Old flies have a robust central oscillator but weaker behavioral rhythms that can be improved by genetic and environmental manipulations

Sleep-wake cycles break down with age, but the causes of this degeneration are not clear. Using a Drosophila model, we addressed the contribution of circadian mechanisms to this age-induced deterioration. We found that in old flies, free-running circadian rhythms (behavioral rhythms assayed in constant darkness) have a longer period and an unstable phase before they eventually degenerate. Surprisingly, rhythms are weaker in light-dark cycles and the circadian-regulated morning peak of activity is diminished under these conditions. On a molecular level, aging results in reduced amplitude of circadian clock gene expression in peripheral tissues. However, oscillations of the clock protein PERIOD (PER) are robust and synchronized among different clock neurons, even in very old, arrhythmic flies. To improve rhythms in old flies, we manipulated environmental conditions, which can have direct effects on behavior, and also tested a role for molecules that act downstream of the clock. Coupling temperature cycles with a light-dark schedule or reducing expression of protein kinase A (PKA) improved behavioral rhythms and consolidated sleep. Our data demonstrate that a robust molecular timekeeping mechanism persists in the central pacemaker of aged flies, and reducing PKA can strengthen behavioral rhythms.     

Circadian rhythm of locomotor activity in the Japanese honeybee, Apis cerana japonica

Abstract.  To reveal circadian characteristics and entrainment mechanisms in the Japanese honeybee Apis cerana japonica , the locomotor-activity rhythm of foragers is investigated under programmed light and temperature conditions. After entrainment to an LD 12 : 12 h photoperiodic regime, free-running rhythms are released in constant dark (DD) or light (LL) conditions with different free-running periods. Under the LD 12 : 12 h regime, activity offset occurs approximately 0.4 h after lights-off transition, assigned to circadian time (Ct) 12.4 h. The phase of activity onset, peak and offset, and activity duration depends on the photoperiodic regimes. The circadian rhythm can be entrained to a 24-h period by exposure to submultiple cycles of LD 6 : 6 h, as if the locomotive rhythm is entrained to LD 18 : 6 h. Phase shifts of delay and advance are observed when perturbing single light pulses are presented during free-running under DD conditions. Temperature compensation of the free-running period is demonstrated under DD and LL conditions. Steady-state entrainment of the locomotor rhythm is achieved with square-wave temperature cycles of 10 °C amplitude, but a 5 °C amplitude fails to entrain.     

Transplanted Drosophila excretory tubules maintain circadian clock cycling out of phase with the host

Circadian rhythms in behaviors and physiological processes are driven by conserved molecular mechanisms involving the rhythmic expression of clock genes in the brains of animals [1]. The persistence of similar molecular rhythms in peripheral tissues in vitro [2] [3] suggests that these tissues contain self-sustained circadian clocks that may be linked to rhythmic physiological functions. It is not known how brain and peripheral clocks are organized into a synchronized timing system; however, it has been assumed that peripheral clocks submit to a master clock in the brain. To address this matter we examined the expression of two clock genes, period (per) and timeless (tim), in host and transplanted abdominal organs of Drosophila. We found that excretory organs in tissue culture display free-running, light-sensitive oscillations in per and tim gene activity indicating that they house self-sustained circadian clocks. To test for humoral factors, we monitored cycling of the TIM protein in excretory tubules transplanted into host flies entrained to an opposite light-dark cycle. We show that the clock protein in the donor tubules cycled out of phase with that in the host tubules, indicating that different organs may cycle independently, despite sharing the same hormonal milieu. We suggest that one way to achieve circadian coordination of physiological sub-systems in higher animals may be through the direct entrainment of light-sensitive clocks by environmental signals.     

Temperature synchronization of the Drosophila circadian clock

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.     

