ENTRAINMENT OF HUMAN ORCADIAN RHYTHMS BY LIGHT-DARK CYCLES: A REASSESSMENT

Abstract— Man is the only eukaryotic organism in which it has been reported that the circadian system cannot be entrained to a 24-h period by a simple light-dark (LD) cycle. In this paper, we reexamine the evidence for that claim and demonstrate that there were some fundamental flaws in the experimental design of the previous studies on which this conclusion was based. We report new studies in which we tested the efficacy of LD cycles in entraining the circadian rhythms of human subjects living in isolation from environmental time cues. We found that the cyclic alternation of light and dark, when applied to human subjects in a comparable way to experiments in other species, was an effective entraining agent. Our results and a critical review of the literature indicate that a LD cycle alone can be an effective environmental synchronizer of the human circadian timing system. Other factors, such as the knowledge of time of day, social contacts, the feeding schedule, and the imposed rest-activity schedule may contribute to stable entrainment, although their relative strengths as synchronizers have yet to be determined.     

PHOTORECEPTIONS IN THE ECLOSION OF THE SILKWORM, Bombyx mort

When silkworms, Bombyx mori , were kept in a photoperiodic regime of LD 8:16 during pupal development, the moths showed overt bimodal eclosion rhythms with one peak in the dark period, and with the other peak at light-on. When the developing pupae were transferred from the photoperiodic cycle to continuous darkness, the eclosion of the dark period persisted with a free-running circadian rhythm, while the eclosion corresponding to the light-on peak was not shown. Vitamin A-deficiency. which brought about a loss of photoreceptive function in compound eyes, significantly reduced the light-on peak, but did not influence the timing of eclosion in the dark period. Covering compound eyes with black wax also reduced the light-on peak. These results show that the eclosion clock of the silkworm is most likely entrained by an extraretinal photoreceptor, but the eclosion at light-on is evoked by an exogenous light effect that is mediated via compound eyes     

Light effects in yeast: persisting oscillations in cell division activity and amino acid transport in cultures of Saccharomyces cerevisiae entrained by light-dark cycles.

Abstract— In addition to the direct inhibitory effects of visible light (cool-white fluorescent, < 3500 lux) on growth and amino acid transport previously reported for cultures of the yeast Saccharomyces cereui-siae (strain Y185 rho⁺) grown at 12°C in medium containing glucose, yeast carbon base, KH₂PO₄, ammonium ion and proline, we have found evidence for endogenous, light-entrainable, self-sustaining circadian and ultradian oscillators underlying both cell division and transport activity. Diurnal light (? 3000 lux) cycles (LD), imposed on cultures previously grown in the dark, phased or synchronized cell number increase to a 24-h period with bud release being confined primarily to the dark intervals (although not necessarily every cell divided during any given division ‘burst’). The observed division or budding rhythm freeran with a circadian period (?26h) only approximating 24 h for a number of days in constant darkness (DD) following prior entrainment by LD, thereby implicating an endogenous circadian clock. Further, a similar light-entrainable circadian rhythm in the uptake of “C-histidine or ¹4C-lysine occurred in nondividing (or very slowly dividing) cultures during the “stationary” (infradian) phase of growth synchronized by a 24-h LD cycle and then released into DD for as long as 10 days. Some experiments revealed a bimodal (ultradian) periodicity in both LD and DD with secondary peaks or shoulders occurring at intervals of ?12h, corresponding approximately to subjective ‘dawn’ and ‘dusk’. Transport in cultures of the Y185 rho^- petite mutant, which lacks cytochromes a/a₃, b and c_l, could not be synchronized by LD cycles, a finding that is consistent with the hypothesis that these chromophores may be primary photoreceptors for synchronization of rhythms in this microorganism.     

The effect of photoperiod and light irradiance on the antioxidant circadian system of two species of crayfish from different latitudes: Procambarus clarkii and P. digueti

This work was carried out to study the antioxidant circadian system of two species of crayfish of different latitude origin. We investigated (1) whether both species possess glutathione circadian rhythms and (2) whether both species' rhythms differ in their ability to synchronize to 24 h cycles. Two batches of Procambarus clarkii and P. digueti were kept in (1) light-dark (LD) 12:12 low irradiance (LI) cycles and then exposed to (2) 72 h of complete darkness, (3) LD 12:12 high irradiance (HI), (4) LD 20:4 LI and (5) LD 20:4 HI for 2 weeks. The midgut and hemolymph were sampled and reduced and oxidized glutathione as well as glutathione reductase and glutathione peroxidase were assayed. Cosinor and analysis of variance revealed differences between both species. Procambarus clarkii robust antioxidant circadian rhythms are able to entrain to all conditions resetting to lights on or off. However, the P. digueti weak circadian glutathione system did not entrain to the LD cycles, showing a random distribution of phases. In this species, LD 12:12 and 20:4 HI evidenced significant daily rhythms indicating a damped circadian antioxidative system that is enhanced by the effect of light. This suggests that each species' photoperiodic history determines the adaptive abilities of the circadian antioxidative mechanisms.     

