Search

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. [ABSTRACT FROM AUTHOR]

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2-null controls expressing EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCNCry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS d power and expression of REMS within total sleep (TS). Wakefulness in SCNCon mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCNCry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS d power, although the SCNCon mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were comparable to WTs. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep. [ABSTRACT FROM AUTHOR]

Almost every facet of our behavior and physiology varies predictably over the course of day and night, anticipating and adapting us to their associated opportunities and challenges. These rhythms are driven by endogenous biological clocks that, when deprived of environmental cues, can continue to oscillate within a period of approximately 1 day, hence circa-dian. Normally, retinal signals synchronize them to the cycle of light and darkness, but disruption of circadian organization, a common feature of modern lifestyles, carries considerable costs to health. Circadian timekeeping pivots around a cell-autonomous molecular clock, widely expressed across tissues. These cellular timers are in turn synchronized by the principal circadian clock of the brain: the hypothalamic suprachiasmatic nucleus (SCN). Intercellular signals make the SCN network a very powerful pacemaker. Previously, neurons were considered the sole SCN timekeepers, with glial cells playing supportive roles. New discoveries have revealed, however, that astrocytes are active partners in SCN network timekeeping, with their cell-autonomous clock regulating extracellular glutamate and GABA concentrations to control circadian cycles of SCN neuronal activity. Here, we introduce circadian timekeeping at the cellular and SCN network levels before focusing on the contributions of astrocytes and their mutual interaction with neurons in circadian control in the brain. [ABSTRACT FROM AUTHOR]

Circadian (approximately daily) rhythms of physiology and behaviour adapt organisms to the alternating environments of day and night. The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian timekeeper of mammals. The mammalian cell-autonomous circadian clock is built around a self-sustaining transcriptional-translational negative feedback loop (TTFL) in which the negative regulators Per and Cry suppress their own expression, which is driven by the positive regulators Clock and Bmal1. Importantly, such TTFL-based clocks are present in all major tissues across the organism, and the SCN is their central co-ordinator. First, we analyse SCN timekeeping at the cell-autonomous and the circuit-based levels of organisation. We consider how molecular-genetic manipulations have been used to probe cell-autonomous timing in the SCN, identifying the integral components of the clock. Second, we consider new approaches that enable real-time monitoring of the activity of these clock components and clock-driven cellular outputs. Finally, we review how intersectional genetic manipulations of the cell-autonomous clockwork can be used to determine how SCN cells interact to generate an ensemble circadian signal. Critically, it is these network-level interactions that confer on the SCN its emergent properties of robustness, light-entrained phase and precision— properties that are essential for its role as the central co-ordinator. Remaining gaps in knowledge include an understanding of how the TTFL proteins behave individually and in complexes: whether particular SCN neuronal populations act as pacemakers, and if so, by which signalling mechanisms, and finally the nature of the recently discovered role of astrocytes within the SCN network. Image 1 • The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals. • The SCN cell-autonomous clock is a transcription-translation negative feedback loop. • Real-time imaging provides a window onto cellular and circuit-level timekeeping. • Critical emergent properties of the SCN network arise by intercellular interactions. • We do not know how clock proteins behave or how SCN cells interact to define time. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non- Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. [ABSTRACT FROM AUTHOR]

Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting have been shown to be largely dependent on both membrane depolarization and intracellular second-messenger signaling. In both of these processes, voltage-gated calcium channels (VGCCs) mediate voltage-dependent calcium influx, which propagates neural impulses by stimulating vesicle fusion and instigates intracellular pathways resulting in clock gene expression. Through the cumulative actions of these processes, the phase of the internal clock is modified to match the light cycle of the external environment. To parse out the distinct roles of the L-type VGCCs, we analyzed mice deficient in Cav1.2 (Cacna1c) in brain tissue. We found that mice deficient in the Cav1.2 channel exhibited a significant reduction in their ability to phase-advance circadian behavior when subjected to a light pulse in the late night. Furthermore, the study revealed that the expression of Cav1.2 mRNA was rhythmic (peaking during the late night) and was regulated by the circadian clock component REV-ERBα. Finally, the induction of clock genes in both the early and late subjective night was affected by the loss of Cav1.2, with reductions in Per2 and Per1 in the early and late night, respectively. In sum, these results reveal a role of the L-type VGCC Cav1.2 in mediating both clock gene expression and phase advances in response to a light pulse in the late night. [ABSTRACT FROM PUBLISHER]

