Ultradian Clock Research Articles

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock.

Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.

Logic of the Temporal Compartmentalization of the Hepatic Metabolic Cycle.

The mammalian liver must cope with various metabolic and physiological changes that normally recur every day and result primarily from rest-activity and fasting-feeding cycles. In this article, I present evidence supporting a temporal compartmentalization of rhythmic hepatic metabolic processes into four main clusters: regulation of energy homeostasis, maintenance of information integrity, immune response, and genetic information flow. I further review literatures and discuss how both the circadian and the newly discovered 12-h ultradian clock work together to regulate these four temporally separated processes in mouse liver, which, interestingly, is largely uncoupled from the liver zonation regulation.

Temporal metabolic partitioning of the yeast and protist cellular networks: the cell is a global scale-invariant (fractal or self-similar) multioscillator.

.Britton Chance, electronics expert when a teenager, became an enthusiastic student of biological oscillations, passing on this enthusiasm to many students and colleagues, including one of us (DL). This historical essay traces BC’s influence through the accumulated work of DL to DL’s many collaborators. The overall temporal organization of mass-energy, information, and signaling networks in yeast in self-synchronized continuous cultures represents, until now, the most characterized example of in vivo elucidation of time structure. Continuous online monitoring of dissolved gases by direct measurement (membrane-inlet mass spectrometry, together with NAD(P)H and flavin fluorescence) gives strain-specific dynamic information from timescales of minutes to hours as does two-photon imaging. The predominantly oscillatory behavior of network components becomes evident, with spontaneously synchronized cellular respiration cycles between discrete periods of increased oxygen consumption (oxidative phase) and decreased oxygen consumption (reductive phase). This temperature-compensated ultradian clock provides coordination, linking temporally partitioned functions by direct feedback loops between the energetic and redox state of the cell and its growing ultrastructure. Multioscillatory outputs in dissolved gases with 13 h, 40 min, and 4 min periods gave statistical self-similarity in power spectral and relative dispersional analyses: i.e., complex nonlinear (chaotic) behavior and a functional scale-free (fractal) network operating simultaneously over several timescales.

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach.

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

Temperature affects frequency but not rhythmicity of nocturnal awakenings in free-living great tits, Parus major

Several bird species display periodic nocturnal sleep–wake patterns, resembling the ultradian rhythms expressed by mammals. Although relatively little is known about the underlying molecular properties of ultradian biological clocks, field observations demonstrate that the frequency of nocturnal awakenings (which may relate to rhythmicity) increases with ambient environmental temperature. To understand how ambient environmental temperature conditions affect the nocturnal sleep–wake pattern of birds, we experimentally heated nestboxes during the night and monitored the frequency and rhythmicity of awakening behaviour of roosting great tits. More than 80% of great tits displayed ultradian rhythmicity in major nocturnal awakenings, with awakenings occurring every 50–170 min. Experimental increases in temperature, on average, 5 °C, caused birds to wake up approximately 30% more frequently over the course of the night, with the strongest temperature effect occurring during the first part of the night. However, the period length of the predominant nocturnal awakening rhythm was unaffected by increased temperature, likely because most additional awakenings were arrhythmic and clustered during the beginning of the night. We suggest that short-duration awakenings elicited primarily during the first part of the night may not be regulated by an ultradian biological clock, and may respond directly to current environmental conditions, such as the risk environment. Longer-duration awakening bouts, which were not affected by experimental heating, may instead be endogenously regulated by an ultradian clock to optimize clock-controlled sleep patterns and sleep homeostasis.

The end effector of circadian heart rate variation: the sinoatrial node pacemaker cell.

Cardiovascular function is regulated by the rhythmicity of circadian, infradian and ultradian clocks. Specific time scales of different cell types drive their functions: circadian gene regulation at hours scale, activation-inactivation cycles of ion channels at millisecond scales, the heart's beating rate at hundreds of millisecond scales, and low frequency autonomic signaling at cycles of tens of seconds. Heart rate and rhythm are modulated by a hierarchical clock system: autonomic signaling from the brain releases neurotransmitters from the vagus and sympathetic nerves to the heart’s pacemaker cells and activate receptors on the cell. These receptors activating ultradian clock functions embedded within pacemaker cells include sarcoplasmic reticulum rhythmic spontaneous Ca2+ cycling, rhythmic ion channel current activation and inactivation, and rhythmic oscillatory mitochondria ATP production. Here we summarize the evidence that intrinsic pacemaker cell mechanisms are the end effector of the hierarchical brain-heart circadian clock system. [BMB Reports 2015; 48(12): 677-684]

