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1.
Osteoarthritis Cartilage ; 23(11): 1981-8, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26521744

ABSTRACT

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).


Subject(s)
Chondrocytes/metabolism , Circadian Clocks/genetics , Cytokines/genetics , DNA/genetics , Gene Expression Regulation , NF-kappa B/genetics , Osteoarthritis/genetics , Animals , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Cells, Cultured , Cytokines/biosynthesis , Disease Models, Animal , Mice , Mice, Transgenic , NF-kappa B/biosynthesis , Osteoarthritis/metabolism , Osteoarthritis/pathology , Reverse Transcriptase Polymerase Chain Reaction
2.
Eur J Neurosci ; 40(3): 2528-40, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24891292

ABSTRACT

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.


Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Cryptochromes/physiology , Receptors, Vasoactive Intestinal Peptide, Type II/physiology , Suprachiasmatic Nucleus/physiology , Animals , Circadian Clocks/genetics , Circadian Rhythm/genetics , Cryptochromes/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Net/physiology , Receptors, Vasoactive Intestinal Peptide, Type II/genetics
3.
J Biol Rhythms ; 29(2): 110-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24682205

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon/genetics , Circadian Rhythm , Mutation , Period Circadian Proteins/genetics , Suprachiasmatic Nucleus/metabolism , Animals , Casein Kinase 1 epsilon/metabolism , Female , Humans , Light , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Male , Mice , Mice, Knockout , Mice, Transgenic , Motor Activity/genetics , Motor Activity/radiation effects , Neurons/metabolism , Organ Culture Techniques , Period Circadian Proteins/metabolism , Suprachiasmatic Nucleus/cytology
4.
J Neuroendocrinol ; 26(1): 2-10, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24329967

ABSTRACT

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.


Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Neural Pathways/physiology , Suprachiasmatic Nucleus/cytology , Suprachiasmatic Nucleus/physiology , Animals , Cryptochromes/physiology , Feedback, Physiological/physiology , Neurons/physiology , Period Circadian Proteins/physiology , Signal Transduction/physiology , Vasoactive Intestinal Peptide/physiology
5.
J Neuroendocrinol ; 20(6): 812-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18601704

ABSTRACT

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.


Subject(s)
Biological Clocks/physiology , Circadian Rhythm/physiology , Suprachiasmatic Nucleus/physiology , Animals , Feedback, Physiological , Humans , Nervous System Diseases/physiopathology , Neurons/metabolism , Sleep/physiology , Suprachiasmatic Nucleus/cytology , Time Factors
6.
Article in English | MEDLINE | ID: mdl-18419265

ABSTRACT

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.


Subject(s)
Circadian Rhythm/genetics , Circadian Rhythm/physiology , Animals , Gene Expression Profiling , Liver/physiology , Mice , Mice, Knockout , Models, Biological , Mutation , Neuropeptides/genetics , Neuropeptides/physiology , Proteasome Endopeptidase Complex/metabolism , Proteome , Receptors, Vasoactive Intestinal Peptide, Type II/deficiency , Receptors, Vasoactive Intestinal Peptide, Type II/genetics , Signal Transduction , Suprachiasmatic Nucleus/physiology
7.
Article in English | MEDLINE | ID: mdl-18522517

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/physiology , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Activity Cycles , Animals , CLOCK Proteins , Casein Kinase 1 epsilon/deficiency , Cricetinae , Cryptochromes , Female , Flavoproteins/genetics , Flavoproteins/physiology , Male , Mesocricetus , Mice , Mice, Knockout , Mice, Mutant Strains , Models, Biological , Mutation , Neurosecretory Systems/physiology , Photoperiod , Seasons , Species Specificity , Trans-Activators/genetics , Trans-Activators/physiology
8.
Hum Reprod ; 21(1): 68-79, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16210390

ABSTRACT

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.


Subject(s)
Circadian Rhythm , Fertility , Receptors, Vasoactive Intestinal Peptide, Type II/genetics , Trans-Activators/genetics , Animals , CLOCK Proteins , Circadian Rhythm/genetics , Estrous Cycle/genetics , Female , Fertility/genetics , Fetus/physiology , Gene Deletion , Infertility, Male/genetics , Litter Size , Male , Mice , Mice, Mutant Strains , Mutation , Uterus/metabolism
9.
J Biol Rhythms ; 20(2): 99-110, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15834107

ABSTRACT

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.


Subject(s)
Biological Clocks/physiology , Circadian Rhythm , Point Mutation , Suprachiasmatic Nucleus/physiology , tau Proteins/genetics , Animals , Base Sequence , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Cell Cycle Proteins , Corpus Striatum/metabolism , Cricetinae , DNA Primers , Immunohistochemistry , In Situ Hybridization , Mesocricetus , Motor Cortex/metabolism , Myocardium/metabolism , Nuclear Proteins/genetics , Period Circadian Proteins , RNA, Messenger/genetics , Suprachiasmatic Nucleus/metabolism , Transcription Factors/genetics
10.
Eur J Neurosci ; 17(11): 822-32, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12814356

ABSTRACT

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.


