Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii

Circadian oscillators are known to regulate the timing of cell division in many organisms. In the case of Chlamydomonas reinhardtii, however, this conclusion has been challenged by several investigators. We have reexamined this issue and find that the division behavior of Chlamydomonas meets all the criteria for circadian rhythmicity: persistence of a cell division rhythm (a) with a period of approximately 24 h under free-running conditions, (b) that is temperature compensated, and (c) which can entrain to light/dark signals. In addition, a mutation that lengthens the circadian period of the phototactic rhythm similarly affects the cell division rhythm. We conclude that a circadian mechanism determines the timing of cell division in Chlamydomonas reinhardtii.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

JNK regulates the photic response of the mammalian circadian clock

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.     

The Effects of Lithium on a Neuronal in Vitro Orcadian Pacemaker

Previous studies have suggested a causal connection between abnormalities of the circadian system and affective disorders. The effectiveness of lithium or rubidium as a treatment for affective disorders and the ability of lithium or rubidium to influence circadian pacemakers has stimulated research into the mechanism of lithium's action on circadian systems. In this study we used a neuronal in vitro circadian pacemaker preparation, the eye of the mollusc Bulla, to examine the cellular effects of lithium and rubidium. Continuous extracellular LiCl application lengthens the period of the circadian rhythm of the Bulla pacemaker in a concentration-dependent manner. Rubidium was found to be more effective than lithium in period lengthening. Stable phase delays were generated by 2-h pulses of 395 mM LiCl applied extracellularly from zeitgeber time (ZT) 5-7 (mid subjective day). Concomitant continuous application of 16 mM LiCl and light (a depolarizing agent) generated period lengthening substantially greater than the arithmetic sum of the modest period lengthening of each treatment alone. Furthermore, LiCl pulses, applied together with depolarizing extracellular KC1 concentrations, yielded an increasing magnitude of phase delays with increasing KC1 concentration. These data suggest that LiCl acts intracellularly on the circadian pacemaker cells by entering through a voltage-dependent channel, most likely a sodium channel.     

Lithium and bipolar disorder: Impacts from molecular to behavioural circadian rhythms

Bipolar disorder (BD) is a severe and common psychiatric disorder. BD pathogenesis, clinical manifestations and relapses are associated with numerous circadian rhythm abnormalities. Lithium (Li) is the first-line treatment in BD, and its therapeutic action has been related to its ability to alter circadian rhythms. We systematically searched the PubMed database until January 2016, aiming to critically examine published studies investigating direct and indirect effects of Li on circadian rhythms. The results, from the 95 retained studies, indicated that Li: acts directly on the molecular clocks; delays the phase of sleep–wakefulness rhythms and the peak elevation of diurnal cycle body temperature; reduces the amplitude and shortens the duration of activity rhythms and lengthens free-running rhythms. Chronic Li treatment stabilizes free-running activity rhythms, by improving day-to-day rhythmicity of the activity, with effects that appear to be dose related. Pharmacogenetics demonstrate several associations of Li’s response with circadian genes (NR1D1, GSK3β, CRY1, ARNTL, TIM, PER2). Finally, Li acts on the retinal-hypothalamic pineal pathway, influencing light sensitivity and melatonin secretion. Li is a highly investigated chronobiologic agent, and although its chronobiological effects are not completely understood, it seems highly likely that they constitute an inherent component of its therapeutic action in the treatment of mood disorders.     

