Drosophila free-running rhythms require intercellular communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Identification of rhythmic subsystems in the circadian cycle of crassulacean acid metabolism under thermoperiodic perturbations

Leaves of the Crassulacean acid metabolism (CAM) plant Kalancho? daigremontiana Hamet et Perrier de la Bathie show overt circadian rhythms in net CO2 uptake, leaf conductance to water and intercellular CO2 concentration, which are entrained by periodic temperature cycles. To probe their sensitivity to thermoperiodic perturbations, intact leaves were exposed to continuous light intensity and temperature cycles with a period of 16 h, applying a set of different baseline temperatures and thermodriver amplitudes. All three overt rhythms were analyzed with respect to their frequency spectra and their phase relations with the thermodriver. For most stimulation protocols, stomatal conductance and net CO2 change were fully or partially entrained by the temperature pulses, while the internal CO2 concentration remained dominated by oscillations in the circadian range. Prolonged time series recorded for up to 22 d in continuous light underline the robustness of these circadian oscillations. This suggests that the overt circadian rhythm of net CO2 uptake in CAM results from the interaction of two coupled original systems: (i) an endogenous cycle of CO2 fixation in the mesophyll, showing very robust periodic activity, and (ii) stomatal movements that respond to environmental stimuli independently of rhythmic processes in the mesophyll, and thus modulate the gas exchange amplitude.     

Drosophila Free-Running Rhythms Require Intercellular Communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

The circadian timing system and reproduction in mammals.

Circadian systems in a wide variety of organisms all appear to include three basic components: 1) biological oscillators that maintain a self-sustained circadian periodicity in the absence of environmental time cues; 2) input pathways that convey environmental information, especially light cues, that can entrain the circadian oscillations to local time; and 3) output pathways that drive overt circadian rhythms, such as the rhythms of locomotor activity and a variety of endocrine rhythms. In mammals, the circadian system is employed in the regulation of reproductive physiology and behavior in two very important ways. 1) In some species, there is a strong circadian component in the timing of ovulation and reproductive behavior, ensuring that these events will occur at a time when the animal is most likely to encounter a potential mate. 2) Many mammals exhibit seasonal reproductive rhythms that are largely under photoperiod regulation; in these species, the circadian system and the pineal gland are crucial components of the mechanism that is used to measure day length. The rhythm of pineal melatonin secretion is driven by a neural pathway that includes the circadian oscillator(s) in the suprachiasmatic nuclei. Melatonin is secreted at night in all mammals, and the duration of each nocturnal episode of melatonin secretion is inversely related to day length. The pineal melatonin rhythm appears to serve as an internal signal that represents day length and that is capable of regulating a variety of seasonal variations in physiology and behavior.     

Extracellular long-term recordings of the isolated accessory medulla,the circadian pacemaker center of the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>, reveal ultradian and hint circadian rhythms

In the cockroach Leucophaea maderae transplantation studies located the circadian pacemaker center, which controls locomotor activity rhythms, to the accessory medulla (AMe), ventromedially to the medulla of the brain’s optic lobes. The AMe is densely innervated via GABA- and manyfold peptide-immunoreactive neurons. They express ultradian action potential oscillations in the gamma frequency range and form phase-locked assemblies of synchronously spiking cells. Peptide application resulted in transient rises of extracellularly recorded activity. It remained unknown whether transient rises in spontaneous electrical activity as a possible indication of peptide release occur in the isolated circadian clock in a rhythmic manner. In extracellular glass electrode recordings of the isolated AMe in constant darkness, which lasted at least 12 h, the distribution of daytime-dependent changes in activity independently of the absolute action potential frequency was examined. Rapid, transient changes in activity preferentially occurred at the mid-subjective night, with a minimum at the middle of the subjective day, hinting the presence of circadian rhythms in the isolated circadian clock. Additionally, ultradian rhythms in activity change that are multiples of a fundamental 2 h period were observed. We hypothesize that circadian rhythms might originate from coupled ultradian oscillations, possibly already at the single cell level.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

BaSAR-A tool in R for frequency detection

Many biological processes are periodic, for example cell cycle expression, circadian rhythms and calcium oscillations. However, measured time series from these processes are commonly short and noisy, and finding frequencies in such data can be challenging. Here we present BaSAR, Bayesian Spectrum Analysis in R, a package for extracting frequency information from time series data. The software uses advanced techniques of Bayesian inference that are well suited for handling typical biological data. The core functions are designed for detecting a single key frequency, without the need for data pre-processing such as detrending. The package is freely available at CRAN - The Comprehensive R Archive Network: http://cran.r-project.org/web/packages/BaSAR.     

