The timing of the human circadian clock is accurately represented by the core body temperature rhythm following phase shifts to a three-cycle light stimulus near the critical zone

A double-stimulus experiment was conducted to evaluate the phase of the underlying circadian clock following light-induced phase shifts of the human circadian system. Circadian phase was assayed by constant routine from the rhythm in core body temperature before and after a three-cycle bright-light stimulus applied near the estimated minimum of the core body temperature rhythm. An identical, consecutive three-cycle light stimulus was then applied, and phase was reassessed. Phase shifts to these consecutive stimuli were no different from those obtained in a previous study following light stimuli applied under steady-state conditions over a range of circadian phases similar to those at which the consecutive stimuli were applied. These data suggest that circadian phase shifts of the core body temperature rhythm in response to a three-cycle stimulus occur within 24 h following the end of the 3-day light stimulus and that this poststimulus temperature rhythm accurately reflects the timing of the underlying circadian clock.     

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.     

Light and serotonin interact in affecting the circadian system of Aplysia

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt rhythm of spontaneous compound action potentials. The current study evaluated the influence of serotonin on light-induced phase shifts of this ocular rhythm. The application of serotonin in combination with light was found to have profound and interactive effects on the magnitude of the resulting phase shifts. Further, the phase shifts that resulted from the interaction between light and serotonin appeared to be phase dependent, i.e., the application of serotonin inhibited the phase shifting effects of light during one part of the circadian cycle but enhanced them during another. Finally, the results show that the interaction between light and serotonin is dependent upon the sequence in which these two treatments are paired. These data, coupled with previous findings, suggest that serotonin may act to modulate light's phase shifting effects on the ocular pacemaker in Aplysia.Abbreviations CAP compound action potential - ASW artificial sea water - CT circadian time - 5-HT serotonin     

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The current study evaluated the effect of FMRFamide on both light and serotonin induced phase shifts of this rhythm. The application of FMRFamide was found to block serotonin induced phase shifts but, by itself, FMRFamide did not cause significant phase shifts. Furthermore, the effects of FMRFamide on light-induced phase shifts appeared to be phase dependent (i.e., the application of FMRFamide inhibited light-induced phase delays but actually enhanced the magnitude of phase advances). As in Aplysia, the eye of Bulla gouldiana also contains a circadian pacemaker. In Bulla, FMRFamide prevented light-induced phase advances and delays. Although FMRFamide alone generated phase dependent phase shifts, it did not cause phase shifts at the phases where it blocked the effects of light. These data demonstrate that FMRFamide can have pronounced modulatory effects on phase shifting inputs to the ocular pacemakers of both Aplysia and Bulla.Abbreviations ASW artificial seawater - CAP compound action potential - CT circadian time - 5-HT serotonin     

Light pulses suppress responsiveness within the mouse photic entrainment pathway

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single "saturating" light pulses ("white" fluorescent light; approximately 1876 [microW; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels < or = 12% of normal. After 2 h, the responsiveness was < or = 42% of normal, and by 4 h, responsiveness had recovered to levels that were < or = 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained < or = 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: I. Cells in Darkness

We have developed protocols for phase shifting the circadian rhythm of Chlamydomonas reinhardtii by light pulses. This paper describes the photobiology of phase-resetting the Chlamydomonas clock by brief (3 seconds to 15 minutes) light pulses administered during a 24 hour dark period. Its action spectrum exhibited two prominent peaks, at 520 and 660 nanometers. The fluence at 520 nanometers required to elicit a 4 hour phase shift was 0.2 millimole photon per square meter, but the pigment that is participating in resetting the clock under these conditions is unknown. The fluence needed at 660 nanomoles to induce a 4 hour phase shift was 0.1 millimole photon per square meter, which is comparable with that needed to induce the typical low fluence rate response of phytochrome in higher plants. However, the phase shift by red light (660 nanometers) was not diminished by subsequent administration of far-red light (730 nanometers), even if the red light pulse was as short as 0.1 second. This constitutes the first report of a regulatory action by red light in Chlamydomonas.     