Fluoxetine normalizes disrupted light-induced entrainment, fragmented ultradian rhythms and altered hippocampal clock gene expression in an animal model of high trait anxiety- and depression-related behavior

ABSTRACTIntroduction Disturbances of circadian rhythms are a key symptom of mood and anxiety disorders. Selective serotonin reuptake inhibitors (SSRIs) - commonly used antidepressant drugs – also modulate aspects of circadian rhythmicity. However, their potential to restore circadian disturbances in depression remains to be investigated.Materials and methods The effects of the SSRI fluoxetine on genetically based, depression-related circadian disruptions at the behavioral and molecular level were examined using mice selectively bred for high anxiety-related and co-segregating depression-like behavior (HAB) and normal anxiety/depression behavior mice (NAB).Results The length of the circadian period was increased in fluoxetine-treated HAB as compared to NAB mice while the number of activity bouts and light-induced entrainment were comparable. No difference in hippocampal Cry2 expression, previously reported to be dysbalanced in untreated HAB mice, was observed, while Per2 and Per3 mRNA levels were higher in HAB mice under fluoxetine treatment.Discussion The present findings provide evidence that fluoxetine treatment normalizes disrupted circadian locomotor activity and clock gene expression in a genetic mouse model of high trait anxiety and depression. An interaction between the molecular mechanisms mediating the antidepressant response to fluoxetine and the endogenous regulation of circadian rhythms in genetically based mood and anxiety disorders is proposed.

Circadian genes, xBmal1 and xNocturnin, modulate the timing and differentiation of somites in Xenopus laevis.

We have been investigating whether xBmal1 and xNocturnin play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (xPeriod1, xPeriod2, xBmal1, and xNocturnin) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In Xenopus, a pair of somites is formed about every 50 minutes from anterior to posterior. We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock. Cyclic expression of genes involved in Notch signaling has been implicated in the somite clock. Disruption of Notch signaling in humans has been linked to skeletal defects in the vertebral column. We found that both depletion (morpholino) and overexpression (mRNA) of xBMAL1 protein (bHLH transcription factor) or xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001). These manipulations also significantly affect expression of a somite clock component (xESR9; p<0.05). We observed opposing effects on somite size. Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.02; p<0.001, respectively). We speculate that circadian genes may play two separate roles during somitogenesis. Depletion and overexpression of xBMAL1 and NOCTURNIN both decrease somite number and influence expression of a somite clock component, suggesting that these proteins may modulate the timing of the somite clock in the undifferentiated presomitic mesoderm. The dosage dependent effects on somite area suggest that xBMAL1 and xNOCTURNIN may also act during somite differentiation to promote myogenesis.

The frequency of the spontaneous behavioral response in Paramecium tetraurelia is simultaneously modulated by both ultradian and circadian rhythms

The behavioral response of Paramecium tetraurelia is due to changes in the direction of swimming, which is initiated by the generation of an action potential that causes the re-orientation of the ciliary beat. The frequency of spontaneous behavioral responses displays an ultradian rhythm, with a periodicity of approximately 50 min. Furthermore, this rhythm is disrupted by the addition of LiCl, due to the interruption of the inositol signaling pathway. The present work is an observation of the frequency of the behavioral response for a 72-h period. The frequency of the behavioral response is influenced by a circadian clock, in that the frequency of behavioral responses changes during a 24-h period, while the periodicity of the ultradian clock remains unchanged. The addition of LiCl also disrupts the circadian rhythm, lengthening the circadian period from 24 h to 27.4 h. However, unlike the ultradian rhythm, where the addition of myo-inositol inhibits the LiCl effect, myo-inositol has no effect on the disruption of the circadian rhythm. In order to ascertain the effect of LiCl on the circadian clock, cells were grown in the presence of indirubin, a compound known to inhibit glycogen synthase kinase 3β (GSK3β); this enzyme is also inhibited by LiCl. When cells are incubated with indirubin for several hours, the circadian rhythm is lengthened to approximately 27 h, while the ultradian rhythm is unaffected. Furthermore, a decrease in GSK3β by RNA interference gives similar results. Therefore, the frequency of the behavioral response in Paramecium appears to be controlled by two different biological rhythms.

