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PubMed1994

Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

Animals↗
PubMed2006

"Clock-scan" protocol for image analysis.

Comparative analysis of extra- and intracellular distributions of protein markers in immunohistochemical and immunofluorescent studies relies on techniques of image analysis. Line or region of interest pixel intensity scans are methods routinely used. However, although having good spatial resolution, linear pixel intensity scans fail to produce integral image of the cellular distribution of the label. On the other hand, the regions of interest scans have good integrative capacity but low spatial resolution. In this work, we describe a "clock-scan" protocol that, when applied to convex objects (such as neuronal cell bodies and the majority of cells in culture), combines advantages and circumnavigates limitations of the above-mentioned techniques. The protocol 1) collects multiple radial pixel intensity profiles scanned from the cell center to the periphery, 2) scales these profiles according to the cell radius measured in the direction of the scan, and finally, 3) averages these individual profiles into one integral radial pixel intensity profile. Because of scaling, the mean pixel intensity profiles produced by the clock-scan protocol depend on neither the cell size nor, within reasonable limits, the cell shape. This allows direct comparison or, if required, averaging or subtraction of profiles of different cells. We have successfully tested the clock-scan protocol in experiments with immunostained dorsal root ganglion neurons. In addition, the protocol seems to be equally applicable for studies in a variety of other preparations.

Animals↗
PubMed1996

Evidence for a central role of transcription in the timing mechanism of a circadian clock.

The retinal circadian clock in the isolated in vitro eye of the marine mollusc Bulla gouldiana exhibits a phase-dependent requirement for transcription. The transcription-sensitive phase extends through most of the subjective day and therefore is substantially longer than the previously reported translation-sensitive phase. Lower concentrations of transcription inhibitors yield a significant dose-dependent lengthening of circadian period. Clock motion can be stopped by a high concentration of the transcription inhibitor 5,6-dichlorobenz-imidazole riboside (DRB) when applied during the sensitive phase; after withdrawal of the inhibitor, motion resumes from the phase at which it was stopped. In a double-pulse experiment, phase shifts to light pulses applied after DRB pulses, and not during the translation-sensitive phase, indicate that the inhibition of transcription has immediate effects on the phase of the clock. These data suggest that DRB-induced phase shifts are independent of translation, which implies that the rate of transcription itself plays a significant role in the mechanism underlying the generation of the circadian cycle.

Animals↗
PubMed2006

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice.

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.

ARNTL Transcription Factors↗
PubMed1996

Acute effects of antisense antagonism of a single peptide neurotransmitter in the circadian clock.

The circadian clock that resides in the suprachiasmatic nucleus (SCN) of the hypothalamus is the major neural pacemaker driving most 24-h rhythms in mammals. Several neurotransmitter peptides are synthesized within this nucleus and communicate rhythmically with other cells in the SCN as well as with cells in other regions of the brain. At the present time, little is known about their role in regulating outputs of the clock. We demonstrate that antisense oligodeoxynucleotides corresponding to the NH2-terminus and the translation start site of vasoactive intestinal peptide (VIP) mRNA infused into the suprachiasmatic region of rats temporarily abolishes the circadian rhythm of corticosterone secretion without influencing stress-related corticosterone secretion in the same animals. Levels of VIP peptide are suppressed 30% on the second day after antisense treatment. These results indicate that a single neuropeptide transmitter in the circadian clock may serve a distinct role in the control of specific circadian rhythms.

Animals↗
PubMed1989

Circadian phototransduction: phase resetting and frequency of the circadian clock of Gonyaulax cells in red light.

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic--rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16 degrees C and 25 degrees C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light.

Animals↗
PubMed1990

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora.

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.

Bacterial Proteins↗
PubMed1992

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.

Animals↗
PubMed1998

Thermoperiodic responses in insects and mites simulated with the double circadian oscillator clock.

The "double circadian oscillator model" for the photoperiodic clock has been used to simulate thermoperiodic responses in insects and mites. Two assumptions have been made: (1) the clock measures cryophase in a similar way to scotophase, and (2) temperature cycles are able to entrain the clock in a similar way to LD cycles. Simulations showed that Assumption 1 causes the "critical cryophase" to be of about equal duration as the critical night length. Assumption 2 is not always needed if diapause incidences in DD are high at low temperatures but low or zero at high temperatures. The latter assumption is needed, however, if high diapause occurs in thermoperiodic cycles in DD, whereas nondiapause occurs in DD with both high and low constant temperatures. The model accounts for the observation that the amplitude of the temperature cycle is important in some insects, whereas the temperature of the cryophase is crucial in others.