Phototaxis by the Dinoflagellate Gymnodinium splendens Lebour*

A microscope-television system was used to monitor quantitatively the behavior of Gymnodinium splendens Lebour in response to light. The predominant behavioral sequence upon stimulation is (a) an initial 2–5 sec cessation of movement (stop-response) followed by (b) positive phototaxis. The action spectra for each response are identical, having maxima at 450 and 280 nm. Upon measuring the percent response to a range of stimulus intensities, it is apparent that a stop-response is not a behavioral prerequisite for phototaxis. An identical circadian rhythm in photoresponsiveness is observed for phototaxis and for the stop-response with greatest light sensitivity occurring during the first 4 hr of the entrained light period. The implication of phototactic sensitivity and the phototactic circadian rhythm in diurnal vertical migration is discussed.     

Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters

Circadian pacemakers respond to light pulses with phase adjustments that allow for daily synchronization to 24-h light-dark cycles. In Syrian hamsters, Mesocricetus auratus, light-induced phase shifts are larger after entrainment to short daylengths (e.g., 10 h light:14 h dark) vs. long daylengths (e.g., 14 h light:10 h dark). The present study assessed whether photoperiodic modulation of phase resetting magnitude extends to nonphotic perturbations of the circadian rhythm and, if so, whether the relationship parallels that of photic responses. Male Syrian hamsters, entrained for 31 days to either short or long daylengths, were transferred to novel wheel running cages for 2 h at times spanning the entire circadian cycle. Phase shifts induced by this stimulus varied with the circadian time of exposure, but the amplitude of the resulting phase response curve was not markedly influenced by photoperiod. Previously reported photoperiodic effects on photic phase resetting were verified under the current paradigm using 15-min light pulses. Photoperiodic modulation of phase resetting magnitude is input specific and may reflect alterations in the transmission of photic stimuli.     

Circadian modulation of light-induced locomotion responses in Drosophila melanogaster

The relationship between light and the circadian system has long been a matter of discussion. Many studies have focused on entrainment of light with the internal biological clock. Light also functions as an environmental stimulus that affects the physiology and behaviour of animals directly. In this study, we used light as an unexpected stimulus for flies at different circadian times. We found that wildtype flies showed circadian changes in light-induced locomotion responses. Elevation of locomotor activity by light occurred during the subjective night, and performance in response to light pulses declined to trough during the subjective day. Moreover, arrhythmic mutants lost the rhythm of locomotion responses to light, with promotion of activity by light in timeless(01)mutants and inhibition of activity by light in Clock(ar)mutants. However, neither ablation of central oscillators nor disturbance of the functional clock inside compound eyes was sufficient to disrupt the rhythm of light responses. We show that, compound eyes, which have been identified as the control point for normal masking (promotion of activity by light), are sufficient but not necessary for paradoxical masking (suppression of activity by light) under high light intensity. This, taken together with the clear difference of light responses in wildtype flies, suggests that two different masking mechanisms may underlie the circadian modulation of light-induced locomotion responses.     

Effects of macromolecule synthesis inhibitors on light-induced phase shift of the circadian rhythm in melatonin release from the cultured pineal organ of a teleost, ayu (Plecoglossus altivelis)

Effects of macromolecule synthesis inhibitors on the light-induced phase shift of the circadian clock in the photoreceptive pineal organ of a teleost, ayu (Plecoglosus altivelis) were investigated using melatonin release as an indicator. A single light pulse during the early- and late-subjective night delayed and advanced the phase of the circadian rhythm in melatonin release, respectively. During the late subjective-night, protein synthesis inhibitor cycloheximide (CHX) delayed the rhythm while RNA synthesis inhibitor 5,6-dichlorobenzimidazole riboside (DRB) had little effect. Light-induced phase advance was diminished by the treatment of CHX but not by DRB. During the early subjective-night, DRB, CHX, light and combination of these (DRB+light, CHX+light) all phase-delayed the rhythm. There were no additive effects of light and DRB or CHX. These results indicate that macromolecule synthesis is somehow involved in generation of circadian oscillation, and that de novo protein synthesis is required for light-induced phase shift of the circadian clock in the ayu pineal organ.     