Interactions between light and circadian rhythms in plant photoperiodism

Abstract— The timing mechanism in plant photoperiodism seems to involve two endogenous circadian rhythms: a light-on (dawn) rhythm and a light-off (dusk) rhythm. Following a period of darkness light may affect flowering without affecting the rhythms, or it may affect flowering by rephasing the rhythms. A hypothesis is presented concerning the mechanism of the interaction between illumination and endogenous rhythms based upon correlations between leaf movements and flowering responses in various photoperiodic treatments. The possible role played by phytochrome in the response is considered in relation to the effects of light quality on the responses.     

CIRCADIAN RHYTHM OF LOCOMOTOR BEHAVIOR IN A POPULATION OF Paramecium multimicronucleatum: ITS CHARACTERISTICS AS DERIVED FROM CIRCADIAN CHANGES IN THE SWIMMING SPEEDS AND THE FREQUENCIES OF AVOIDING RESPONSE AMONG INDIVIDUAL CELLS

Abstract The locomotor behavior of Paramecium is determined by two components: swimming speed and the frequency of avoiding responses. The circadian oscillations of these two components were examined in order to interpret characteristics of the circadian locomotor rhythm, previously found in a Paramecium population using an originally defined parameter, "traverse frequency" (Hasegawa et al ., 1978, 1982). In our present study, a modified version of the previously developed, fully computerized, close-up video/photoamplifier system was used. Results indicate that individual specimens swam fast and unidirectionally during the day, while at night, in a light–dark cycle, they swam slowly and frequently turned. This oscillatory pattern was sustained in continuous darkness, where fluctuation in the frequency of avoiding responses was a dominant characteristic. The time structure of the "random walk" of Paramecium behavior was also examined by constructing and stochastically testing histograms of the interval times between specimens consecutively traversing beneath an observation point. Statistical analyses of observation data indicated that the circadian organization of the two components by individual specimens resulted in a circadian accumulation/dispersal rhythm of the entire population. It was concluded that the circadian "traverse frequency" rhythm principally represented this circadian accumulation/dispersal rhythm.     

IN VIVO CHLOROPHYLL a FLUORESCENCE TRANSIENTS AND THE CIRCADIAN RHYTHM OF PHOTOSYNTHESIS IN GONYAULAX POLYEDRA

Abstract— The intensity of chlorophyll a fluorescence during the early part of fluorescence induction at O , initial fluorescence, and P, peak fluorescence, was higher during the day phase of the circadian cycle than during the night phase in continuous light (LL) conditions and was positively correlated with the rate of oxygen evolution. The circadian rhythm in fluorescence in LL persisted in the presence of 10μM 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which blocks electron flow from photo-system (PS) II in photosynthesis. The rhythmic changes in fluorescence intensity are consistent with a lower rate constant for radiationless transitions during the day phase than during the night phase of the circadian rhythmicity. The circadian changes in the intensity of fluorescence were abolished at 77K, which may indicate the importance of structural changes in membranes in circadian oscillations.     

RHYTHMIC REGULATION OF THE LIGHT-HARVESTING CHLOROPHYLL alb PROTEIN and THE SMALL SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE mRNA IN RYE SEEDLINGS

In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.     

Crayfish Procambarus clarkii shows circadian variations in different parameters of the GSH cycle

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant-darkness conditions for 24 or 72 h after they had spent 15 days under light-dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric method. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.     

CIRCADIAN RHYTHM OF EXTRARETINAL PHOTOSENSITIVITY IN HATCHLING ALLIGATORS, ALLIGATOR MISSISSIPPIENSIS

Hatchling American alligators, Alligator mississippiensis , display a diel rhythm of extra-retinal (non-pineal) photosensitivity. There is a bimodal pattern in the threshold fluence rate required for initiating and effecting extra-retinally-mediated phototaclic responses. Significantly greater threshold fluence rates are required in the photophase than the scotophasc of a 12 h light: 12 h dark cycle. Under constant darkness there continues an endogenous, apparently circadian rhythm of extra-retinal photosensitivity. In all cases there is a significantly greater photosensitivity at lower temperatures.     

