A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies.

Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system (CNS) function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour.

Catabolic cytokines disrupt the circadian clock and the expression of clock-controlled genes in cartilage via an NFкB-dependent pathway.

OBJECTIVE: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes. METHODS: Ex vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NFкB pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms. RESULTS: Exposure to IL-1ß severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1ß was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NFкB signalling was involved in the effect of IL-1ß on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNFα had little impact on the circadian rhythm and clock gene expression in cartilage. CONCLUSION: In our experimental system (young healthy mouse cartilage), we demonstrate that IL-1ß (but not TNFα) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to pro-inflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA).

Differential contributions of intra-cellular and inter-cellular mechanisms to the spatial and temporal architecture of the suprachiasmatic nucleus circadian circuitry in wild-type, cryptochrome-null and vasoactive intestinal peptide receptor 2-null mutant mice.

To serve as a robust internal circadian clock, the cell-autonomous molecular and electrophysiological activities of the individual neurons of the mammalian suprachiasmatic nucleus (SCN) are coordinated in time and neuroanatomical space. Although the contributions of the chemical and electrical interconnections between neurons are essential to this circuit-level orchestration, the features upon which they operate to confer robustness to the ensemble signal are not known. To address this, we applied several methods to deconstruct the interactions between the spatial and temporal organisation of circadian oscillations in organotypic slices from mice with circadian abnormalities. We studied the SCN of mice lacking Cryptochrome genes (Cry1 and Cry2), which are essential for cell-autonomous oscillation, and the SCN of mice lacking the vasoactive intestinal peptide receptor 2 (VPAC2-null), which is necessary for circuit-level integration, in order to map biological mechanisms to the revealed oscillatory features. The SCN of wild-type mice showed a strong link between the temporal rhythm of the bioluminescence profiles of PER2::LUC and regularly repeated spatially organised oscillation. The Cry-null SCN had stable spatial organisation but lacked temporal organisation, whereas in VPAC2-null SCN some specimens exhibited temporal organisation in the absence of spatial organisation. The results indicated that spatial and temporal organisation were separable, that they may have different mechanistic origins (cell-autonomous vs. interneuronal signaling) and that both were necessary to maintain robust and organised circadian rhythms throughout the SCN. This study therefore provided evidence that the coherent emergent properties of the neuronal circuitry, revealed in the spatially organised clusters, were essential to the pacemaking function of the SCN.

The Tau mutation of casein kinase 1ε sets the period of the mammalian pacemaker via regulation of Period1 or Period2 clock proteins.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker in mammals, coordinating daily metabolic and physiological rhythms with the cycle of sleep and wakefulness. SCN neurons define circadian time via an auto-regulatory feedback loop in which the activation of Period (Per) and Cryptochrome genes is periodically suppressed by their own protein products. Casein kinase 1 (CK1) enzymes have a critical role in circadian pacemaking because they phosphorylate PER proteins and thereby direct their proteasomal degradation. In human pedigrees, individual mutations in either hCK1 or hPER2 lead to advanced sleep phase disorders, whereas in rodents, the Tau mutation of CK1 epsilon (CK1ε (Tau)) accelerates rest-activity cycles and shortens the period of the SCN molecular pacemaker. Biochemical analyses of recombinant PER proteins in cultured cells and endogenous proteins in peripheral tissues have identified PER1 and PER2, but not PER3, as direct substrates of CK1ε. The purpose of this study, therefore, was to determine the relative contributions of endogenous PER proteins to the period-accelerating effects of CK1ε (Tau), both in vivo and in vitro. CK1ε (Tau) mice were mated onto Per1-, Per2-, and Per1-Per2 (Per1/2) double-null backgrounds, in all cases carrying the Per1-luciferase bioluminescent circadian reporter gene. Mice lacking both PER1 and PER2 were behaviorally arrhythmic, confirming the inadequacy of PER3 as a circadian factor. Individual loss of either PER1 or PER2 had no significant effect on the circadian period or quality of wheel-running behavior, and CK1ε (Tau) accelerated behavioral rhythms in both Per1- and Per2-null mice. CK1ε (Tau) also accelerated in vitro molecular pacemaking in SCN lacking either PER1 or PER2, with a greater effect in PER2-dependent (i.e., Per1-null) SCN than in PER1-dependent slices. In double-null slices, some SCN were arrhythmic, whereas others exhibited transient rhythms, which trended nonsignificantly toward a shorter period. Both short-period and long-period rhythms could be identified in individual SCN neurons imaged by charge-coupled device camera. CK1ε (Tau) had no effect, however, on SCN-level or individual neuronal rhythms in the absence of PER1 and PER2. Thus, the CK1ε (Tau) allele has divergent actions, acting via both endogenous PER1 and PER2, but not PER3 protein, to mediate its circadian actions in vivo. Moreover, PER-independent cellular oscillations may contribute to pacemaking, but they are unstable and imprecise, and are not affected by the Tau mutation.

Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell-autonomous and emergent, circuit-level properties.

Cry1 circadian phase in vitro: wrapped up with an E-box.

The circadian timing of gene expression is determined by transcriptional regulation through upstream response elements present throughout the genome. Central to this regulation are the actions of a core group of transcriptional activators and repressors, which act through, and are themselves regulated by, a small set of canonical circadian response elements. Among these, the E-box (CACGTG) is crucial for daytime transcriptional activity. The mammalian Period (Per1-3) and Cryptochrome (Cry1-2) genes are E-box-regulated genes, but in peripheral tissues peak Cry1 mRNA expression is delayed by several hours relative to that of Per. It has been proposed that this delay originates from interactions between the proximal E-box and retinoic acid-related orphan receptor response elements (RORE) present in the Cry1 promoter. By using real-time luciferase reporter assays in NIH3T3 cells the authors show here that a proximal 47-bp E-box containing region of the Cry1 promoter is both necessary and sufficient to drive circadian Cry1 transcription with an appropriate phase delay (around 4 h) relative to Per2. The results therefore suggest that, at least in this in vitro model of the clock, RORE are not necessary for the appropriate circadian regulation of Cry1 expression and rather suggest that sequences surrounding the proximal E-boxes confer gene-specific circadian phasing.

Two decades of circadian time.

Circadian rhythms coordinate our physiology at a fundamental level. Over the last 20 years, we have witnessed a paradigm shift in our perception of what the clocks driving such rhythms actually are, moving from 'black boxes' to talking about autoregulatory transcriptional/post-translational feedback loops with identified molecular components. We also now know that the pacemaker of the suprachiasmatic nuclei (SCN) is not our only clock but quite the opposite because circadian clocks abound in our bodies, driving local rhythms of cellular metabolism, and synchronised to each other and to solar time, by cues from the SCN. This discovery of dispersed local clocks has far-reaching implications for understanding our physiology and the pathological consequences of clock dysfunction, revealing that clocks are critical in a variety of metabolic and neurological conditions, all of which have long-term morbidity attributable to them. Without the currently available molecular framework, these insights would have not have been possible. In the circadian future, a growing appreciation of the systems-level functioning of these clocks and their various cerebral and visceral outputs, will likely stimulate the development of novel therapies for major illnesses.

[Interactions between clock gene mutation, circadian phenotype and tumor growth in mice]. / Interactions entre mutation des gènes horloge, phénotype circadien et croissance tumorale chez la Souris.

The relation between circadian physiology (rest-activity and body temperature) and the growth of a grafted tumor (Glasgow osteosarcoma-GOS) was investigated in the mice with mutation of clock gene (ClockDelta19(-)) or gene controlled by the clock (Vpac(-/-)). Circadian rhythms in temperature and activity were stable, with an approximately 24-h period in all the mice synchronized by the alternation of 12 h of light and 12 h of darkness (LD 12:12). Following exposure to constant darkness (DD), both rhythms persisted in ClockDelta19(-), yet with a lengthening of the period by 4.5 h compared to wild type. In DD, the amplitude increased by 45.9% for the temperature rhythm (p<0.001) and by 17.4% for the activity one (p=0.08) as compared to LD 12:12 in ClockDelta19(-). The improvement of circadian coordination and/or the lengthening of the circadian period observed in ClockDelta19(-) kept in DD was associated with a moderate slowing down of tumor growth. Although the exposure to DD ablated the activity and temperature rhythms in Vpac(-/-), no modification in tumor growth was observed as compared to wide type or Vpac(-/-) in LD 12:12. Major alternations of circadian physiology can result from interactions between photoperiodic environment and mutation of clock gene or gene controlled by the clock. In these conditions, we have shown that the alternation of the circadian phenotype does not seem to constitute an essential determinant of the growth of a grafted tumor.