Light and temperature entrainment of a locomotor rhythm in honeybees

Abstract. The circadian locomotor (walking) rhythms of forager honeybees (Apis mellifera ligustica L.) were entrained to eight different 24 h light-dark cycles. The phases of activity onset, peak activity, and offset were correlated with the lights-off transition, suggesting lights-off as the primary zeitgeber for the rhythm. Further support for this hypothesis was provided by LD 1:23 experiments, in which entrainment occurred when the light pulse was situated at the end, but not at the beginning, of the subjective photophase. Steady-state entrainment of the locomotor rhythm was achieved with square-wave temperature cycles of 10^oC amplitude under constant dark: most of the activity occurred within the early thermophase. Smaller amplitude temperature cycles yielded relative coordination of the rhythm. Interactions of temperature and light-dark cycles resulted in entrainment patterns different from those elicited in response to either cycle alone or those formed by a simple combination of the two separate responses. Furthermore, temperature cycles having amplitudes insufficient for entrainment of the rhythm nevertheless modified the pattern of entrainment to light - dark cycles, suggesting a synergism of light and temperature effects on the underlying circadian clock system.     

Cryptochrome-positive and -negative clock neurons in Drosophila entrain differentially to light and temperature

The blue-light photoreceptive protein Cryptochrome (CRY) plays an important role in the light synchronization of the Drosophila circadian clock. Previously, we found that among the approximately 150 clock neurons, many but not all neurons express CRY. We speculated that the CRY-positive pacemaker neurons may be especially important for light entrainment, whereas the CRY-negative neurons may be important for other environmental cues, for example, temperature. To investigate this hypothesis, we tested the entrainability of the clock neurons to out-of-phase light and temperature cycles. When light-dark or light-dim light cycles were shifted by 12 h with respect to temperature cycles, behavioral rhythms of wild-type flies were re-entrained by the light cycles. In this condition, we found that TIMELESS (TIM) level was strongly influenced by the temperature cycles in many CRY-negative clock neurons, suggesting that the CRY-negative neurons have higher sensitivity to temperature. Under the same conditions, cry-null mutants entrained to the temperature cycles or very slowly re-entrained to light-dark cycles. Our results suggest that there are 2 types of clock neurons having differential sensitivities to light and temperature, and CRY is a key component for the preferential entrainment to light.     

Significance of Nonparametric Light Effects in Entrainment of Circadian Rhythms in Owl Monkeys (Aotus Lemurinus Griseimembra) by Light-Dark Cycles

In a total of 12 adult Colombian owl monkeys, Aotus lemurinus griseimembra, the significance of nonparametric light effects for the entrainment of the circadian system by light-dark (LD) cycles was studied by carrying out (a) phase-response experiments testing the phase-shifting effect of 30-min light pulses (LPs) of 250 lx applied at various phases of the free-running circadian activity rhythm (LL 0.2 lx) and (b) synchronization experiments testing the entraining effect of 24-h single LP photoperiods consisting of 30-min L of 80 lx and 23.5-h D of 0.5 lx (sP 0.5) and skeleton photoperiods consisting of two 30-min LPs of 80 lx, given against a background illuminance of 0.5 lx either symmetrically at 12-h intervals (PP 12:12) or asymmetrically at 9- and 15-h intervals (PP 9:15). The phase-response characteristics in Aotus, as evidenced by the phase-response curve, generally correspond to those of nocturnal rodents, proving that this neotropical simian primate chronobiologically is a genuine nocturnal species. When free-running with a spontaneous period close to 24 h (24.3 ± 0.1 h), the PP 12:12 produced entrainment in only two of five owl monkeys, whereas the sP 0.5 entrained four of them. The PP 9:15, however, brought about stable entrainment of the circadian rhythms of locomotor activity, feeding activity, and core temperature in all animals tested (n = 8). Changes in phase position of the activity time with the endogenous rhythm entrained by a PP 12:12, by an sP 0.5, or by a PP 9:15 give evidence that both LPs of a skeleton photoperiod contribute to the phase setting of the circadian system. When free-running with a considerably lengthened spontaneous period (τ ≥ 25.5 h), even the sP 0.5 and the PP 9:15 failed to entrain the owl monkeys' circadian rhythms, whereas a 24-h photoperiod with a very long LP of 3 h caused entrainment. The results indicate that in Aotus lemurinus griseimembra, in addition to the nonparametric light effects, parametric light effects play a significant role in the entrainment of circadian rhythms by LD cycles.     