A fluorescence spotlight on the clockwork development and metabolism of bone

Biological phenomena that exhibit periodic activity are often referred as biorhythms or biological clocks. Among these, circadian rhythms, cyclic patterns reflecting a 24-h cycle, are the most obvious in many physiological activities including bone growth and metabolism. In the late 1990s, several clock genes were isolated and their primary structures and functions were identified. The feedback loop model of transcriptional factors was proposed to work as a circadian core oscillator not only in the suprachiasmatic nuclei of the anterior hypothalamus, which is recognized as the mammalian central clock, but also in various peripheral tissues including cartilage and bone. Looking back to embryonic development, the fundamental architecture of skeletal patterning is regulated by ultradian clocks that are defined as biorhythms that cycle more than once every 24 h. As post-genomic approaches, transcriptome analysis by micro-array and bioimaging assays to detect luminescent and fluorescent signals have been exploited to uncover a more comprehensive set of genes and spatio-temporal regulation of the clockwork machinery in animal models. In this review paper, we provide an overview of topics related to these molecular clocks in skeletal biology and medicine, and discuss how fluorescence imaging approaches can contribute to widening our views of this realm of biomedical science.

Ultradian rhythm in the intestine of Caenorhabditis elegans is controlled by the C-terminal region of the FLR-1 ion channel and the hydrophobic domain of the FLR-4 protein kinase

Defecation behavior in Caenorhabditis elegans is driven by an endogenous ultradian clock in the intestine. Its periods are positively regulated by FLR-1, an ion channel of the epithelial sodium channel/degenerin superfamily, and FLR-4, a protein kinase with a hydrophobic domain at the carboxyl terminus. FLR-1 has many putative phosphorylation sites in the C-terminal intracellular region. This structure implies that the periods may be regulated by the phosphorylation of FLR-1 by FLR-4, but it remains to be clarified. Here, we show that a truncated FLR-1 lacking the C-terminal intracellular region resulted in longer periods, suggesting that this region is involved in the negative regulation of defecation cycle periods. Contrary to our expectation, FLR-4 was still necessary for the function of the truncated FLR-1. Furthermore, FLR-4 containing a kinase-dead mutation or lacking the whole kinase domain was sufficient for normal defecation cycle periods. FLR-4 was necessary for the stable expression of FLR-1::GFP, and its hydrophobic domain was sufficient also for this function. FLR-1 and FLR-4 are often colocalized in the plasma membrane. These data showed an unexpected role of FLR-4: its hydrophobic domain stabilizes the FLR-1 ion channel, a key regulator of defecation cycle periods in the intestine.

The frequency of the behavioral response in Paramecium tetraurelia displays an ultradian rhythm: a regulatory role for the inositol signaling pathway

Paramecium tetraurelia displays a behavioral response to environmental stimuli that involves changes in the direction and speed of swimming. This paper demonstrates that there are periodic variations in the frequency of behavioral responses of individual cells. The frequency of the backward swimming response undergoes repeated oscillations with a period (tau) between 40 and 50 minutes; these oscillations are only slightly affected by the temperature at which the cells have been grown. These data indicate the presence of an ultradian clock that underlies this cellular process. However, growing cells in the presence of low concentrations of LiCl perturbs the ultradian rhythm, with the periodicity being reduced to approximately 11 minutes. The LiCl effect is seen within 10–45 minutes, depending on the concentration of LiCl used, and is completely reversible. In order to determine if the LiCl effect was due to a depletion of inositol, cells were grown in 10 mM myo-inositol for 24 hours prior to the addition of LiCl. It was found that the presence of excess inositol inhibited the perturbation of the periodicity caused by the LiCl. These results signify that Paramecium tetraurelia possesses an endogenous ultradian clock that influences the strength of the behavioral response, and the inositol signaling pathway is most likely involved in the generation of the ultradian rhythm. Therefore, the Paramecium behavioral response is an excellent model system for elucidation of the molecular mechanisms underlying the ultradian rhythms.

Individual behavioural rhythmicity is linked to social motivation in Japanese quail

This experiment explored links between individual social traits and behavioural rhythms by comparing the feeding activity rhythms of HSR (high social reinstatement) Japanese quail that will cover long distances to reach conspecifics, to those of LSR (low social reinstatement) quail that will cover only short distances to reach conspecifics. We evaluated the functioning of their circadian and ultradian clocks under constant darkness (DD), and then under photoperiodic conditions (LD). Our results revealed that, under DD conditions, the circadian periods of HSR quail were closer to 24 h than those of LSR quail. Under LD conditions, the daily rhythm of HSR quail was clearer than that of LSR quail. HSR quail synchronized faster and better to the environmental LD cycle than LSR quail. Our results also showed that, under DD conditions, more LSR than HSR quail expressed ultradian rhythmic phenotypes and that the ultradian rhythm of LSR quail was clearer than that of LSR quail. Under LD conditions, these differences between HSR and LSR quail showed the same tendencies. LSR quail seem to present a more functional ultradian system, and thus could possibly synchronise better on an ultradian environmental cue than HSR quail. Our study demonstrated a link between individual rhythmicity and social traits at an intra-specific level. It revealed complex relationships between ultradian, circadian, and daily rhythms and level of social motivation in Japanese quail.

