Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock

Biological rhythms are driven in mammals by a central circadian clock located in the suprachiasmatic nucleus (SCN). Light-induced phase shifting of this clock is correlated with phosphorylation of CREB at Ser133 in the SCN. Here, we characterize phosphorylation of CREB at Ser142 and describe its contribution to the entrainment of the clock. In the SCN, light and glutamate strongly induce CREB Ser142 phosphorylation. To determine the physiological relevance of phosphorylation at Ser142, we generated a mouse mutant, CREB(S142A), lacking this phosphorylation site. Light-induced phase shifts of locomotion and expression of c-Fos and mPer1 in the SCN are significantly attenuated in CREB(S142A) mutants. Our findings provide genetic evidence that CREB Ser142 phosphorylation is involved in the entrainment of the mammalian clock and reveal a novel phosphorylation-dependent regulation of CREB activity.     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

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.     

Melatonin affects circadian rhythmicity in lizards

Summary Subcutaneous Silastic capsules which continuously released melatonin at low rates (10 g/day) either (i) lengthened the period of the freerunning activity rhythm in two iguanid lizard species (Sceloporus occidentalis andS. olivaceus) exposed to either continuous illumination or continuous darkness or (ii) induced arrhythmicity inS. olivaceus exposed to continuous illumination. These results and previous observations that two melatonin synthesizing sites in lizards, the pineal organ and lateral eyes, are involved in circadian rhythmicity suggest that melatonin may be a chemical messenger between the pineal or eyes and other elements of the circadian system.     

Altered circadian rhythm reentrainment to light phase shifts in rats with low levels of brain angiotensinogen

In this study, we aimed to investigate the adaptation of blood pressure (BP), heart rate (HR), and locomotor activity (LA) circadian rhythms to light cycle shift in transgenic rats with a deficit in brain angiotensin [TGR(ASrAOGEN)]. BP, HR, and LA were measured by telemetry. After baseline recordings (bLD), the light cycle was inverted by prolonging the light by 12 h and thereafter the dark period by 12 h, resulting in inverted dark-light (DL) or light-dark (LD) cycles. Toward that end, a 24-h dark was maintained for 14 days (free-running conditions). When light cycle was changed from bLD to DL, the acrophases (peak time of curve fitting) of BP, HR, and LA shifted to the new dark period in both SD and TGR(ASrAOGEN) rats. However, the readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. The LA acrophases changed similarly in both strains. When light cycle was changed from DL to LD by prolonging the dark period by 12 h, the reentrainment of BP and LA occurred faster than the previous shift in both strains. The readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. In free-running conditions, the circadian rhythms of the investigated parameters adapted in TGR(ASrAOGEN) and SD rats in a similar manner. These results demonstrate that the brain RAS plays an important role in mediating the effects of light cycle shifts on the circadian variation of BP and HR. The adaptive behavior of cardiovascular circadian rhythms depends on the initial direction of light-dark changes.     

Quantification of circadian phase shifts with the cross-correlation technique

This paper concerns the applicability of the cross-correlation technique for the assessment of shifts of the circadian system (e.g., caused by night work). Melatonin and cortisol profiles of 52 healthy young men were ascertained during two 24 h phase assessment procedures. The first was performed after three consecutive day shifts, and the second was performed one week later on 24 men again after three day shifts and on 28 men after three night shifts, where adaptation to night work was accelerated by bright light. The cross-correlation technique that relies on the processing of all the measured data of a whole profile, as compared to the differences between temporal parameters determined with a conventional method, provided reliable estimates of the phase shifts. Its applicability is restricted to time series with similar profiles assessed at different times and to observation periods of a full diurnal cycle (in the case of substantial shifts) with equally distributed measures, but it is applicable to raw data and available in common statistical packages (e.g., SPSS, SAS, BMDP).     

Calcium channels mediate phase shifts of the Bulla circadian pacemaker

1. Light-induced phase advances of the activity rhythm of the Bulla ocular circadian pacemaker are blocked when the extracellular calcium concentration is reduced with EGTA to 0.13 microM. Phase advances are also blocked in low calcium solutions without EGTA [( Ca] less than 50 microM). 2. The dependence of light-induced phase delays on extracellular calcium concentration in EGTA-free seawater was determined. Phase delays are blocked at calcium concentrations below 400 microM, and reduced at concentrations of 1 mM and 3.5 mM (relative to shifts in normal ASW, [Ca] = 10 mM). Phase delays are also reduced and blocked at calcium concentrations higher than normal (60 mM and 110 mM, respectively). 3. Low calcium EGTA also blocked both phase delays and phase advances induced by pulses of depolarizing high K+ seawater. Low calcium EGTA pulses presented alone at the same times did not generate significant phase shifts. 4. The organic calcium channel antagonists verapamil, diltiazem and nitrendipine as well as the inorganic calcium channel antagonists La3+, Co2+, Cd2+, and Mn2+ were applied along with light pulses, however, the treated eyes were either phase shifted by these substances, or these substances were found to be toxic. 5. The inorganic calcium channel antagonist Ni2+ blocked both light-induced phase delays and advances at a concentration of 5 mM. Ni2+ applied alone did not generate significant phase shifts. Phase delays induced by high K+ seawater were blocked in the presence of 50 mM Ni2+ but not in 5 mM Ni2+. The light-induced CAP activity of the putative pacemaker cells was not inhibited by Ni2+, suggesting that its blocking action was probably via its known role as a calcium channel antagonist.     