Twofold effect of blue light on a circadian rhythm in Acetabularia

Abstract

The circadian chloroplast migration in Acetabularia mediterranea was monitored by continuously measuring the transmission of the cells near the apex. Under continuous red light the amplitude of the rhythm decreased rapidly within a few days. However, circadian changes of chloroplast density were still detectable even after 28 days of red light, indicating the persistence of the rhythm. When blue light was added after red light preirradiation of several days phase shifts were observed which were expressed as advances as well as delays. The period of the rhythm proved to be strongly dependent on the intensity of the continuous blue light which was given in addition to red light. Different red light intensities did not change the period. The occurrence of both effects indicates that the sensory transduction of blue light photoreception in Acetabularia works in two different ways: quanta counting processes and processes of light intensity measurement.     

Modeling the molecular regulatory mechanism of circadian rhythms in Drosophila

Thanks to genetic and biochemical advances on the molecular mechanism of circadian rhythms in Drosophila, theoretical models closely related to experimental observations can be considered for the regulatory mechanism of the circadian clock in this organism. Modeling is based on the autoregulatory negative feedback exerted by a complex between PER and TIM proteins on the expression of per and tim genes. The model predicts the occurrence of sustained circadian oscillations in continuous darkness. When incorporating light-induced TIM degradation, the model accounts for damping of oscillations in constant light, entrainment of the rhythm by light-dark cycles of varying period or photoperiod, and phase shifting by light pulses. The model further provides a molecular dynamical explanation for the permanent or transient suppression of circadian rhythmicity triggered in a variety of organisms by a critical pulse of light. Finally, the model shows that to produce a robust rhythm the various clock genes must be expressed at the appropriate levels since sustained oscillations only occur in a precise range of parameter values. BioEssays 22:84-93, 2000.     

Mutants with Altered Sensitivity to a Calmodulin Antagonist Affect the Circadian Clock in Neurospora Crassa

Two newly isolated mutant strains of Neurospora crassa, cpz-1 and cpz-2, were hypersensitive to chlorpromazine with respect to mycelial growth but responded differently to the drug with respect to the circadian conidiation rhythm. In the wild type, chlorpromazine caused shortening of the period length of the conidiation rhythm. Pulse treatment with the drug shifted the phase and inhibited light-induced phase shifting in Neurospora. By contrast to the wild type, the cpz-2 strain was resistant to these inhibitory effects of chlorpromazine. Inhibition of cpz-2 function by chlorpromazine affected three different parameters of circadian conidiation rhythm, namely, period length, phase and light-induced phase shifting. These results indicate that the cpz-2 gene must be involved in or related closely to the clock mechanism of Neurospora. By contrast, the cpz-1 strain was hypersensitive to chlorpromazine with respect to the circadian conidiation rhythm.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: II. Illuminated Cells

The action spectrum for resetting the phase of the circadian clock in Chlamydomonas reinhardtii is different depending upon whether the light stimuli are presented to cells that were in darkness versus dim illumination before stimulation. In this report, we show that phase resetting of illuminated cells appears to be mediated by components of the photosynthetic apparatus. This conclusion is based upon the action spectrum for phase-shifting illuminated cells (which looks like that for photosynthesis) and upon the fact that inhibitors of photosynthetic electron transport also inhibit the light-induced phase shift of illuminated cells. Both of these characteristics differ from that of cells taken from darkness. We, therefore, believe that at least two resetting pathways for this circadian clock exist and that both of these pathways are ecologically significant.     