Crayfish Procambarus clarkii retina and nervous system exhibit antioxidant circadian rhythms coupled with metabolic and luminous daily cycles

Based on previous work in which we proposed midgut as a putative peripheral oscillator responsible for circadian reduced glutathione (GSH) crayfish status, herein we investigated the retina and optic lobe-brain (OL-B) circadian GSH system and its ability to deal with reactive oxygen species (ROS) produced as a consequence of metabolic rhythms and light variations. We characterized daily and antioxidant circadian variations of the different parameters of the glutathione system, including GSH, oxidized glutathione (GSSG), glutathione reductase (GR) and glutathione peroxidase (GPx), as well as metabolic and lipoperoxidative circadian oscillations in retina and OL-B, determining internal and external GSH-system synchrony. The results demonstrate statistically significant bi- and unimodal daily and circadian rhythms in all GSH-cycle parameters, substrates and enzymes in OL-B and retina, as well as an apparent direct effect of light on these rhythms, especially in the retina. The luminous condition appears to stimulate the GSH system to antagonize ROS and lipid peroxidation (LPO) daily and circadian rhythms occurring in both structures, oscillating with higher LPO under dark conditions. We suggest that the difference in the effect of light on GSH rhythmic mechanisms of both structures for antagonizing ROS could be due to differences in glutathione-system coupling strength with the circadian clock.     

Circadian rhythms of the L-ascorbic acid level in Euglena and spinach

Plant defenses against photo-oxidative stress have been studied almost exclusively with respect to stress responses, and little is known about how non-enzymic antioxidants change under constant conditions without a time cue or an environmental stress. Here, we show that, in both the flagellated alga Euglena gracilis Z and the angiosperm Spinacia oleracea L., the potent antioxidant L-ascorbic acid (Asc) displays a circadian rhythm with a maximum at subjective midday, a physiological state reflecting that attained at noon under daily light/dark cycles. Thus, photosynthetic organisms can maximize antioxidant levels in anticipation of midday, when photo-oxidative stress is most severe. These results may partly explain the in-phase circadian UV-C resistance rhythm recently identified in the alga. However, the Asc, but not the resistance, rhythm wanes in continuous darkness. This suggests the presence of persistent circadian rhythms in the levels of other antioxidants in continuous darkness, which may account for the UV-C resistance rhythm.     

Effects of European mistletoe (Viscum album L. subsp. album) extracts on activity rhythms of the Syrian hamsters (Mesocricetus auratus)

Antitumor drugs may have some significant nervous system side-effects such as disrupted sleep, eating and drinking patterns. European Mistletoe has been investigated for many decades for its potential use against cancer. To test the hypothesis that the non-antineoplastic effects of mistletoe might be mediated by the actions on the circadian timing system, we have applied mistletoe extracts and vehicle and have measured locomotor activity, feeding and drinking rhythms under constant darkness. Four groups (vehicle, 20 mg kg(-1) ip injection, 40 mg kg(-1) ip injection and 6 g kg(-1) oral administration) were performed for both heat-treated and freeze-dried extracts. None of the administrations changed the locomotor activity, feeding and drinking rhythms in the groups except for 40 mg kg(-1) freeze-dried injected group. The locomotor activity levels decreased in 40 mg kg(-1) freeze-dried extract injected group. These results suggest that, side-effects of mistletoe on circadian timing system of the Syrian hamster depend on the dose and the preparation method of the extract.     

Circadian gating of photoinduction of commitment to cell-cycle transitions in relation to photoperiodic control of cell reproduction in Euglena

A novel type of circadian and photoperiodic control of the cell division cycle was found in photoautotrophic Euglena gracilis. When algae entrained to 24 h light-dark (LD) cycles (14 h L) were transferred to continuous darkness (DD) at the eighth hour of the final LD photoperiod, cell-cycle transition was arrested in phase G1, S or G2. The subsequent exposure of these dark-arrested cells to a 6 h light-break allowed the dark-arrested cells to undergo cell-cycle progression in DD, in a manner dependent on the circadian phase; maximum photoinduction occurred around dusk. Inhibitor experiments suggested that the photoinduced commitment of G2 cells to cell division required light for a signal originating in noncyclic photosynthetic electron transport (PET), particularly cytochrome b6-f but not for the metabolic energy required by the process. The fact that the circadian rhythm of photoinduction ran out-of-phase from that of noncyclic PET signaling suggests that the site of regulation by the former rhythm is downstream of noncyclic PET. The occurrence of maximum photoinduction around dusk suggests that the 'external coincidence' model of photoperiodic induction describes the activation of the photoinductive phase. Further evidence supporting this hypothesis is the relationship between cell reproduction and day length; the resulting sigmoidal curve indicates a combined effect of photosynthesizing period and circadian stimulation around dusk. Circadian control is shown to be an integral part of the mechanism for 24 h LD cycle-induced synchronous cell division.     

Determination of creatinine-related urinary uracil excretion in children by high-performance liquid chromatography

Excretion rates of uracil and thymine in children (n = 140) and circadian rhythms of urinary uracil excretion (n = 9) were investigated by reversed-phase high-performance liquid chromatography with ultraviolet detection. Excretion values were related to urinary creatinine determined by Jaffé's method. Creatinine-related uracil excretion was not dependent on age or sex. The values seemed to be distributed according to a Gaussian graph in both school children and those in hospital. The intra-individual range was 1.32-23.70 mg uracil per g creatinine over a four-day period in one subject. Uracil excretion seems to be somewhat lower during the night.     

