Exotic photoperiods induce and entrain split circadian activity rhythms in hamsters

The split circadian activity rhythm that emerges in hamsters after prolonged exposure to constant light has been a theoretical cornerstone of a multioscillator view of the mammalian circadian pacemaker. The present study demonstrates a novel method for splitting hamster circadian rhythms and entraining them to exotic light:dark cycles. Male Syrian hamsters previously maintained on a 14-h day and 10-h night were exposed to a second 5-h dark phase in the afternoon. The 10-h night was progressively shortened until animals experienced two 5-h dark phases beginning 10 h apart. Most hamsters responded by splitting their activity rhythms into two components associated with the afternoon and nighttime dark phases, respectively. Each activity component was entrained to this light:dark:light:dark cycle. Transfer of split hamsters to constant darkness resulted in rapid joining of the two activity components with the afternoon component associated with onset of the fused rhythm. In constant light, the nighttime component corresponded to activity onset of the fused rhythm, but splitting emerged again at an interval characteristic for this species. The results place constraints on multi-oscillator models of circadian rhythms and offer opportunities to characterize the properties of constituent circadian oscillators and their interactions.     

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.     

Characterization of transcriptional oscillation of an Arabidopsis homolog of PnC401 related to photoperiodic induction of flowering in Pharbitis nil

AtC401 is an Arabidopsis homolog of PnC401 that is related to photoperiodic induction of flowering in Pharbitis nil. These genes show free-running rhythms. To study the free-running rhythm of AtC401, we fused a firefly luciferase reporter to the AtC401 promoter and transformed it into Arabidopsis plants. The observed bioluminescence oscillated under continuous light and continuous dark only with sucrose supplementation. The free-running period of bioluminescence was temperature-compensated between 22 degrees C and 30 degrees C. Light-pulse experiments under continuous darkness produced a phase-response curve typical of circadian rhythms. We conclude that rhythmic expression of AtC401 is controlled by a circadian oscillator.     

Time-restricted feeding entrains daily rhythms of energy metabolism in mice

Energy metabolism, oxygen consumption rate (VO2), and respiratory quotient (RQ) in mice were monitored continuously throughout 12:12-h light-dark cycles before, during, and after time-restricted feeding (RF). Mice fed ad libitum showed robust daily rhythms in both parameters: high during the dark phase and low during the light phase. The daily profile of energy metabolism in mice under daytime-only feeding was reversed at the beginning of the first fasting night. A few days after daytime-only feeding began, RF also reversed the circadian core body temperature rhythm. Moreover, RF for 6 consecutive days shifted the phases of circadian expression patterns of clock genes in liver significantly by 8-10 h. When mice were fed a high-fat (HF) diet ad libitum, the daily rhythm of RQ dampened day by day and disappeared on the sixth day of RF, whereas VO2 showed a robust daily rhythm. Mice fed HF only in the daytime had reversed VO2 and RQ rhythms. Similarly, mice fed HF only in the daytime significantly phase shifted the clock gene expression in liver, whereas ad libitum feeding with HF had no significant effect on the expression phases of liver clock genes. These results suggested that VO2 is a sensitive indicator of entrainment in the mouse liver. Moreover, physiologically, it can be determined without any surgery or constraint. On the basis of these results, we hypothesize that a change in the daily VO2 rhythm, independent of the energy source, might drive phase shifts of circadian oscillators in peripheral tissues, at least in the liver.     