Synthesis of mRNA is Required for Light-Induced Phase Shifting of the Circadian Conidiation Rhythm in Neurospora crassa

The process of light-induced phase shifting was investigatedin Neurospora crassa using a liquid culture system and a combinationof treatment with a nucleoside analogue and light. 5-Azacytidineinhibited the light-induced phase shifting at all phases thatwere sensitive to light. Electrophoresis of proteins that weresynthesized in a translation system in vitro showed that 5-azacytidineinhibited the synthesis of most mRNAs. The inhibition of mRNAsynthesis was correlated with the inhibition of light-inducedphase shifting. An excess of cytidine completely overcame theinhibition by 5-azacytidine of both light-induced phase shiftingand mRNA synthesis. Other analogues, namely, 6-azauridine and6-methylpurine, failed to inhibit either the light-induced phaseshifting or the synthesis of mRNA. Two-dimensional gel electrophoresisshowed that the levels of expression of nine mRNAs were affectedby light within 30 min after irradiation. By contrast, the oscillatorof the circadian clock was not affected by pulse treatment with5-azacytidine alone because such treatment failed to shift thephase of the circadian rhythm at any phase. These results indicatethat newly synthesized mRNA(s) is required during the processof signal transduction, from the light-perceiving system tothe circadian clock, for light-induced phase shifting in Neurospora. (Received October 17, 1994; Accepted January 23, 1995)     

Extracellular pH is under circadian control in Gonyaulax polyedra and forms a metabolic feedback loop

This study investigates the relationship between the circadian clock and metabolism based on recordings of the extracellular pH in cultures of the marine dinoflagellate, Gonyaulax polyedra. In light-dark cycles, pH of the medium rises during the light phase and declines in the dark. The amplitude of this pH-rhythm correlates with light intensity, indicating photosynthesis (and respiration) as the driving force. The recorded extracellular pH changes probably reflect the need to control intracellular pH in spite of pH-modifying reactions. The daily pH-changes are under control of the circadian clock because they continue to oscillate with a circa-24 h period in constant light, albeit with a smaller amplitude. Similar to other circadian output rhythms, the pH rhythm depends (amplitude and phase) on nitrate levels in the medium. Both the bioluminescence and the pH rhythm can also be shifted by extracellular pH-changes although Gonyaulax is rarely exposed to significant pH changes in its marine ecosystems (except for highly dense algal blooms). Because intracellular proton levels are both affecting circadian input and output they form a feedback loop with the Gonyaulax circadian system indicating complex interactions between metabolism and the circadian clock.     

ELF3 modulates resetting of the circadian clock in Arabidopsis

The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.     

Binocular contributions to the responsiveness and integrative capacity of the circadian rhythm system to light

The retinohypothalamic tract (RHT), a monosynaptic retinal projection to the SCN, is the major path by which light entrains the circadian system to the external photoperiod. The circadian system of rodents effectively integrates or counts photons, and the magnitude of the rhythm phase response is proportional to the total energy of the photic stimulus. In the present studies, responsiveness to light and integrative capacity of the circadian system were tested in hamsters after reduction of retinal photoreceptor input by 50%. At CT 19, animals in constant darkness with or without unilateral retinal occlusion were exposed to 1 of 6 irradiances of 5-min white-light pulses ranging from 0.0011 to 70 microW/cm(2) or 5 white-light pulses of 0.6 microW/cm(2) with durations ranging from 0.25 to 150.0 min. Assessment of light-induced circadian rhythm phase response and Fos expression in the SCN by these animals revealed that a 50% reduction in input from photoreceptors stimulated directly with light caused a decrease in responsiveness to the longest duration and highest irradiance pulses presented. Despite this effect, both the magnitude of Fos induction in the SCN and phase-shift response remained directly proportional to the total energy in the photic stimuli. The results support the view that a reciprocal relationship between stimulus irradiance and duration persists despite the 50% reduction in retinal photoreceptor input. The mechanism of integration neither resides in the retina nor in the RHT.     

Light activates the adrenal gland: timing of gene expression and glucocorticoid release

Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamo-adenohypophysial axis. The abolishment after SCN lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The magnitude of corticostereone response is dose dependently correlated with the light intensity. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.