Non-stationary time series and the robustness of circadian rhythms

Circadian rhythms are regular oscillations in the value of behavioral and physiological variables of organisms that recur on a daily basis. The purpose of this study was to evaluate the extent of non-stationarity of circadian rhythms over several days, to determine how damaging is the violation of the assumption of stationarity in the analysis of circadian rhythms, and to formalize the concept of "rhythm robustness" as an index of oscillatory ("weak") stationarity. Simulated (computer-generated) and experimental data sets (rhythms of body temperature and running-wheel activity in several rodent species) were analysed. Tests of stationarity based on the variance of the daily means and the variance of the daily variances revealed that most experimental data sets are not stationary. Analysis of linear trends indicated that significant trends are rare in experimental data sets. Although the non-stationarity of the experimental data sets reduced the spectral energy of the Enright periodogram used to assess rhythmicity, detection of circadian rhythmicity was not prevented in any of the rhythmic data sets. The results of the various analyses allow the inference that, after high-frequency noise is filtered out, the value of the periodogram's Q(P) statistic reflects the extent of stationarity of the time series. Thus, the "robustness" of a circadian rhythm (i.e. the magnitude of the empirical Q(P) value as compared to the Q(P) value associated with a perfectly rhythmic time series) can serve as an index of the stationarity of the rhythm.     

Locomotor activity rhythms in cave fishes from Chapada Diamantina, northeastern Brazil (Teleostei: Siluriformes)

Previous studies on the locomotor activity of troglobitic (exclusively subterranean) species have shown that circadian rhythmicity may be reduced in populations evolving in the absence of zeitgebers such as daily cycles of light and temperature; therefore, circadian activity rhythms, although not infradian nor ultradian rhythms, seem to have been selected by external, ecological factors. We studied the locomotor activity of a highly specialized Heptapteridae catfish (undescribed genus and species) from Chapada Diamantina, NE Brazil, compared to another specialized Brazilian troglobitic heptapterid, Taunayia sp. Locomotor activity was continuously measured in the laboratory with an infra-red photocell system. Seven specimens of the new genus were tested, each one during 14 consecutive days according to the following schedule: three days in DD → seven days in LD (12:12 h) → four days in DD. Data were submitted both to fast Fourier transform periodogram followed by Siegel's test of significance and Lombs - Scargle periodogram techniques in order to identify spectral composition of the time series. In general, results were similar to those obtained for Taunayia sp.: (a) for most specimens, absence of significant circadian components in locomotor activity under DD; (b) for all specimens, significant circadian components under LD, with higher levels of activity during the dark phase, as expected for species belonging to nocturnal epigean taxa; (c) for most specimens, no residual oscillations recorded when free-running conditions were reinstalled. Circadian locomotor activity detected under LD may thus be interpreted as a direct, masking effect of the LD cycle. This suggests a pattern for highly specialized troglobitic species, isolated for a long time in the subterranean habitat, with a progressive reduction of circadian time-keeping mechanisms. Our studies also demonstrate the potential of subterranean organisms for investigation of the origin, evolution, functioning and genetics of circadian rhthmicity.     

The bear circadian clock doesn’t ‘sleep’ during winter dormancy

Background

Most biological functions are synchronized to the environmental light:dark cycle via a circadian timekeeping system. Bears exhibit shallow torpor combined with metabolic suppression during winter dormancy. We sought to confirm that free-running circadian rhythms of body temperature (Tb) and activity were expressed in torpid grizzly (brown) bears and that they were functionally responsive to environmental light. We also measured activity and ambient light exposures in denning wild bears to determine if rhythms were evident and what the photic conditions of their natural dens were. Lastly, we used cultured skin fibroblasts obtained from captive torpid bears to assess molecular clock operation in peripheral tissues. Circadian parameters were estimated using robust wavelet transforms and maximum entropy spectral analyses.