Glycogen synthase kinase 3beta as a likely target for the action of lithium on circadian clocks

Although lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder, the mechanisms underlying its therapeutic action are still unclear. Together with its mood-stabilizing effects, lithium is also known to influence the circadian clocks of several organisms including man. Circadian rhythms are altered in patients with bipolar disorder, and it is believed that these rhythms may play an important role in disease mechanisms. It is therefore possible that some of the therapeutic actions of lithium may be related to its effect on circadian clocks. Identifying the targets for lithium's action on circadian clocks would therefore be important both for understanding the mechanisms of its therapeutic effect and also in further understanding disease mechanisms in bipolar disorders. Using Drosophila melanogaster as a model system, we show that long-term administration of lithium results in lengthening of the free-running period (τ) of circadian locomotor activity rhythm of flies in constant darkness (DD). This effect occurs at concentrations similar to the plasma levels of lithium used in the treatment of bipolar disorder. The lithium-treated flies also show reduced activity of one of the previously reported targets of lithium action, Glycogen Synthase Kinase 3β (GSK 3β). GSK 3β has been shown to be involved in the regulation of circadian clocks as the down regulation of this protein results in an elongation of τ. The τ elongation resembles the effect seen with lithium administration in a number of organisms including man, and taken together with the earlier observations our results suggest that lithium inhibits the activity of GSK 3β to produce its effect on circadian clocks.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

Tau differences between short-day responsive and short-day nonresponsive white-footed mice (Peromyscus leucopus) do not affect reproductive photoresponsiveness

In laboratory-bred rodent populations, intraspecific variation in circadian system organization is a known cause of individual variation in reproductive photoresponsiveness. The authors sought to determine whether circadian system variation accounted for individual variation in reproductive photoresponsiveness in a single, highly genetically variable population of Peromyscus leucopus recently derived from the wild. Running-wheel activity patterns of male and female mice, aged 70 to 90 days, from artificially selected lines of reproductively photoresponsive (R) and nonresponsive (NR) lines were monitored under short-day photoperiod (8 h light, 16 h dark), long-day photoperiod (16 h light, 8 h dark), and constant darkness (DD). NR mice displayed a significantly longer mean free-running period (24.08 h) in DD compared with R mice (23.75 h), due in large part to a difference between NR and R females (24.25 h vs. 23.74 h, respectively). All other entrainment characteristics (alpha, phase angle of activity) under short days, long days, and DD were similar between R and NR mice. Variation in free-running period and entrainment characteristics has been shown to affect photoresponsiveness in other rodent species by altering the manner in which the circadian system interprets short days. To determine whether variation in photoresponsiveness in P. leucopus is due to differences in free-running period instead of variation downstream from the central circadian clock in the pathway controlling photoresponsiveness, the authors exposed young R and NR mice to DD and measured the effect on reproductive organ development. If variation in free-running period affected how the circadian system of mice interpreted short days, then both R and NR mice exposed to DD should have exhibited a delay in gonadal development. Only R mice exhibited pubertal delay in DD. NR mice exhibited large paired testes, paired seminal vesicles, paired ovaries, and uterine weight typical of mice nonresponsive to short days, whereas R mice exhibited reproductive organ weight typical of mice responsive to short days. These data suggest that despite significant differences in free-running period between R and NR mice, individual variation in photoresponsiveness is not due to differences in how the circadian systems of R and NR mice interpret the LD cycle.     

The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

The methamphetamine-sensitive circadian oscillator (MASCO) in mice

The suprachiasmatic nucleus (SCN) orchestrates synchrony among many peripheral oscillators and is required for circadian rhythms of locomotor activity and many physiological processes. However, the unique effects of methamphetamine (MAP) on circadian behavior suggest the presence of an SCN-independent, methamphetamine-sensitive circadian oscillator (MASCO). Substantial data collected using rat models show that chronic methamphetamine dramatically lengthens circadian period of locomotor activity rhythms and induces rhythms in animals lacking an SCN. However, the anatomical substrate and the molecular components of the MASCO are unknown. The response to MAP is less well studied in mice, a model that would provide the genetic tools to probe the molecular components of this extra-SCN oscillator. The authors tested the effects of chronic MAP on 2 strains of intact and SCN-lesioned mice in constant dark and constant light. Furthermore, they applied various MAP availability schedules to SCN-lesioned mice to confirm the circadian nature of the underlying oscillator. The results indicate that this oscillator has circadian properties. In intact mice, the MASCO interacts with the SCN in a manner that is strain, sex, and dose dependent. In SCN-lesioned mice, it induces robust free-running locomotor rhythmicity, which persists for up to 14 cycles after methamphetamine is withdrawn. In the future, localization of the MASCO and characterization of its underlying molecular mechanism, as well as its interactions with other oscillators in the body, will be essential to a complete understanding of the organization of the mammalian circadian system.     

