On the ontogeny of the motor circadian rhythm in crayfish

In this paper we attack the problem of understanding the localization of the main structures involved in the motor circadian rhythm of crayfish by analysing its ontogeny. We present experimental results giving the properties of this rhythm in young and adult crayfish. Then we construct a mathematical model (based on a previous one for the electroretinogram circadian rhythm in the same species) simulating those properties. In the process of constructing the model we clarified and made precise various hypotheses about the biological structures involved in them and about the characteristics of the oscillators present in those structures. We also formulate some hypotheses about the general properties of circadian rhythms. Finally, we propose some experiments suggested by the mathematical model.     

Pinealectomy and LL Abolished Circadian Perching Rhythms but Did Not Alter Circannual Reproductive or Fattening Rhythms in Finches

In the subtropical finch, spotted munia (Lonchura punctulata) circannual rhythms (of gonads, fattening, feeding) have been demonstrated in an information-free environment of continuous illumination (LL), rendering it an ideal model for research on the physiology of the circannual clock. In an attempt to understand the involvement, if any, of the circadian system in the genesis of circannual rhythms, we studied the effect of pinealectomy (LL 15 lux) and strong continuous illumination (LL 300 lux), both known to abolish circadian rhythms, on the circadian perch-hopping rhythm and on the circannual rhythm of reproduction and fattening in the same birds. While both pinealectomy and LL 300 lux treatments abolished the circadian rhythm of motor activity, they had no effect on the circannual rhythms of gonadal size and fattening. If the endogenous circadian rhythm in perch-hopping can be taken to reflect the circadian clock mechanism associated with gonadal functioning, present results suggest that circannual rhythm of reproduction in spotted munia is independent of circadian events.     

Pinealectomy and LL Abolished Circadian Perching Rhythms but Did Not Alter Circannual Reproductive or Fattening Rhythms in Finches

In the subtropical finch, spotted munia (Lonchura punctulata) circannual rhythms (of gonads, fattening, feeding) have been demonstrated in an information-free environment of continuous illumination (LL), rendering it an ideal model for research on the physiology of the circannual clock. In an attempt to understand the involvement, if any, of the circadian system in the genesis of circannual rhythms, we studied the effect of pinealectomy (LL 15 lux) and strong continuous illumination (LL 300 lux), both known to abolish circadian rhythms, on the circadian perch-hopping rhythm and on the circannual rhythm of reproduction and fattening in the same birds. While both pinealectomy and LL 300 lux treatments abolished the circadian rhythm of motor activity, they had no effect on the circannual rhythms of gonadal size and fattening. If the endogenous circadian rhythm in perch-hopping can be taken to reflect the circadian clock mechanism associated with gonadal functioning, present results suggest that circannual rhythm of reproduction in spotted munia is independent of circadian events.     

A phase response curve for circannual rhythm in the varied carpet beetle <Emphasis Type="Italic">Anthrenus verbasci</Emphasis>

We know that entrainment, a stable phase relationship with an environmental cycle, must be established for a biological clock to function properly. Phase response curves (PRCs), which are plots of phase shifts that result as a function of the phase of a stimulus, have been created to examine the mode of entrainment. In circadian rhythms, single-light pulse PRCs have been obtained by giving a light pulse to various phases of a free-running rhythm under continuous darkness. This successfully explains the entrainment to light-dark cycles. Some organisms show circannual rhythms. In some of these, changes in photoperiod entrain the circannual rhythms. However, no single-pulse PRCs have been created. Here we show the PRC to a long-day pulse superimposed for 4 weeks over constant short days in the circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci. Because the shape of that PRC closely resembles that of the Type 0 PRC with large phase shifts in circadian rhythms, we suggest that an oscillator having a common feature in the phase response with the circadian clock, produces a circannual rhythm.     

A new mutation affecting FRQ-less rhythms in the circadian system of Neurospora crassa

We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq(10) knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.     

Thymidine incorporation by cultured chick pineal glands is not subject to adrenergic regulation

Norepinephrine is known to play a role in regulating the circadian rhythms of serotonin N-acetyltransferase activity and melatonin formation in the chick pineal gland. We have recently demonstrated that the cultured chick pineal exhibits a circadian rhythm in the incorporation of thymidine. In this study we show that this latter rhythm is not subject to adrenergic control.     