Results

Captive grizzly bears housed in constant darkness during winter dormancy expressed circadian rhythms of activity and Tb. The rhythm period of juvenile bears was significantly shorter than that of adult bears. However, the period of activity rhythms in adult captive bears was virtually identical to that of adult wild denning bears as was the strength of the activity rhythms. Similar to what has been found in other mammals, a single light exposure during the bear’s active period delayed subsequent activity onsets whereas these were advanced when light was applied during the bear’s inactive period. Lastly, in vitro studies confirmed the expression of molecular circadian rhythms with a period comparable to the bear’s own behavioral rhythms.

Conclusions

Based on these findings we conclude that the circadian system is functional in torpid bears and their peripheral tissues even when housed in constant darkness, is responsive to phase-shifting effects of light, and therefore, is a normal facet of torpid bear physiology.

     

Rhythms of mammalian body temperature can sustain peripheral circadian clocks

BACKGROUND: Low-amplitude temperature oscillations can entrain the phase of circadian rhythms in several unicellular and multicellular organisms, including Neurospora and Drosophila. Because mammalian body temperature is subject to circadian variations of 1 degrees C-4 degrees C, we wished to determine whether these temperature cycles could serve as a Zeitgeber for circadian gene expression in peripheral cell types. RESULTS: In RAT1 fibroblasts cultured in vitro, circadian gene expression could be established by a square wave temperature rhythm with a (Delta)T of 4 degrees C (12 hr 37 degrees C/12 hr 33 degrees C). To examine whether natural body temperature rhythms can also affect circadian gene expression, we first measured core body temperature cycles in the peritoneal cavities of mice by radiotelemetry. We then reproduced these rhythms with high precision in the liquid medium of cultured fibroblasts for several days by means of a homemade computer-driven incubator. While these "in vivo" temperature rhythms were incapable of establishing circadian gene expression de novo, they could maintain previously induced rhythms for multiple days; by contrast, the rhythms of control cells kept at constant temperature rapidly dampened. Moreover, circadian oscillations of environmental temperature could reentrain circadian clocks in the livers of mice, probably via the changes they imposed upon both body temperature and feeding behavior. Interestingly, these changes in ambient temperature did not affect the phase of the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. CONCLUSIONS: We postulate that both endogenous and environmental temperature cycles can participate in the synchronization of peripheral clocks in mammals.     

Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine

Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial ‘miniguts’), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis and disease.KEY WORDS: Circadian rhythm, Intestinal organoid, PERIOD2, R-spondin, RU486     

Diurnal rhythm of the total antioxidant activity of saliva of children with chronic viral hepatitis

The diurinal rhythms of a total antioxidant activity of a saliva were investigated depending on activity of the inflammatory process in a liver of children with chronic virus hepatitis HBV and HCV. The circadian rhythms were found desynchronized, which was showed in lowering of the mesor value, oscillations amplitude, and a rhythm inversion and transformation, as an additional biorhythmological criterion of the presence of the chronic hepatitis of a viral etiology. A tendency of a circadian rhythm to remain with a daily acrophasis but with a law level of amplitude oscillations and a mesor was to be observed after the treatment of children with inactive HBV. By an active HBV a circadian rhythm with it's inversion has been renewed. It was determined the remaining of an ultradian rhythm with increasing level of amplitude oscillations after the treatment of inactive HCV. The law amplitude oscillations and a insignificant increasing of a meror level were characteristic for the circadian rhythm of total AOA of the children with active HBV. It was proved the possibility of usage of the biorhythmological parameters of diurinal rhythms of total AOA as a criteria of efficiency of the conducted therapy for the given group with disease children.     