Algorithms↗
PubMed1999

A model of molecular circadian clocks: multiple mechanisms for phase shifting and a requirement for strong nonlinear interactions.

A fundamental question in the field of circadian rhythms concerns the biochemical and molecular nature of the oscillator. There is strong evidence that circadian oscillators are cell autonomous and rely on periodic gene expression. In Drosophila, Neurospora, Aplysia, and vertebrates, circadian oscillators are thought to be based on molecular autoregulatory loops composed of transcription, translation, and negative feedback by proteins on nuclear transcription. By studying a mathematical model of molecular clocks based on this general concept, the authors sought to determine which features such clocks must have to generate robust and stable oscillations and to allow entrainment by external stimuli such as light. The model produced circadian oscillations as an emergent property even though a time delay in protein synthesis and rate constants of the feedback loop were much shorter than 24 h. Along with the delay in protein production, strong nonlinear interactions in macromolecular synthesis and nuclear feedback appeared to be required for the model to show well-behaved oscillatory behavior. Realistic phase-shifting patterns induced by external stimuli could be achieved by multiple mechanisms-namely, up- and downward perturbations of protein or mRNA synthesis or degradation rates. The model makes testable predictions about interactions between clock elements and mechanisms of entrainment and may help to understand the functions of the intricate molecular interactions governing circadian rhythmogenesis.

Animals↗
PubMed2006

The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks.

BACKGROUND: Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure. RESULTS: We developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ2 transiently induces Cry1, Cry2, and Roralpha mRNA expressions and that 15d-PGJ2-induced entrainment signaling pathway is PPAR-gamma--and MAPKs (ERK, JNK, p38MAPK)-independent. CONCLUSION: Here, we identified 15d-PGJ2 as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings.

Animals↗
PubMed2006

Altered expression of circadian clock gene, mPer1, in mouse brain and kidney under morphine dependence and withdrawal.

Every physiological function in the human body exhibits some form of circadian rhythmicity. Under pathological conditions, however, circadian rhythmicity may be disrupted. Patients infected with HIV or addicted to drugs of abuse often suffer from sleep disorders and altered circadian rhythms. Early studies in Drosophila suggested that drug seeking behavior might be related to the expression of certain circadian clock genes. Our previous research showed that conditioned place preference with morphine treatment was altered in mice lacking the Period-1 (mPer1) circadian clock gene. Thus, we sought to investigate whether morphine treatment could alter the expression of mPer1, especially in brain regions outside the SCN and in peripheral tissues. Our results using Western blot analysis showed that the mPER1 immunoreactivity exhibited a strong circadian rhythm in the brains of the control (Con), morphine-dependent (MD), and morphine-withdrawal (MW) mice. However, the phase of the circadian rhythm of mPER1 expression in the brains of MD mice significantly differed from that of the Con mice (p < 0.05). In contrast to mPER1 expression in the brain, the circadian rhythm of mPER1 immunoreactivity in the kidneys was abolished after morphine administration, whereas the Con mice maintained robust circadian rhythmicity of mPER1 in the kidney. Therefore, the effect of morphine on the circadian clock gene mPer1 may vary among different organs, resulting in desynchronization of circadian function between the SCN and peripheral organs.

Journal Article↗
PubMed1999

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

Animals↗
PubMed2001

Physical and inflammatory stressors elevate circadian clock gene mPer1 mRNA levels in the paraventricular nucleus of the mouse.

Stress induces secretion of corticosterone through activation of the hypothalamic-pituitary-adrenal axis. This corticosterone secretion is thought to be controlled by a circadian clock in the suprachiasmatic nucleus (SCN). The hypothalamic paraventricular nucleus (PVN) receives convergent information from both stress and the circadian clock. Recent reports demonstrate that mammalian orthologs (Per1, Per2, and Per3) of the Drosophila clock gene Period are expressed in the SCN, PVN, and peripheral tissues. In this experiment, we examined the effect of physical and inflammatory stressors on mPer gene expression in the SCN, PVN, and liver. Forced swimming, immobilization, and lipopolysaccharide injection elevated mPer1 gene expression in the PVN but not in the SCN or liver. A stress-induced increase in mPer1 expression was observed in the corticotropin-releasing factor-positive cells of the PVN; however, the stressors used in this study did not affect mPer2 expression in the PVN, SCN, or liver. The present study suggests that a stress-induced disturbance of circadian corticosterone secretion may be associated with the stress-induced expression of mPer1 mRNA in the PVN.