Daily Rhythms of P‐glycoprotein Expression in Mice

Recent studies have shown the gene expression of several transporters to be circadian rhythmic. However, it remains to be elucidated whether the expression of P‐glycoprotein, which is involved in the transport of many medications, undergoes 24 h rhythmicity. To address this issue, we investigated daily profiles of P‐glycoprotein mRNA and protein levels in peripheral mouse tissues. In the liver and intestine, but not in the kidney, Abcb1a mRNA expression showed clear 24 h rhythmicity. On the other hand, Abcb1b and Abcb4, the other P‐glycoprotein genes, did not exhibit significant rhythmic expression in the studied tissues. In the intestine, levels of whole P‐glycoprotein also exhibited a daily rhythm, with a peak occurring in the latter half of the light phase and a trough at the onset of the light phase. Consistent with the day‐night change of P‐glycoprotein level, the ex vivo accumulation of digoxin, an Abcb1a P‐glycoprotein substrate, into the intestinal segments at the onset of dark phase was significantly lower than it was at the onset of the light phase. Thus, Abcb1a P‐glycoprotein expression, and apparently its function, are 24 h rhythmic at least in mouse intestine tissue. This circadian variation might be involved in various chronopharmacological phenomena.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

Rhythms in glutamine synthetase activity, energy charge, and glutamine in sunflower roots

Roots of sunflower plants (Helianthus annuus L. var. Mammoth Russian) subjected to L12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the `light phase' and one in the `dark phase.' Root energy charge (EC = ATP+½ADP/ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.

Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms.

     

Synchronization of Mating Reactivity Rhythms in Populations of Paramecium bursaria

Cell populations of Paramecium bursaria show arhythmic mating reactivity after exposure to constant light (LL) for more than 2 wk. After this arhythmic population is exposed to darkness for 9 h, the mating reactivity rhythm of the cell population reappears. The phases of rhythms in individual cells are synchronized to each other. When the arhythmic population in constant light is exposed to dark pulses of various durations, the first peak of the recovered mating reactivity rhythm appears 6 h after the end of the dark pulse. Thus, in the case of dark pulses to cells in LL, the transition from dark to light sets the phase of the subsequent mating reactivity rhythm. When an arhythmic population in LL is transferred to constant darkness (DD), a rhythm of mating reactivity also appears and, in this case, the first peak of the rhythm occurs 18 h after the LL to DD transition. Therefore, arhythmic populations of cells in LL can be synchronized by either a dark pulse or by transition to continuous darkness. When the arhythmic populations in LL were transferred to various light/dark (LD) cycles, the mating reactivity rhythms entrained to LD cycles of 18 to 30 h in duration. Finally, mating rhythms can also be synchronized by treatment with puromycin (400 μg/ml for 6–18 h).     

The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

Circadian rhythms in the green sunfish retina

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.     

Circadian rhythmicity in dopamine content of mammalian retina: role of the photoreceptors

Dopamine, the predominant retinal catecholamine, is a neurotransmitter and neuromodulator known to regulate light-adaptive retinal processes. Because dopamine influences several rhythmic events in the retina it is also a candidate for a retinal circadian signal. Using high performance liquid chromatography (HPLC), we have tested whether dopamine and its breakdown products are rhythmic in Royal College of Surgeons (RCS) rats with normal and dystrophic retinas. In both normal and mutant animals entrained to a 12-h light/12-h dark cycle, we found robust daily rhythms of dopamine and its two major metabolites. To address circadian rhythmicity of dopamine content, rats were entrained to light/dark cycles and released into constant darkness, using the circadian rhythm of wheel-running activity as a marker of each individual's circadian phase. Circadian rhythms of dopamine and metabolite content persisted in both wild type and retinally degenerate animals held for two weeks in constant darkness. Our results demonstrate for the first time clear circadian rhythms of dopamine content and turnover in a free-running mammal, and suggest that rods and cones are not required for dopamine rhythmicity.     

Age-related effects on the biological clock and its behavioral output in a primate