Genetic and molecular analysis of the central and peripheral circadian clockwork of mice.

A hierarchy of interacting, tissue-based clocks controls circadian physiology and behavior in mammals. Preeminent are the suprachiasmatic nuclei (SCN): central hypothalamic pacemakers synchronized to solar time via retinal afferents and in turn responsible for internal synchronization of other clocks present in major organ systems. The SCN and peripheral clocks share essentially the same cellular timing mechanism. This consists of autoregulatory transcriptional/posttranslational feedback loops in which the Period (Per) and Cryptochrome (Cry) "clock" genes are negatively regulated by their protein products. Here, we review recent studies directed at understanding the molecular and cellular bases to the mammalian clock. At the cellular level, we demonstrate the role of F-box protein Fbxl3 (characterized by the afterhours mutation) in directing the proteasomal degradation of Cry and thereby controlling negative feedback and circadian period of the molecular loops. Within SCN neural circuitry, we describe how neuropeptidergic signaling by VIP synchronizes and sustains the cellular clocks. At the hypothalamic level, signaling via a different SCN neuropeptide, prokineticin, is not required for pacemaking but is necessary for control of circadian behavior. Finally, we consider how metabolic pathways are coordinated in time, focusing on liver function and the role of glucocorticoid signals in driving the circadian transcriptome and proteome.

The biology of the circadian Ck1epsilon tau mutation in mice and Syrian hamsters: a tale of two species.

The tau mutation in the Syrian hamster resides in the enzyme casein kinase 1 epsilon (CK1epsilon), resulting in a dramatic acceleration of wheel-running activity cycles to about 20 hours. tau also impacts growth, energy, metabolism, feeding behavior, and circadian mechanisms underpinning seasonal timing, causing accelerated reproductive and neuroendocrine responses to photoperiodic changes. Modeling and experimental studies suggest that tau acts as a gain of function on specific residues of PER, consistent with hamster studies showing accelerated degradation of PER in the suprachiasmatic nucleus in the early circadian night. We have created null and tau mutants of Ck1epsilon in mice. Circadian period lengthens in CK1epsilon(/), whereas CK1epsilon(tau/tau) shortens circadian period of behavior in vivo in a manner nearly identical to that of the Syrian hamster. CK1epsilon(tau/tau) also accelerates molecular oscillations in peripheral tissues, demonstrating its global circadian role. CK1epsilon(tau) acts by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Our studies reveal that tau acts as a gain-of-function mutation, to accelerate degradation of PERIOD proteins. tau has consistent effects in both hamsters and mice on the circadian organization of behavior and metabolism, highlighting the global impact of this mutation on mammalian clockwork in brain and periphery.

Do corticosteroids damage the brain?

Corticosteroids are an essential component of the body's homeostatic system. In common with other such systems, this implies that corticosteroid levels in blood and, more importantly, in the tissues remain within an optimal range. It also implies that this range may vary according to circumstance. Lack of corticosteroids, such as untreated Addison's disease, can be fatal in humans. In this review, we are principally concerned with excess or disturbed patterns of circulating corticosteroids in the longer or shorter term, and the effects they have on the brain.

Developmental and reproductive performance in circadian mutant mice.

BACKGROUND: Genes underlying circadian rhythm generation are expressed in many tissues. We explore a role for circadian rhythms in the timing and efficacy of mouse reproduction and development using a genetic approach. METHODS: We compare fecundity in Clock(Delta19) mutant mice (a dominant-negative protein essential for circadian rhythm activity) and in Vipr2-/- null mutant mice (affecting the generation and output of the circadian rhythm of the hypothalamic suprachiasmatic nucleus) with wild type (WT) litter mates under both a 12 h:12 h light:dark cycle and continuous darkness. RESULTS: Uteri from Clock(Delta19) mice show no circadian rhythm and Vipr2-/- mice show a phase-advanced rhythm compared to WT uteri. In neither mutant line were homozygous or heterozygous fetuses lethal. Sexually mature adults of both mutant lines showed mildly reduced male in vivo (but not in vitro) fertility and irregular estrous cycles exacerbated by continuous darkness. However, pregnancy rates and neonatal litter sizes were not affected. The Clock(Delta19) mutant line was distinguishable from the Vipr2-/- null mutant line in showing more peri-natal delivery problems and very poor survival of offspring to weaning. CONCLUSIONS: A fully functional central and peripheral circadian clock is not essential for reproduction and development to term, but has critical roles peri-natally and post-partum.

