhythms, which persist in constant conditions and can be entrained to environmental rhythms, light drives rhythmic behavior and brain function, especially in nocturnal laboratory rodents. In recent decades, research has made great advances in the elucidation of the molecular circadian clockwork and circadian light perception. This review summarizes the role of light and the circadian clock in rhythmic brain function, with a focus on the complex interaction between the different components of the mammalian circadian system. Furthermore, chronodisruption as a consequence of light at night, genetic manipulation, and neurodegenerative diseases is briefly discussed.

Adult Neurogenesis under Control of the Circadian System.

The mammalian circadian system is a hierarchically organized system, which controls a 24-h periodicity in a wide variety of body and brain functions and physiological processes. There is increasing evidence that the circadian system modulates the complex multistep process of adult neurogenesis, which is crucial for brain plasticity. This modulatory effect may be exercised via rhythmic systemic factors including neurotransmitters, hormones and neurotrophic factors as well as rhythmic behavior and physiology or via intrinsic factors within the neural progenitor cells such as the redox state and clock genes/molecular clockwork. In this review, we discuss the role of the circadian system for adult neurogenesis at both the systemic and the cellular levels. Better understanding of the role of the circadian system in modulation of adult neurogenesis can help develop new treatment strategies to improve the cognitive deterioration associated with chronodisruption due to detrimental light regimes or neurodegenerative diseases.

Real-Time Monitoring of Circadian Rhythms in the Eye.

The mammalian eye harbors a full circadian system that controls several physiologically relevant functions within this organ. During the last two decades a few laboratories have developed transgenic animal models in which circadian rhythms can be monitored in real time using luciferase activity. The most famous transgenic mouse to record bioluminescence rhythms from different tissues and organs is the PERIOD2::LUCIFERASE (PER2::LUC) mouse developed by the Takahashi laboratory in early 2000. Since then, several studies have used this mouse model to dissect the mammalian circadian system by monitoring the circadian rhythm in the brain, the eye, and in many other peripheral organs and tissues. This chapter describes the methodology to record and analyze bioluminescence rhythms from the retina, retinal pigment epithelium, and cornea of PER2::LUC mice.