Temperature entrainment of the circadian cuticle deposition rhythm in Drosophila melanogaster

The cuticle deposition rhythm, which is observed in the apodeme of the furca in the thorax, is controlled by a peripheral circadian clock in the epidermal cells and entrained to light-dark (LD) cycles via CRYPTOCHROME (CRY) in Drosophila melanogaster. In the present study, we examined the effects of temperature (TC) cycles and the combination of LD and TC cycles on entrainment of the cuticle deposition rhythm. The rhythm was entrained to TC cycles, whose period was 28 h. In T = 21 and 24 h, the rhythm was entrained to TC cycles in some individuals. CRY is not necessary for temperature entrainment of the cuticle deposition rhythm because the rhythm in cry(b) (lacking functional CRY) was entrained to TC cycles. Temperature entrainment of the rhythm was achieved even when the thoraxes or furcae were cultured in vitro, suggesting that the mechanism for temperature entrainment is independent of the central clock in the brain and the site of the thermoreception resides in the epidermal cells. When LD and TC cycles with different periods were applied, the rhythm was entrained to LD cycles with a slight influence of TC cycles. Thus, the LD cycle is a stronger zeitgeber than the TC cycle. The variance of the number of the cuticle layers decreased in the flies kept under LD and TC cycles with the same period in which the thermophase coincided with the photophase. Therefore, we conclude that LD and TC cycles synergistically entrain the rhythm. Synergistic effects of LD and TC cycles on entrainment were also observed even when the thoraxes were cultured in vitro, suggesting that the light and temperature information is integrated within the peripheral circadian system.     

Circadian clock properties of fruit flies Drosophila melanogaster exhibiting early and late emergence chronotypes

The role of circadian clocks in timing daily behaviors is widely acknowledged, and while empirical evidence suggests that clock period is correlated with the preferred phase of a rhythmic behavior (chronotype), other clock properties have also been hypothesized to underlie chronotype variation. Here, we report that fruit fly Drosophila melanogaster populations exhibiting evening emergence chronotype (late) are characterized by higher incidence of behavioral arrhythmicity in constant dim light, wider range of entrainment, reduced rates of re-entrainment to simulated jet-lag and higher amplitude of both entrained and free-running rhythms as compared to those exhibiting morning emergence chronotype (early). Our results thus highlight the role of circadian clock properties such as zeitgeber sensitivity, amplitude and coupling in driving chronotype variation.     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness     

Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.     

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.     

Photic desynchronization of two subgroups of circadian oscillators in a network model of the suprachiasmatic nucleus with dispersed coupling strengths

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is composed of thousands of neuronal oscillators expressing different intrinsic periods. These oscillators form a coupled network with a free-running period around 24 h in constant darkness and entrainable to the external light-dark cycle (T cycle). Coupling plays an important role in setting the period of the network and its range of entrainment. Experiments in rats have shown that two subgroups of oscillators within the SCN, a ventrolateral (VL) subgroup that receives photic input and a dorsomedial (DM) subgroup that is coupled to VL, can be desynchronized under a short (22-h) T cycle, with VL entrained to the cycle and DM free-running. We use a modified Goodwin model to understand how entrainment of the subgroups to short (22-h) and long (26-h) T cycles is influenced by light intensity, the proportion of neurons that receives photic input, and coupling heterogeneity. We find that the model's critical value for the proportion of photically-sensitive neurons is in accord with actual experimental estimates, while the model's inclusion of dispersed coupling can account for the experimental observation that VL and DM desynchronize more readily under the 22-h than under the 26-h T cycle. Heterogeneous intercellular coupling within the SCN is likely central to the generation of complex behavioral patterns.     