Egg-laying rhythm in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Extensive research has been carried out to understand how circadian clocks regulate various physiological processes in organisms. The discovery of clock genes and the molecular clockwork has helped researchers to understand the possible role of these genes in regulating various metabolic processes. In Drosophila melanogaster, many studies have shown that the basic architecture of circadian clocks is multi-oscillatory. In nature, different neuronal subgroups in the brain of D. melanogaster have been demonstrated to control different circadian behavioural rhythms or different aspects of the same circadian rhythm. Among the circadian phenomena that have been studied so far in Drosophila, the egg-laying rhythm is unique, and relatively less explored. Unlike most other circadian rhythms, the egg-laying rhythm is rhythmic under constant light conditions, and the endogenous or free-running period of the rhythm is greater than those of most other rhythms. Although the clock genes and neurons required for the persistence of adult emergence and activity/rest rhythms have been studied extensively, those underlying the circadian egg-laying rhythm still remain largely unknown. In this review, we discuss our current understanding of the circadian egg-laying rhythm in D. melanogaster, and the possible molecular and physiological mechanisms that control the rhythmic output of the egg-laying process.     

Circadian rhythm mutants of the prokaryoticSynechococcus RF-1

Synechococcus RF-1 established circadian rhythms in nitrogen fixation and leucine uptake when growing in a diurnal light/dark regimen. The rhythms persisted in subsequent uniform light/light conditions. In order to analyze the circadian rhythm at the genetic level, mutants were induced by N-methyl-N-nitro-N-nitrosoguanidine and then isolated by procedures with the circadian nitrogen-fixing rhythm as a selecton marker. Characterization of the mutants with respect to the circadian rhythm indicated that some mutants were abnormal only in the nitrogen-fixing rhythm, while some simultaneously lost the ability to establish the nitrogen-fixing and leucine-uptake rhythms. The physiological properties of the circadian rhythm were compared. The genetic potential of the mutants that were abnormal in both rhythms is emphasized.     

Extracellular low pH affects circadian rhythm expression in human primary fibroblasts

Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47 ± 0.03 h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60 ± 0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability.     

Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.     

Rats with minimal hepatic encephalopathy show reduced cGMP-dependent protein kinase activity in hypothalamus correlating with circadian rhythms alterations

Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light.     

Electrophysiology of the circadian pacemaker in mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

Electrophysiology of the Circadian Pacemaker in Mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

A phase dynamics model of human circadian rhythms

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.     

On analyzing circadian rhythms data using nonlinear mixed models with harmonic terms

Wang, Ke, and Brown (2003, Biometrics59, 804-812) developed a smoothing-based approach for modeling circadian rhythms with random effects. Their approach is flexible in that fixed and random covariates can affect both the amplitude and phase shift of a nonparametrically smoothed periodic function. In motivating their approach, Wang et al. stated that a simple sinusoidal function is too restrictive. In addition, they stated that "although adding harmonics can improve the fit, it is difficult to decide how many harmonics to include in the model, and the results are difficult to interpret." We disagree with the notion that harmonic models cannot be a useful tool in modeling longitudinal circadian rhythm data. In this note, we show how nonlinear mixed models with harmonic terms allow for a simple and flexible alternative to Wang et al.'s approach. We show how to choose the number of harmonics using penalized likelihood to flexibly model circadian rhythms and to estimate the effect of covariates on the rhythms. We fit harmonic models to the cortisol circadian rhythm data presented by Wang et al. to illustrate our approach. Furthermore, we evaluate the properties of our procedure with a small simulation study. The proposed parametric approach provides an alternative to Wang et al.'s semiparametric approach and has the added advantage of being easy to implement in most statistical software packages.     

Temperature can entrain egg laying rhythm of Drosophila but may not be a stronger zeitgeber than light

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.     

Circadian Stomatal Rhythms in Epidermal Peels from Vicia faba

Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 30°C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population.     

Chronic Administration of Imipramine and Lithium Changes the Phase-Angle Relationship Between the Activity and Core Body Temperature Circadian Rhythms in Rats

Evidence suggests that there is an association between the pathophysiology of depression and a disturbance of circadian rhythms. Accordingly, attention has focused on the possible effects of antidepressants on circadian rhythms. In the present study, we examined the effects of chronic administration of two clinically effective antidepressant agents, imipramine and lithium, on several circadian rhythms in the rat. Activity, core body temperature, and drinking rhythms were assessed in constant darkness (DD) and light-dark (LD) conditions. In DD, lithium significantly lengthened the circadian period of the activity, temperature, and drinking rhythms, while imipramine had no effect. In LD, both drugs significantly delayed the phase of the activity rhythm, but did not change that of the other two rhythms. As a result, the phase-angle differences between the activity and temperature rhythms significantly increased. Neither lithium nor imipramine produced any effect on the resynchronization of these rhythms after an 8-h delay in the LD cycle. These results indicate that although both drugs produced different effects on the circadian period of individual rhythms, both caused a relative phase advance of the temperature rhythm as compared to the activity rhythm, and this effect may be related to the similarity in their antidepressant effects. (Chronobiology International, 13(4), 251-259, 1996)