In humans, activity rhythms become fragmented and attenuated in the elderly. This suggests an alteration of the circadian system per se that could in turn affect the expression of biological rhythms. In primates, very few studies have analyzed the effect of aging on the circadian system. The mouse lemur provides a unique model of aging in non-human primates. To assess the effect of aging on the circadian system of this primate, we recorded the circadian and daily rhythms of locomotor activity of mouse lemurs of various ages. We also examined age-related changes in the daily rhythm of immunoreactivities for vasoactive intestinal polypeptide (VIP) and arginine-vasopressin (AVP) in suprachiasmatic nucleus neurons (SCN), two major peptides of the biological clock. Compared to adult animals, aged mouse lemurs showed a significant increase in daytime activity and an advanced activity onset. Moreover, when maintained in constant dim red light, aged animals exhibited a shortening of the free-running period compared to adult animals. In adults, AVP immunoreactivity (ir) peaked during the second part of the day, and VIP ir peaked during the night. In aged mouse lemurs, the peaks of AVP ir and VIP ir were significantly shifted with no change in amplitude. AVP ir was most intense at the beginning of the night; whereas, VIP ir peaked at the beginning of the daytime. A weakened oscillator could account for the rhythmic disorders often observed in the elderly. Changes in the daily rhythms of AVP ir and VIP ir may affect the ability of the SCN to transmit rhythmic information to other neural target sites, and thereby modify the expression of some biological rhythms.     

Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Photoperiod Differentially Affects the Expression of Three mPer Genes in the Mouse Heart

To investigate the mechanism that controls circadian rhythms in mammalian peripheral tissues, we housed mice in short days (6 h light: 18 h dark) or long days (18 h light: 6 h dark) and examined the rhythmic expression patterns of the mammalian clock genes mPer1 , mPer2 and mPer3 and a clock-controlled gene Dbp in the mouse heart. Northern blot analyses showed that peak levels of mPer1 mRNA expression in long days were about 50 % higher than those in short days. On the contrary the amplitude of the mPer2 mRNA peak in long days was about 25 % lower than that in short days. We could not find any effect of photoperiod on either the amplitude or waveform of the rhythms of mPer3 and Dbp mRNAs. Photoperiod differentially affected the expression of three mPer genes even in a peripheral tissue of mice.     

CIRCADIAN GROWTH RHYTHM IN JUVENILE SPOROPHYTES OF LAMINARIALES (PHAEOPHYTA)1

A circadian rhythm in growth was detected by computer-aided image analysis in 3–4-cm-long, juvenile sporophytes of the kelp species Pterygophora California Rupr. and in seven Laminaria spp. In P. californica, the free-running rhythm occurred in continuous white fluorescent light, had a period of 26 h at 10°or 15°C, and persisted for at least 2 weeks in white or blue light. The rhythm became insignificant in continuous green or red light after 3 cycles. Synchronization by white light-dark regimes, e.g. by 16 h light per day, resulted in an entrained period of 24 h and in a shift of the circadian growth minimum into the middle of the light phase. A morning growth peak represented the decreasing portion of the circadian growth curve, and an evening peak the increasing portion. The circadian growth peak was not visible during the dark phase, because growth rate decreased immediately after the onset of darkness. At night, some growth still occurred at 16 or 12 h light per day, whereas growth stopped completely at 8 h light per day, as in continuous darkness. During 11 days of darkness, the thallus area became reduced by 3.5%, but growth rate recovered in subsequent light–dark cycles, and the circadian growth rhythm reappeared in subsequent continuous light.     

Ontogeny of melatonin, Per2 and E4bp4 light responsiveness in the chicken embryonic pineal gland.

The chicken pineal gland possesses the capacity to generate circadian oscillations, is able to synchronize to external light:dark cycles and can generate an hormonal output--melatonin. We examined the light responses of the chicken pineal gland and its effects on melatonin and Per2, Bmal1 and E4bp4 expression in 19-day old embryos and hatchlings during the dark phase, subjective light phase and in constant darkness. Expression of Per2 and E4bp4 were rhythmic under light:dark conditions, but the rhythms of E4bp4 and Bmal1 mRNA did not persist in constant darkness in 19-day old embryos. Per2 mRNA expression persisted in constant darkness, but with a reduced amplitude. Per2 expression was inducible by light only during the subjective day. Melatonin release was inhibited by light only at end of the dark phase and during the subjective light phase in embryos. Our data demonstrate that the embryonic avian pineal pacemaker is light sensitive and can generate rhythmic output, however the effects of light were diminished in chick embryos in compared to hatchlings.