Modeling the circadian clock: from molecular mechanism to physiological disorders

Based on genetic and biochemical advances on the molecular mechanism of circadian rhythms, a computational model for the mammalian circadian clock is used to examine the dynamical bases of circadian-clock-related physiological disorders in humans. Entrainment by the light-dark cycle with a phase advance or a phase delay is associated with the Familial advanced sleep phase syndrome (FASPS) or the Delayed sleep phase syndrome (DSPS), respectively. Lack of entrainment corresponding to the occurrence of quasiperiodic oscillations with or without phase jump can be associated with the non-24 h sleep-wake syndrome. In the close vicinity of the entrainment domain, the model uncovers the possibility of infradian oscillations of very long period. Perturbation in the form of chronic jet lag, as used in mice, prevents entrainment of the circadian clock and results in chaotic or quasiperiodic oscillations. It is important to clarify the conditions for entrainment and for its failure because dysfunctions of the circadian clock may lead to physiological disorders, which pertain not only to the sleep-wake cycle but also to mood and cancer.     

Modeling the mammalian circadian clock: sensitivity analysis and multiplicity of oscillatory mechanisms

We extend the study of a computational model recently proposed for the mammalian circadian clock (Proc. Natl Acad. Sci. USA 100 (2003) 7051). The model, based on the intertwined positive and negative regulatory loops involving the Per, Cry, Bmal1, and Clock genes, can give rise to sustained circadian oscillations in conditions of continuous darkness. These limit cycle oscillations correspond to circadian rhythms autonomously generated by suprachiasmatic nuclei and by some peripheral tissues. By using different sets of parameter values producing circadian oscillations, we compare the effect of the various parameters and show that both the occurrence and the period of the oscillations are generally most sensitive to parameters related to synthesis or degradation of Bmal1 mRNA and BMAL1 protein. The mechanism of circadian oscillations relies on the formation of an inactive complex between PER and CRY and the activators CLOCK and BMAL1 that enhance Per and Cry expression. Bifurcation diagrams and computer simulations nevertheless indicate the possible existence of a second source of oscillatory behavior. Thus, sustained oscillations might arise from the sole negative autoregulation of Bmal1 expression. This second oscillatory mechanism may not be functional in physiological conditions, and its period need not necessarily be circadian. When incorporating the light-induced expression of the Per gene, the model accounts for entrainment of the oscillations by light-dark (LD) cycles. Long-term suppression of circadian oscillations by a single light pulse can occur in the model when a stable steady state coexists with a stable limit cycle. The phase of the oscillations upon entrainment in LD critically depends on the parameters that govern the level of CRY protein. Small changes in the parameters governing CRY levels can shift the peak in Per mRNA from the L to the D phase, or can prevent entrainment. The results are discussed in relation to physiological disorders of the sleep-wake cycle linked to perturbations of the human circadian clock, such as the familial advanced sleep phase syndrome or the non-24h sleep-wake syndrome.     

Nonphotic entrainment in humans?

Although light is accepted as the dominant zeitgeber for entrainment of the human circadian system, there is evidence that nonphotic stimuli may play a role. This review critically assesses the current evidence in support of nonphotic entrainment in humans. Studies involving manipulations of sleep-wake schedules, exercise, mealtimes, and social stimuli are re-examined, bearing in mind the fact that the human circadian clock is sensitive to very dim light and has a free-running period very close to 24 h. Because of light confounds, the study of totally blind subjects with free-running circadian rhythms represents the ideal model to investigate the effects of nonphotic stimuli on circadian phase and period. Strong support for nonphotic entrainment in humans has already come from the study of a few blind subjects with entrained circadian rhythms. However, in these studies the nonphotic stimulus(i) responsible was not identified. The effect of appropriately timed exercise or exogenous melatonin represents the best proof to date of an effect of nonphotic stimuli on human circadian timing. Phase-response curves for both exercise and melatonin have been constructed. Given the powerful effect of feeding as a circadian zeitgeber in various nonhuman species, studies of meal timing are recommended. In conclusion, the available evidence indicates that it remains worthwhile to continue to study nonphotic effects on human circadian timing to identify treatment strategies for shift workers and transmeridian travelers as well as for the blind and possibly the elderly.