Stochastic models for circadian rhythms: effect of molecular noise on periodic and chaotic behaviour

Circadian rhythms are endogenous oscillations that occur with a period close to 24 h in nearly all living organisms. These rhythms originate from the negative autoregulation of gene expression. Deterministic models based on such genetic regulatory processes account for the occurrence of circadian rhythms in constant environmental conditions (e.g., constant darkness), for entrainment of these rhythms by light-dark cycles, and for their phase-shifting by light pulses. When the numbers of protein and mRNA molecules involved in the oscillations are small, as may occur in cellular conditions, it becomes necessary to resort to stochastic simulations to assess the influence of molecular noise on circadian oscillations. We address the effect of molecular noise by considering the stochastic version of a deterministic model previously proposed for circadian oscillations of the PER and TIM proteins and their mRNAs in Drosophila. The model is based on repression of the per and tim genes by a complex between the PER and TIM proteins. Numerical simulations of the stochastic version of the model are performed by means of the Gillespie method. The predictions of the stochastic approach compare well with those of the deterministic model with respect both to sustained oscillations of the limit cycle type and to the influence of the proximity from a bifurcation point beyond which the system evolves to stable steady state. Stochastic simulations indicate that robust circadian oscillations can emerge at the cellular level even when the maximum numbers of mRNA and protein molecules involved in the oscillations are of the order of only a few tens or hundreds. The stochastic model also reproduces the evolution to a strange attractor in conditions where the deterministic PER-TIM model admits chaotic behaviour. The difference between periodic oscillations of the limit cycle type and aperiodic oscillations (i.e. chaos) persists in the presence of molecular noise, as shown by means of Poincaré sections. The progressive obliteration of periodicity observed as the number of molecules decreases can thus be distinguished from the aperiodicity originating from chaotic dynamics. As long as the numbers of molecules involved in the oscillations remain sufficiently large (of the order of a few tens or hundreds, or more), stochastic models therefore provide good agreement with the predictions of the deterministic model for circadian rhythms.     

Circadian and Ultradian Rhythms in Blood Glucose and Plasma Insulin of Healthy Adults

The circadian and ultradian variations of blood glucose and plasma insulin have been characterized individually and as a group phenomenon in five healthy young adults studied while adhering as closely as possible to their usual routine of sleep, activity, meal content and timing. Three complementary methods were used to analyze the data: displaying raw data as a function of time; cosinor method according to Nelson and Halberg; and time series analyses as proposed by De Prins and Malbecq. The subjects were studied in the laboratory and their life routine were controlled, but very close to that of their habitual routine. They had mainly ultradian rhythms of blood glucose (mainly about 6 hr) and circadian rhythms of immunoreactive insulin (I.R.I.). Blood glucose ultradian rhythms seem to be mainly but not exclusively mealtime dependent, while I.R.I, circadian rhythms appear to be primarily endogenous in origin. Therefore, the role played by insulin in the control of blood glucose levels seems to be programmed on a circadian basis rather than by a time independent feedback phenomenon as postulated by the conventional homeostatic hypothesis. The advantage of this chronophysiologic approach is to consider circadian rhythms of both I.R.I. and insulin effectiveness as an adaptive phenomenon able to maintain blood sugar changes in the ultradian domain of rhythms.     

Adaptive significance of a circadian clock: temporal segregation of activities reduces intrinsic competitive inferiority in Drosophila parasitoids

Most organisms show self-sustained circadian oscillations or biological clocks which control their daily fluctuations in behavioural and physiological activities. While extensive progress has been made in understanding the molecular mechanisms of biological clocks, there have been few clear demonstrations of the fitness value of endogenous rhythms. This study investigated the adaptive significance of circadian rhythms in a Drosophila parasitoid community. The activity rhythms of three sympatric Drosophila parasitoids are out of phase, the competitively inferior parasitoid species being active earlier than the superior competitor. This temporal segregation appears at least partially determined by endogenous periods of the clock which also vary between species and which correlate the time of activity. This earlier activity of the inferior competitor significantly reduces its intrinsic competitive disadvantage when multiparasitism occurs, thus suggesting that natural selection acting on the phase of the rhythm could substantially deviate the endogenous period from the optimal ca. 24 h period. This study demonstrates that temporal segregation of competing species could be endogenously controlled, which undoubtedly favours their coexistence in nature and also shows how natural selection can act on biological clocks to shape daily activity patterns.