The tau mutation in the Syrian hamster differentially reprograms the circadian clock in the SCN and peripheral tissues.

The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are synchronized to the solar day by the light-dark cycle, and in turn, they coordinate circadian oscillations in peripheral tissues. The tau mutation in the Syrian hamster is caused by a point mutation leading to a deficiency in the ability of Casein Kinase 1epsilon to phosphorylate its targets, including circadian PER proteins. How this accelerates circadian period in neural tissues is not known, nor is its impact on peripheral circadian oscillators established. We show that this mutation has no effect on per mRNA expression nor the nuclear accumulation of PER proteins in the SCN. It does, however, accelerate the clearance of PER proteins from the nucleus to an extent sufficient to explain the shortened circadian period of behavioral rhythms. The mutation also has novel, unanticipated consequences for circadian timing in the periphery, including tissue-specific phase advances and/or reduced amplitude of circadian gene expression. The results suggest that the tau mutation accelerates a specific phase, during mid-late subjective night of the SCN circadian feedback loop, rather than cause a global compression of the entire cycle. This reprogrammed output from the clock is associated with peripheral desynchrony, which in turn could account for impaired growth and metabolic efficiency of the mutant.

Disrupted daily activity/rest cycles in relation to daily cortisol rhythms of home-dwelling patients with early Alzheimer's dementia.

Disturbed sleep cycles are the principal cause of institutionalization in dementia, and therefore represent a major clinical problem. They may arise from dysfunction within the circadian clock of the hypothalamus that times and consolidates wakefulness, or from neuropathology in output pathways and/or target sites of the clock specifically controlling sleep and wakefulness. To determine the relationship of disturbed activity cycles to other circadian clock-controlled rhythms, cross-sectional and longitudinal actigraphy and serial sampling of saliva were used to compare the impact of early Alzheimer's dementia on activity/rest and cortisol rhythms in home-dwelling subjects. Mildly demented subjects had daily activity rhythms comparable to those of healthy age-matched subjects. Moderately demented subjects exhibited a range of disturbances of the activity/rest cycle, with reduced stability, increased fragmentation and loss of amplitude. Within the moderately demented group, however, the degree of circadian disruption was not correlated with the individual severity of dementia. All groups of subjects, mild, moderate with normal activity cycles and moderate with abnormal activity cycles, exhibited robust daily profiles of salivary cortisol, with highest levels in the morning (08:00 h) and an evening nadir (20:00-24:00 h). Salivary cortisol levels tended to be higher in the moderately demented subjects in the afternoon, but this effect was not specific to those with abnormal activity/rest patterns. The actimetric data confirm that deterioration of activity/rest cycles is a common and progressive feature in home-dwelling Alzheimer's patients, occurring early in the disease but after the measurable onset of dementia. The maintenance of highly rhythmic daily cortisol profiles in association with disturbed activity profiles, both on an individual and on a group basis, demonstrates that loss of circadian control to activity/rest cycles is not a consequence of global circadian disruption in early dementia. Rather, pathology may develop in discrete elements of the circadian clockwork and/or its output systems that control activity/rest, sleep and wakefulness. Further characterization of this pathology will facilitate more effective management of sleep patterns in home-dwelling demented patients.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

Expression of mCLOCK and other circadian clock-relevant proteins in the mouse suprachiasmatic nuclei.