The possible signaling role of prokineticin 2 (PK2) and its receptor, prokineticin receptor 2 (PKR2), on female reproduction was investigated. First, the expression of PKR2 and its co-localization with estrogen receptor (ERα) in the hypothalamus was examined. Sexually dimorphic expression of PKR2 in the preoptic area of the hypothalamus was observed. Compared to the male mice, there was more widespread PKR2 expression in the preoptic area of the hypothalamus in the female mice. The likely co-expression of PKR2 and ERα in the preoptic area of the hypothalamus was observed. The estrous cycles in female PK2-null, and PKR2-null heterozygous mice, as well as in PK2-null and PKR2-null compound heterozygous mice were examined. Loss of one copy of PK2 or PKR2 gene caused elongated and irregular estrous cycle in the female mice. The alterations in the estrous cycle were more pronounced in PK2-null and PKR2-null compound heterozygous mice. Consistent with these observations, administration of a small molecule PK2 receptor antagonist led to temporary blocking of estrous cycle at the proestrous phase in female mice. The administration of PKR2 antagonist was found to blunt the circulating LH levels. Taken together, these studies indicate PK2 signaling is required for the maintenance of normal female estrous cycles. [ABSTRACT FROM AUTHOR]

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2-/-) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2-/- mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2-/- mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock. [ABSTRACT FROM AUTHOR]

Sleep disturbances in neurological disorders have a devastating impact on patient and carer alike. However, their pathological origin is unknown. Here we show that patients with Huntington's disease (HD) have disrupted night-day activity patterns. This disruption was mirrored in a transgenic model of HD (R6/2 mice) in which daytime activity increased and nocturnal activity fell, eventually leading to the complete disintegration of circadian behavior. The behavioral disturbance was accompanied by marked disruption of expression of the circadian clock genes mPer2 and mBmal1 in the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in the brain. The circadian peak of expression of mPer2 was prematurely truncated, and the mRNA levels of mBmal1 were attenuated and failed to exhibit a significant circadian oscillation. Circadian cycles of gene expression in the motor cortex and striatum, markers of behavioral activation in wild-type mice, were also suppressed in the R6/2 mice, providing a neural correlate of the disturbed activity cycles. Increased daytime activity was also associated with reduced SCN expression of prokineticin 2, a transcriptional target of mBmal1 encoding a neuropeptide that normally suppresses daytime activity in nocturnal mammals. Together, these molecular abnormalities could explain the pathophysiological changes in circadian behavior. We propose that circadian sleep disturbances are an important pathological feature of HD, that they arise from pathology within the SCN molecular oscillation, and that their treatment will bring appreciable benefits to HD patients. [ABSTRACT FROM AUTHOR]