Animals↗
PubMed1980

Protein synthesis requirement of the Aplysia circadian clock. Tested by active and inactive derivatives of the inhibitor anisomycin.

1. The circadian rhythm of compound action potential frequency recorded from the isolated eye of Aplysia in culture medium and darkness was subjected to 6 h pulse treatments with either anisomycin, a protein synthesis inhibitor, or inactive derivatives of anisomycin. 2. Anisomycin caused phase-dependent phase shifts of the rhythm as expected from previous experiments, but none of the derivative molecules caused phase shifts or perturbed the rhythm. 3. Anisomycin inhibited eye-protein synthesis by 75% at the concentrations (10(-6) M) used in the phase shifting experiments but none of the derivatives inhibited synthesis. 4. Only those molecules that actually inhibited protein synthesis caused phase shifts of the clock, although the inactive derivatives differed from anisomycin by only an acetyl group. 5. The results strengthen the conclusion that the inhibition of protein synthesis caused by anisomycin is important in perturbing the timing of the circadian clock and not some other characteristic effect of the inhibitor molecule. Together with the results from other systems, these findings imply that the daily synthesis of protein is a general requirement for circadian clocks.

Animals↗
PubMed2006

Atomic-based stabilization for laser-pumped atomic clocks.

We describe a novel technique for stabilizing frequency shifts in laser-interrogated vapor-cell atomic clocks. The method suppresses frequency shifts due to changes in the laser frequency, intensity, and modulation index as well as atomic vapor density. The clock operating parameters are monitored by using the atoms themselves, rather than by using conventional schemes for laser frequency and cell temperature control. The experiment is realized using a chip-scale atomic clock. The novel atomic-based stabilization approach results in a simpler setup and improved long-term performance.

Journal Article↗
PubMed2002

Light-dependent changes in the chick pineal temperature and the expression of cHsp90 alpha gene: a potential contribution of in vivo temperature change to the photic-entrainment of the chick pineal circadian clock.

The circadian clock is entrained to the diurnal alteration of environmental conditions such as light and temperature, but the molecular mechanism underlying the entrainment is not fully understood. In the present study, we employed a differential display-based screening for a set of genes that are induced by light in the chick pineal gland, a structure of the central clock entrainable to both light and temperature changes. We found that the level of the mRNA encoding chicken heat shock protein 90 alpha (cHSP90 alpha) was rapidly elevated in the pineal gland within a 5-min exposure of chicks to light. Furthermore, the pineal cHsp90 alpha mRNA was expressed rhythmically under both 12-hr light/12-hr dark (LD) cycles and constant dark (DD) conditions. The total amount of the pineal cHSP90 alpha protein was, however, kept at nearly constant levels under LD cycles, and immunohistochemical analyses of the pineal cHSP90 alpha showed invariable localization at the cytoplasm throughout the day. In vivo measurement of the chick pineal temperature demonstrated its light-dependent and time-of-day-dependent change, and the profile was very similar to that of the pineal cHSP90 alpha mRNA level. These observations suggest that the in vivo temperature change regulates the expression of temperature-responsive genes including cHSP 90 alpha in the pineal gland. The temperature change may induce a phase-shift of the pineal clock, thereby facilitating its efficient entrainment to environmental LD cycles.

Animals↗
PubMed2006

Characterization of peripheral circadian clocks in adipose tissues.

First described in the suprachiasmatic nucleus, circadian clocks have since been found in several peripheral tissues. Although obesity has been associated with dysregulated circadian expression profiles of leptin, adiponectin, and other fat-derived cytokines, there have been no comprehensive analyses of the circadian clock machinery in adipose depots. In this study, we show robust and coordinated expression of circadian oscillator genes (Npas2, Bmal1, Per1-3, and Cry1-2) and clock-controlled downstream genes (Rev-erb alpha, Rev-erb beta, Dbp, E4bp4, Stra13, and Id2) in murine brown, inguinal, and epididymal (BAT, iWAT, and eWAT) adipose tissues. These results correlated with respective gene expression in liver and the serum markers of circadian function. Through Affymetrix microarray analysis, we identified 650 genes that shared circadian expression profiles in BAT, iWAT, and liver. Furthermore, we have demonstrated that temporally restricted feeding causes a coordinated phase-shift in circadian expression of the major oscillator genes and their downstream targets in adipose tissues. The presence of circadian oscillator genes in fat has significant metabolic implications, and their characterization may have potential therapeutic relevance with respect to the pathogenesis and treatment of diseases such as obesity, type 2 diabetes, and the metabolic syndrome.

Adiponectin↗