Tissue-specific function of Period3 in circadian rhythmicity

The mammalian circadian system is composed of multiple central and peripheral clocks that are temporally coordinated to synchronize physiology and behavior with environmental cycles. Mammals have three homologs of the circadian Period gene (Per1, 2, 3). While numerous studies have demonstrated that Per1 and Per2 are necessary for molecular timekeeping and light responsiveness in the master circadian clock in the suprachiasmatic nuclei (SCN), the function of Per3 has been elusive. In the current study, we investigated the role of Per3 in circadian timekeeping in central and peripheral oscillators by analyzing PER2::LUCIFERASE expression in tissues explanted from C57BL/6J wild-type and Per3^−/− mice. We observed shortening of the periods in some tissues from Per3^−/− mice compared to wild-types. Importantly, the periods were not altered in other tissues, including the SCN, in Per3^−/− mice. We also found that Per3-dependent shortening of endogenous periods resulted in advanced phases of those tissues, demonstrating that the in vitro phenotype is also present in vivo. Our data demonstrate that Per3 is important for endogenous timekeeping in specific tissues and those tissue-specific changes in endogenous periods result in internal misalignment of circadian clocks in Per3^−/− mice. Taken together, our studies demonstrate that Per3 is a key player in the mammalian circadian system.     

Serotonin modulates a photic response in circadian locomotor rhythmicity of adults of the blow fly, Calliphora vicina

Abstract. The present experiments were undertaken to explore a role for serotonin (5-hydroxytryptamine, 5-HT) in modulating photic signal transduction in photoreceptors of the blow fly, Calliphora vicina. Injection of p-chlorophenylalanine (pCPA) into the haemolymph appeared to reduce sensitivity to the photic effects of constant ‘bright’ light (LL hyperactivity and circadian arrhythmicity). After drug injection in bright LL, flies continued with a free-running rhythm as in constant darkness (DD) or with a lengthened period τ as in ‘dim’ LL. When 5-HT was injected into flies kept in dim LL, they became hyperactive and arrhythmic as in bright LL. This finding suggests a potential role for serotonin as mediator of circadian changes in the insect visual system including extraretinal photoreceptors.     

MHPG circadian rhythmicity in blood of healthy subjects

An earlier study showed that plasma concentrations of total 3-methoxy-4-hydroxyphenylglycol (MHPG), the major metabolite of norepinephrine, display a circadian rhythm in 6 male healthy subjects. In the previous study, the period of the rhythm was not fixed to 24 h thereby undermining the reliability of the cosinor parameter estimates. The present study extends the findings to a larger group of 12 clinically healthy male volunteers. Plasma total MHPG concentrations were determined every 3h for one full day. The data were fitted to a cosinor model fixing the period of the putative MHPG rhythm at 24 h. Several estimation techniques were utilized including Fourier analysis and time domain analysis with 4 variations. It is concluded that a circadian rhythm indeed characterizes MHPG blood concentrations. The concordance among the various parameter estimates is discussed.     

Diazepam phase shifts the circadian clock of the field mouseMus booduga

Single 2h administration of diazepam (benzodiazepine) in 3.5% ethanol solution was found to evoke advance and delay phase shifts in the locomotor activity rhythm in the field mouseMus booduga. Through such pulsed administration of diazepam at various phases of circadian rhythm a phase response curve could be constructed. Phase advance occurred during early subjective day (CT 2) and phase delays were observed in the remaining phases. The shape of the diazepam phase response curve is similar to the general shape of the phase response curves generated by intraperitoneal injections of other benzodiazepines in hamsters. The phase shifting action of diazepam may be explained by its agonistic action on the neurotransmitter gamma-aminobutyric acid.     

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.     

Exercise elicits phase shifts and acute alterations of melatonin that vary with circadian phase

To examine the immediate phase-shifting effects of high-intensity exercise of a practical duration (1 h) on human circadian phase, five groups of healthy men 20-30 yr of age participated in studies involving no exercise or exposure to morning, afternoon, evening, or nocturnal exercise. Except during scheduled sleep/dark and exercise periods, subjects remained under modified constant routine conditions allowing a sleep period and including constant posture, knowledge of clock time, and exposure to dim light intensities averaging (+/-SD) 42 +/- 19 lx. The nocturnal onset of plasma melatonin secretion was used as a marker of circadian phase. A phase response curve was used to summarize the phase-shifting effects of exercise as a function of the timing of exercise. A significant effect of time of day on circadian phase shifts was observed (P < 0.004). Over the interval from the melatonin onset before exercise to the first onset after exercise, circadian phase was significantly advanced in the evening exercise group by 30 +/- 15 min (SE) compared with the phase delays observed in the no-exercise group (-25 +/- 14 min, P < 0.05). Phase shifts in response to evening exercise exposure were attenuated on the second day after exercise exposure and no longer significantly different from phase shifts observed in the absence of exercise. Unanticipated transient elevations of melatonin levels were observed in response to nocturnal exercise and in some evening exercise subjects. Taken together with the results from previous studies in humans and diurnal rodents, the current results suggest that 1) a longer duration of exercise exposure and/or repeated daily exposure to exercise may be necessary for reliable phase-shifting of the human circadian system and that 2) early evening exercise of high intensity may induce phase advances relevant for nonphotic entrainment of the human circadian system.