Circadian timing in mammals is based upon the cell-autonomous clockwork located in the suprachiasmatic nuclei (SCN) of the hypothalamus. It is thought to involve interlocked feedback loops in which periodic transcriptional drive to core clock genes is mediated by CLOCK/BMAL1 heterodimers. Negative-feedback actions of the encoded proteins PER and CRY terminate this phase of the cycle. In lower species, rhythmic abundance of the mCLOCK homologue initiates the subsequent cycle. By contrast, it is proposed that the new circadian cycle in mammals is triggered by indirect, positive transcriptional actions leading to a subsequent surge in BMAL1. The aim of this study was to test predictions made by this model concerning the behaviour of the native clock factor mCLOCK in the mouse SCN. Using in situ hybridization, immunocytochemistry, Western blotting and immunoprecipitation, we demonstrate constitutive expression of mCLOCK as a nuclear antigen in the SCN. mCLOCK forms alternating, periodic associations with either mBMAL1 or the negative regulators mPER and mCRY. The results confirm predictions made by the "two-loop" model of the mouse clock, and further highlight the role of interlocked cycles of positive and negative transcriptional regulatory complexes at the heart of the circadian clockwork.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

Expression of clock gene products in the suprachiasmatic nucleus in relation to circadian behaviour.

Circadian timing within the suprachiasmatic nucleus (SCN) is modelled around cell-autonomous, autoregulatory transcriptional/post-translational feedback loops, in which protein products of canonical clock genes Period and Cryptochrome periodically oppose transcription driven by CLOCK:BMAL complexes. Consistent with this model, mCLOCK is a nuclear antigen constitutively expressed in mouse SCN, whereas nuclear mPER and mCRY are expressed rhythmically. Peaking in late subjective day, mPER and mCRY form heteromeric complexes with mCLOCK, completing the negative feedback loop as levels of mPer and mCry mRNA decline. Circadian resetting by light or non-photic resetting (mediated by neuropeptide Y) involves acute up- and down-regulation of mPer mRNA, respectively. Expression of Per mRNA also peaks in subjective day in the SCN of the ground squirrel, indicating common clock and entrainment mechanisms for nocturnal and diurnal species. Oscillation within the SCN is dependent on intercellular signals, in so far as genetic ablation of the VPAC2 receptor for vasoactive intestinal polypeptide (VIP) suspends SCN circadian gene expression. The pervasive effect of the SCN on peripheral physiology is underscored by cDNA microarray analysis of the circadian gene expression in liver, which involves ca. 10% of the genome and almost all aspects of cell function. Moreover, the same molecular regulatory mechanisms driving the SCN appear also to underpin peripheral cycles.

Differential resynchronisation of circadian clock gene expression within the suprachiasmatic nuclei of mice subjected to experimental jet lag.

Disruption of the circadian timing system arising from travel between time zones ("jet lag") and rotational shift work impairs mental and physical performance and severely compromises long-term health. Circadian disruption is more severe during adaptation to advances in local time, because the circadian clock takes much longer to phase advance than delay. The recent identification of mammalian circadian clock genes now makes it possible to examine time zone adjustments from the perspective of molecular events within the suprachiasmatic nucleus (SCN), the principal circadian oscillator. Current models of the clockwork posit interlocked transcriptional/post-translational feedback loops based on the light-sensitive Period (Per) genes and the Cryptochrome (Cry) genes, which are indirectly regulated by light. We show that circadian cycles of mPer expression in the mouse SCN react rapidly to an advance in the lighting schedule, whereas rhythmic mCry1 expression advances more slowly, in parallel to the gradual resetting of the activity-rest cycle. In contrast, during a delay in local time the mPer and mCry cycles react rapidly, completing the 6 hr shift together by the second cycle, in parallel with the activity-rest cycle. These results reveal the potential for dissociation of mPer and mCry expression within the central oscillator during circadian resetting and a differential molecular response of the clock during advance and delay resetting. They highlight the indirect photic regulation of mCry1 as a potentially rate-limiting factor in behavioral adjustment to time zone transitions.

Cycle of period gene expression in a diurnal mammal (Spermophilus tridecemlineatus): implications for nonphotic phase shifting.

Ground squirrels, Spermophilus tridecemlineatus, were kept in a 12:12 h light-dark cycle. As expected for a diurnal species, their locomotor activity occurred almost entirely in the daytime. Expression of mPer1 and mPer2 in the suprachiasmatic nucleus was studied at six time points by in situ hybridization. For both these genes, mRNA was highest in the first part of the subjective day (about zeitgeber time 5). This is close to the time when mPer1 and mPer2 expression is maximal in nocturnal rodents. These results have implications for understanding nonphotic phase response curves in diurnal species and thereby for guiding research on nonphotic phase shifting in people.