Interaction of the retina with suprachiasmatic pacemakers in the control of circadian behavior

The suprachiasmatic nucleus (SCN) is the central circadian pacemaker governing the circadian rhythm of locomotor activity in mammals. The mammalian retina also contains circadian oscillators, but their roles are unknown. To test whether the retina influences circadian rhythms of locomotor behavior, the authors compared the activity of bilaterally enucleated hamsters with the activity of intact controls held in constant darkness (DD). Enucleated hamsters showed a broader range of free-running periods (tau) than did intact hamsters held for the same length of time in DD. This effect was independent of the age at enucleation (on postnatal days 1, 7, or 28). The average tau of intact animals kept in DD from days 7 or 28 was significantly longer than that of intact animals kept in DD from day 1 or any of the enucleated groups. This indicates that early exposure to light-dark cycles lengthens the tau and that the eye is required to maintain this effect even in DD. These data suggest that hypothalamic circadian pacemakers may interact continuously with the retina to determine the tau of locomotor activity. Enucleation caused a large decrease in glial fibrillary acidic protein in the SCN but has no (or slight) effects on calbindin, neuropeptide Y, vasopressin, or vasoactive intestinal polypeptide, which suggests that enucleation does not produce major damage to the SCN, an interpretation that is supported by the fact that enucleated animals retain robust circadian rhythmicity. The presence of an intact retina appears to contribute to system-level circadian organization in mammals perhaps as a consequence of interaction between its circadian oscillators and those in the SCN.     

Circadian clock functioning is linked to acute stress reactivity in rats

At least two major physiological systems are involved in the adaptation of the organism to environmental challenges: the circadian system and the stress reaction. This study addressed the possibility that interindividual differences in stress sensitivity and in the functioning of the circadian system are related. At 2 months of age, corticosterone secretion in response to a 20-min restraint stress was assessed in 9 Sprague-Dawley rats for which running wheel activity was recorded as a rhythmic behavioral marker of the circadian clock. Two weeks later, the adaptive response of the circadian system to an abrupt shift in the light:dark (LD) cycle was assessed in those rats using a jet-lag paradigm. Finally, after resynchronization to the new LD cycle, rats were transferred to constant darkness to assess the free-running period of their circadian rhythm of running-wheel activity. Results indicate that stress-induced corticosterone secretion was (1) positively correlated with the number of days to resynchronize the circadian activity rhythm to the new LD cycle, and with the value of its free-running period, and (2) negatively correlated with the intensity of daily locomotor activity. Those data, emphasizing the interactions between the stress response of an organism and the functioning of its circadian system, could explain interindividual differences in humans' susceptibility to shift work or other circadian-related disorders.     

Probing the circadian oscillator of a mammal by two-pulse perturbations

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129-136, 2000)     

Pineal and hypothalamic pacemakers: Their role in regulating circadian rhythmicity in Japanese quail

Summary Neither pinealectomy nor administration of melatoninvia silastic capsules had any effect on free-running circadian rhythms of locomotor activity in Japanese quail (Coturnix coturnix japonica). The quail, like the chicken, therefore differs from sparrows and starlings in which pinealectomy dramatically disrupts free-running rhythms. Nevertheless, it seems unlikely that there are fundamental differences in circadian organisation within the Class Aves. The effects of lesions within the supraoptic region (SOR) of the hypothalamus were similar to those which follow the ablation of the suprachiasmatic nuclei (SCN) in sparrows, rats and hamsters, causing the breakdown of free-running rhythms of locomotor activity, but not necessarily an arrhythmic state. The SOR and SCN appear then to have homologous functions in birds and mammals. Differences in circadian organisation, such as the degree of influence of the pineal gland and the particular photoreceptors used for entrainment, may therefore be modifications peripheral to the fundamental components of the circadian clock.Abbreviations POR preoptic area - SOR supraoptic region - SCN suprachiasmatic nuclei     