TEMPERATURE-SENSITIVITY OF LIGHT-INDUCED PHASE SHIFTING OF THE CIRCADIAN CLOCK OF NEUROSPORA

Two new mutants of Neurospora craasa , designated hth-1 and hth-2 , have been isolated which allow clear expression of the circadian conidiation rhythm at high temperature (36°C). Both strains showed single-gene segregation and produced similar phenotypes but mapped to different genetic loci. These mutants allowed an analysis of the effect of temperature on (1) light-induced phase-shifting of the circadian rhythm, (2) period length of rhythm, and (3) growth rate. The amplitude of the phase response curve to light was drastically reduced as the temperature was increased from 25°C to 34°C. Phase advances were decreased more than phase delays. As previously reported (Sargent et al. , 1966), the period length of the rhythm is temperature-compensated below 30°C ( Q ₁₀˜ 1) but not well-compensated above 30°C ( Q ₁₀ 1.3–1.7). The decrease in amplitude of the light phase response curve occurred in both temperature ranges. Furthermore, the Q ₁₀ value was lowered by addition of yeast extract in the high temperature range but not in the low range. Q ₁₀ values for growth rate also differed in these strains both in the low temperature range (25–30°C) and the high temperature range (30–34°C).     

Circadian rhythms of resistance to UV-C and UV-B radiation in Euglena as related to "escape from light" and "resistance to light"

Radiation-induced stress, either from visible or UV light, is strongest at midday. We found that, in the absence of stress or time cues, Euglena gracilis Z was the most resistant to UV-C and UV-B at subjective midday, whether judged from immediate or reproductive survival. The circadian UV-resistance rhythms were free-running in stationary cultures under 1-h light/1-h dark cycles or continuous darkness, indicating that cell-cycle dependent DNA susceptibility to UV was not involved. We moreover examined what was the primary cause of the circadian UV resistance, estimated as the immediate cell survival. The half-maximal lethal dose (LD(50)) of UV-C at subjective midday (the most resistant phase) was 156 J/m(2), which is approximately 3-fold that at subjective midnight. The same was true for UV-B, except the LD(50) was approximately 13-fold that of UV-C. Temperature during UV irradiation had little effect, indicating that survival was not mediated via enzymatic reactions. Non-enzymatic antioxidants were added 5 min before UV irradiation. Dimethylsulfoxide (a hydroxyl radical scavenger) increased survival after UV-B, but had little effect after UV-C; conversely, sodium ascorbate increased survival after UV-C, but not after UV-B. These findings suggest that circadian rhythms of resistance to UVs involve a common mechanism for maximizing non-enzymatic antioxidative capacity at subjective midday, but the specific antioxidants differ.     

PHOTOCHEMICAL REACTIVITY OF MOLECULES TRAPPED IN SOL-GEL GLASSES*

The effects of different wavelengths of light on period, phase shifting, entrainment and after-effects of the circadian clock of the motile marine dinoflagellate Gonyaulax polyedra are described. Phase shifting and entrainment by light can be explained by the action of a single blue sensitive light input pathway. However, tonic effects of light on the period under free-running conditions, and also after-effects on period resulting from single 4 h light exposures, appear to involve two input pathways with different absorption and temperature characteristics. These results suggest different mechanisms for the control of phase and period of the circadian clock.     

EXTRARETINAL PHOTORECEFTION IN INSECTS

Abstract— Extraretinal photoreceptors are widespread among insects and function in the photoperiodic control of development and in the entrainment of circadian rhythms. The effects of light on the daily and seasonal regulation of brain neuroendocrine activity are mediated solely through extraretinal photoreceptors. In primitive insects, the eyes participate in the entrainment of nonendocrine circadian rhythms such as the locomotor rhythm. In more advanced forms, however, extraretinal pathways appear to be the only pathway for the entrainment of all rhythms thus far examined. But even in this latter case, the eyes sometimes effect a masking of the expression of the overt rhythm. An exact localization of the extraretinal receptors has not been accomplished, but in all studies to date they appear to be associated with the cerebral lobe region of the brain. Action spectra for photoperiodic responses have been determined for a number of insects. In general the responses are maximally sensitive in the blue with a marked decline in the red although exceptions do exist. Complete action spectra for circadian responses have been determined only for two insects. In both cases a plateau of sensitivity extends through the blue with a steep drop at longer wavelengths. From the action spectra data, the extraretinal receptors appear to have a threshold sensitivity less than 3 times 10^-2 J/m². The pigment nature of the receptor is unknown although it appears not to be a carotenoid derivative.