Filipski, Elisabeth, Verdun M. King, Marie-Christine Etienne, XiaoMei Li, Bruno Claustrat, Teresa G. Granda, Gérard Milano, Michael H. Hastings, and Francis Lévi. Persistent twenty-four hour changes in liver and bone marrow despite suprachiasmatic nuclei ablation in mice. Am J Physiol Regul Integr Comp Physiol 287: R844-R851, 2004. First published June 24, 2004; 10.1152/ajpregu. 00085.2004.—Rest-activity or cortisol rhythms can be altered in cancer patients, a condition that may impair the benefits from a timed delivery of anticancer treatments. In rodents, the circadian pattern in rest-activity is suppressed by the destruction of the suprachiasmatic nuclei (SCN) in the hypothalamus. We sought whether such ablation would result in a similar alteration of cellular rhythms known to be relevant for anticancer drug chronopharmacology. The SCN of 77 B6D2F1 mice synchronized with 12 h of light and 12 h of darkness were destroyed by electrocoagulation [SCN(-)], while 34 animals were sham operated. Activity and body temperature were recorded by telemetry. Blood and organs were sampled at one of six circadian times for determinations of serum corticosterone concentration, blood leukocyte count, reduced glutathione (GSH), and dihydropyrimidine dehydrogenase (DPD) mRNA expression in liver and cell cycle phase distribution of bone marrow cells. Sham-operated mice displayed significant 24-h rhythms in rest-activity and body temperature, whereas such rhythms were found in none and in 15% of the SCN(-) mice, respectively. SCN lesions markedly altered the rhythmic pat- terns in serum corticosterone and liver GSH, which became nonsinu- soidal. Liver DPD mRNA expression and bone marrow cell cycle phase distribution displayed similar 24-h sinusoidal patterns in sham- operated and SCN(-) mice. These results support the existence of another light-dark entrainable pacemaker that can coordinate cellular functions in peripheral organs. They suggest that the delivery of anticancer treatments at an optimal time of day may still be beneficial, despite suppressed rest-activity or cortisol rhythms. [ABSTRACT FROM AUTHOR]

Siberian hamsters accumulate fat reserves in long photoperiods, but show a long-term decrease in food intake and body weight when exposed to a short winter photoperiod. The aim of this study was to determine the role of central melanocortin 3/4 receptors (MC3/4-R) in generating this chronic catabolic state by investigating the effects of SHU9119, a MC3/4-R antagonist, on food intake and associated behaviours. In adult male hamsters, intra-cerebroventricular infusions of SHU9119 significantly increased food intake in a dose-dependent manner. The time course of action was slow, food intake being increased between 4 and 24 h after intra-cerebroventricular administration. A similar degree of increase in food intake occurred in fat hamsters in long days and in lean hamsters chronically exposed to short days. Intra-cerebroventricular treatment with MTII (a MC3/4-R agonist) significantly decreased food intake for up to 24 h after treatment, and SHU9119 reversed these suppressive effects between 4 and 24 h after treatment, a similar time course to that observed when SHU9119 was administered alone. We conclude that endogenous melanocortin peptides acting via MC3/4-R are involved in the regulation of food intake in hamsters in both anabolic and catabolic states, but these acute studies do not provide evidence that increased activity of this hypothalamic system underlies the seasonal decrease in food intake that contributes to the long-term catabolic state in short days. [Copyright &y& Elsevier]

Summary Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period ( Per ) and Cryptochrome ( Cry ) following nuclear entry of their protein products is the basis of circadian oscillation [ 1, 2 ]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [ 3, 4 ]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [ 5–7 ]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock. [ABSTRACT FROM AUTHOR]

Background: Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions.Results: Using two independent tissue sampling and hybridization regimes, we show that ∼9% of the 2122 genes studied show robust circadian cycling in the liver. These transcripts were categorized by their phase of abundance, defining clusters of day- and night-related genes, and also by the function of their products. Circadian regulation of genes was tissue specific, insofar as novel rhythmic liver genes were not necessarily rhythmic in the brain, even when expressed in the SCN. The rhythmic transcriptome in the periphery is, nevertheless, dependent on the SCN because surgical ablation of the SCN severely dampened or destroyed completely the cyclical expression of both canonical circadian genes and novel genes identified by microarray analysis.Conclusions: Temporally complex, circadian programming of the transcriptome in a peripheral organ is imposed across a wide range of core cellular functions and is dependent on an interaction between intrinsic, tissue-specific factors and extrinsic regulation by the SCN central pacemaker. [Copyright &y& Elsevier]