Dissociation of ultradian and circadian phenotypes in female and male siberian hamsters

Three experiments addressed whether pronounced alterations in the circadian system yielded concomitant changes in ultradian timing. Female Siberian hamsters were housed in a 16L:8D photoperiod after being subjected to a disruptive phase-shifting protocol that produced 3 distinct permanent circadian phenotypes: some hamsters entrained their circadian rhythms (CRs) with predominantly nocturnal locomotor activity (ENTR), others displayed free-running CRs (FR), and a third cohort was circadian arrhythmic (ARR). The period of the ultradian locomotor rhythm (UR) did not differ among the 3 circadian phenotypes; neuroendocrine generation of URs remains viable in the absence of coherent circadian organization and appears to be mediated by substrates functionally and anatomically distinct from those that generate CRs. Pronounced light-dark differences in several UR characteristics in ENTR hamsters were completely absent in circadian arrhythmic hamsters. The disruptive phase-shifting protocol may compromise direct visual input to ultradian oscillators but more likely indirectly affects URs by interrupting visual afference to the circadian system. Additional experiments documented that deuterium oxide and constant light, each of which substantially lengthened the period of free-running CRs, failed to change the period of concurrently monitored URs. The resistance of URs to deuteration contrasts with the slowing of virtually all other biological timing processes, including CRs. Considered together, the present results point to the existence of separable control mechanisms for generation of circadian and ultradian rhythms.     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Locomotor Activity Rhythms in Cave Catfishes, Genus Taunayia, from Eastern Brazil (Teleostei: Siluriformes: Heptapterinae)

We studied the locomotor rhythmicity in heptapterine catfishes, genus Taunayia, under free-running conditions (DD) and LD cycles (12:12). Taunayia sp., anophthalmic and depigmented undescribed species from a cave in northeastern Brazil, is the fourth Brazilian troglobitic catfish studied with focus on circadian rhythms. Weak free-running rhythmicity, with absence of significant circadian components, was observed for this species when compared to the epigean, eyed relatives. On the other hand, the studied troglobitic catfishes in general presented significant circadian rhythms under LD cycles, with activity peaks in the night phase probably corresponding to nocturnal activity pattern inherited from their epigean ancestors. However, no residual oscillations were observed after transition from LD to DD. This indicates masking of activity by light-dark cycles. Regression of circadian rhythmicity in the stable, permanently dark subterranean habitat was also observed for other cave fishes. Such regression corroborates the notion that circadian rhythmicity is mainly selected in the epigean environment by ecological factors, namely daily cycles of light and/or temperature.     

Modeling the differential fitness of cyanobacterial strains whose circadian oscillators have different free-running periods: comparing the mutual inhibition and substrate depletion hypotheses

In a recent experimental study, Ouyang et al. (1998, Proc. Natl. Acad. Sci. U.S.A.95, 8660-8664) have shown that, in direct competition, cyanobacterial strains whose circadian clocks have free-running periods (FRPs) which match the period of an imposed light/dark (LD) cycle exclude strains whose FRPs are out of resonance with the LD cycle. These differences in competitive fitness are observed despite the lack of measurable differences in monoculture growth rates between the strains. Here we show that the experimental results are consistent with a mathematical model in which cells rhythmically produce a metabolic inhibitor to which they display a sensitivity modulated by their circadian rhythm. We argue that models in which there is a circadian modulation of nutrient uptake kinetics cannot account for the results of these experiments. We discuss possible experiments to further characterize this phenomenon. The experimental protocol we propose can be used to distinguish between mutual inhibition and substrate depletion as underlying causes of the competitive advantage of circadian resonance.