Subject(s)
Circadian Rhythm/genetics , Circadian Rhythm/physiology , Gene Expression Regulation , Receptors, Vasoactive Intestinal Peptide/genetics , Receptors, Vasoactive Intestinal Peptide/metabolism , Suprachiasmatic Nucleus/metabolism , Animals , Animals, Newborn , Behavior, Animal , Brain Tissue Transplantation/methods , Cell Count , Cell Cycle Proteins , Chi-Square Distribution , Humans , Immunohistochemistry , Male , Mice , Mice, Transgenic/metabolism , Motor Activity/genetics , Motor Activity/physiology , Neurophysins/metabolism , Nuclear Proteins/metabolism , Peptide PHI/metabolism , Period Circadian Proteins , Photic Stimulation , Receptors, Vasoactive Intestinal Peptide, Type II , Statistics, Nonparametric , Suprachiasmatic Nucleus/cytology , Suprachiasmatic Nucleus/transplantation , Time Factors , Transcription Factors , beta-Galactosidase/metabolism
11.
J Neuroendocrinol ; 15(4): 329-34, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12622829

ABSTRACT

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.


Subject(s)
Drosophila Proteins , Eye Proteins , Gene Expression Regulation/physiology , Photoreceptor Cells, Invertebrate , Suprachiasmatic Nucleus/physiology , Trans-Activators/genetics , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Biological Clocks/genetics , Biological Clocks/physiology , Blotting, Western , CLOCK Proteins , Cell Cycle Proteins , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Cryptochromes , Feedback/physiology , Flavoproteins/genetics , Male , Mice , Models, Biological , Nuclear Proteins/genetics , Period Circadian Proteins , Receptors, G-Protein-Coupled , Time , Transcription Factors/genetics , Transcription, Genetic/physiology
12.
Eur J Neurosci ; 17(4): 822-32, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12603272

ABSTRACT

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.


Subject(s)
Circadian Rhythm/physiology , Mice, Transgenic/physiology , Receptors, Vasoactive Intestinal Peptide/genetics , Suprachiasmatic Nucleus/physiology , Animals , Brain Tissue Transplantation , Cell Count , Cell Cycle Proteins , Chi-Square Distribution , Electrolysis/methods , Humans , Immunohistochemistry/methods , Mice , Motor Activity/genetics , Motor Activity/physiology , Neurophysins/metabolism , Nuclear Proteins/metabolism , Peptide PHI/metabolism , Period Circadian Proteins , Receptors, Vasoactive Intestinal Peptide, Type II , Statistics, Nonparametric , Suprachiasmatic Nucleus/transplantation , Transcription Factors , beta-Galactosidase/metabolism
13.
Novartis Found Symp ; 253: 203-17; discussion 102-9, 218-22, 281-4, 2003.
Article in English | MEDLINE | ID: mdl-14712923

ABSTRACT

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.


Subject(s)
Circadian Rhythm/genetics , Circadian Rhythm/physiology , Suprachiasmatic Nucleus/physiology , Trans-Activators/genetics , Trans-Activators/physiology , Animals , CLOCK Proteins , Circadian Rhythm/radiation effects , Feedback , Light , Mammals , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction
14.
J Neurosci ; 22(17): 7326-30, 2002 Sep 01.
Article in English | MEDLINE | ID: mdl-12196553

ABSTRACT

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.


Subject(s)
Circadian Rhythm , Drosophila Proteins , Eye Proteins , Gene Expression Regulation , Jet Lag Syndrome/physiopathology , Photoreceptor Cells, Invertebrate , Suprachiasmatic Nucleus/physiopathology , Analysis of Variance , Animals , Biological Clocks , Cell Cycle Proteins , Cell Nucleus/metabolism , Circadian Rhythm/physiology , Cryptochromes , Disease Models, Animal , Flavoproteins/genetics , Flavoproteins/metabolism , In Situ Hybridization , Jet Lag Syndrome/pathology , Male , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , Periodicity , Photoperiod , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled , Suprachiasmatic Nucleus/pathology , Time Factors , Transcription Factors
15.
J Biol Rhythms ; 16(5): 471-8, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11669420

ABSTRACT

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.


Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation/physiology , Nuclear Proteins/genetics , Periodicity , Sciuridae/physiology , Animals , Brain Chemistry/physiology , Cell Cycle Proteins , Electrophysiology , In Situ Hybridization , Period Circadian Proteins , RNA, Messenger/biosynthesis , Suprachiasmatic Nucleus/metabolism , Transcription Factors
16.
Chronobiol Int ; 18(3): 559-65, 2001 May.
Article in English | MEDLINE | ID: mdl-11475423

ABSTRACT

The expression of circadian clock genes was investigated in the suprachiasmatic nuclei (SCN) of young adult and old laboratory mice. Samples were taken at two time points, which corresponded to the expected maximum (circadian time 7 [CT7]) or minimum (CT21) of mPer mRNA expression. Whereas the young mice had a stable and well-synchronized circadian activity/rest cycle, the rhythms of old animals were less stable and were phase advanced. The expression of mPerl mRNA and mPer2 mRNA was rhythmic in both groups, with peak values at CT7. The levels of mClock and mCry1 mRNA were not different depending on the time of day and did not vary with age. In contrast, an age-dependent difference was found in the case of mPer2 (but not mPerl) mRNA expression, with the maximum at CT7 significantly lower in old mice. The decreased expression of mPer2 may be relevant for the observed differences in the overt activity rhythm of aged mice.


Subject(s)
Aging , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Suprachiasmatic Nucleus/metabolism , Age Factors , Animals , Cell Cycle Proteins , Circadian Rhythm , Male , Mice , Period Circadian Proteins , RNA, Messenger/metabolism , Time Factors , Transcription Factors
17.
Neuron ; 30(2): 525-36, 2001 May.
Article in English | MEDLINE | ID: mdl-11395012

ABSTRACT

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.


Subject(s)
Circadian Rhythm/genetics , Motor Activity/physiology , Nuclear Proteins/physiology , Suprachiasmatic Nucleus/physiology , Animals , Biological Clocks , Brain/metabolism , Cell Cycle Proteins , Cloning, Molecular , Gene Expression Regulation , Genomic Library , Genotype , Homozygote , Mice , Mice, Knockout , Molecular Sequence Data , Motor Activity/genetics , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Period Circadian Proteins , Periodicity , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors , Transcription, Genetic
18.
Brain Res Gene Expr Patterns ; 1(1): 27-31, 2001 Aug.
Article in English | MEDLINE | ID: mdl-15018816

ABSTRACT

Non-photic clock-resetting events (arousal and locomotor activity) in the subjective day reduced expression of Period genes in the suprachiasmatic nucleus of hamsters. This decrease was attenuated by a 30-min light pulse occurring during the last 0.5 h of 3.5 h of confinement to a novel running wheel. This provides an example at the molecular level of an interaction between different modalities of synchronizing agents.

19.
Eur J Neurosci ; 12(12): 4557-61, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11122368

ABSTRACT

Precise temporal regulation of transcription is pivotal to the role of the mammalian pineal gland as a transducer of circadian and seasonal information. The circadian clock genes Per1 and Per2 encode factors implicated in temporally gated transcriptional programmes in brain and pituitary. Here we show that the nocturnal circadian expression of Per1 and Per2 in the rat pineal gland parallels that of serotonin N-acetyltransferase (NAT) mRNA, which encodes the rate-limiting enzyme of melatonin biosynthesis. This rhythm is dependent upon an intact sympathetic innervation. Increases in rPer1 (r indicates rat) and rPer2, as well as rNAT, expression during subjective night were blocked completely by superior cervical ganglionectomy (SCGX). In SCGX rats, the beta-adrenergic receptor agonist isoproterenol rapidly induced the rPer1 mRNA with dynamics very similar to its effect on rNAT mRNA. In contrast, isoproterenol was without effect on expression of rPer2 mRNA. These findings demonstrate that circadian pineal expression of both rPer1 and rPer2 is controlled by sympathetic afferent innervation, but whereas beta-adrenergic signalling regulates rPer1 and rNAT, an alternative route mediates sympathetic regulation over rPer2 expression.


Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation/physiology , Nuclear Proteins/genetics , Pineal Gland/physiology , Superior Cervical Ganglion/physiology , Transcription, Genetic/physiology , Animals , Arylamine N-Acetyltransferase/genetics , Biological Clocks , Cell Cycle Proteins , Circadian Rhythm/genetics , Darkness , Ganglionectomy , Light , Male , Period Circadian Proteins , RNA, Messenger/genetics , Rats , Rats, Wistar , Time Factors , Transcription Factors
20.
Eur J Neurosci ; 12(8): 2856-64, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10971628

ABSTRACT

The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights-on. These rhythms are circadian in nature as they continue approximately under free-running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.


Subject(s)
Circadian Rhythm/physiology , Nuclear Proteins/metabolism , Seasons , Suprachiasmatic Nucleus/physiology , Animals , Blotting, Western , Cell Cycle Proteins , Cerebral Cortex/chemistry , Cerebral Cortex/physiology , Cricetinae , Female , Lighting , Male , Nuclear Proteins/analysis , Period Circadian Proteins , Phodopus , Pituitary Gland, Anterior/physiology , Suprachiasmatic Nucleus/chemistry , Transcription Factors/analysis , Transcription Factors/metabolism , Tuber Cinereum/physiology
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