Computational Analysis of Mammalian Cell Division Gated by a Circadian Clock: Quantized Cell Cycles and Cell Size Control

Cell cycle and circadian rhythms are conserved from cyanobacteria to humans with robust cyclic features. Recently, molecular links between these two cyclic processes have been discovered. Core clock transcription factors, Bmal1 and Clock (Clk), directly regulate Wee1 kinase, which inhibits entry into the mitosis. We investigate the effect of this connection on the timing of mammalian cell cycle processes with computational modeling tools. We connect a minimal model of circadian rhythms, which consists of transcription-translation feedback loops, with a modified mammalian cell cycle model from Novak and Tyson (2004). As we vary the mass doubling time (MDT) of the cell cycle, stochastic simulations reveal quantized cell cycles when the activity of Wee1 is influenced by clock components. The quantized cell cycles disappear in the absence of coupling or when the strength of this link is reduced. More intriguingly, our simulations indicate that the circadian clock triggers critical size control in the mammalian cell cycle. A periodic brake on the cell cycle progress via Wee1 enforces size control when the MDT is quite different from the circadian period. No size control is observed in the absence of coupling. The issue of size control in the mammalian system is debatable, whereas it is well established in yeast. It is possible that the size control is more readily observed in cell lines that contain circadian rhythms, since not all cell types have a circadian clock. This would be analogous to an ultradian clock intertwined with quantized cell cycles (and possibly cell size control) in yeast. We present the first coupled model between the mammalian cell cycle and circadian rhythms that reveals quantized cell cycles and cell size control influenced by the clock.

Modeling the Hes1 Oscillator

Somitogenesis describes the segmentation of vertebrate embryonic bodies, which is thought to be induced by ultradian clocks (i.e., clocks with relatively short cycles compared to circadian clocks). One candidate for such a clock is the bHLH factor Hes1, forming dimers which repress the transcription of its own encoding gene. Most models for such small autoregulative networks are based on delay equations where a Hill function represents the regulation of transcription. The aim of the present paper is to estimate the Hill coefficient in the switch of an Hes1 oscillator and to suggest a more detailed model of the autoregulative network. The promoter of Hes1 consists of three to four binding sites for Hes1 dimers. Using the sparse data from literature, we find, in contrast to other statements in literature, that there is not much evidence for synergistic binding in the regulatory region of Hes1, and that the Hill coefficient is about three. As a model for the negative feedback loop, we use a Goodwin system and find sustained oscillations for systems with a large enough number of linear differential equations. By a suitable variation of the number of equations, we provide a rational lower bound for the Hill coefficient for such a system. Our results suggest that there exist additional nonlinear processes outside of the regulatory region of Hes1.

The ultradian clock: not to be confused with the cell cycle

The ultradian clock: not to be confused with the cell cycle

Intestinal calcium waves coordinate a behavioral motor program in C. elegans

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans, Genetics 124 (1990) 855–872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C. elegans, J. Neurosci. 14 (1994) 1953–1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA 92 (1995), 10317–10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50 s behavioral rhythm in C. elegans, Cell 98 (1999) 757–767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca 2+ dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca 2+-wave propagation in the intestine. Lack of the Ca 2+-wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca 2+-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca 2+-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans.

Ultradian metronome: timekeeper for orchestration of cellular coherence

Dynamic intracellular spatial and temporal organization emerges from spontaneous synchronization of a massive array of weakly coupled oscillators; the majority of subcellular processes are implicated in this integrated expression of cellular physiology. Evidence for this view comes mainly from studies of Saccharomyces cerevisiae growing in self-synchronized continuous cultures, in which a temperature-compensated ultradian clock (period of approximately 40 min) couples fermentation with redox state in addition to the transcriptome and cell-division-cycle progression. Functions for ultradian clocks have also been determined in other yeasts (e.g. Schizosaccharomyces pombe and Candida utilis), seven protists (e.g. Acanthamoeba castellanii and Paramecium tetraurelia), as well as cultured mammalian cells. We suggest that ultradian timekeeping is a basic universal necessity for coordinated intracellular coherence.

An ultradian clock shapes genome expression in yeast.

Cell biology has traditionally focused on where rather than when things happen inside cells. The fundamental biochemical activities of the cell have been sorted into spatial domains, with functions allotted to subcellular compartments that can either promote or inhibit molecular interaction. Important exceptions to this prominence of three dimensions have arisen in the areas of cell-division cycle research and studies of circadian (≈24-hr) rhythm. Here the emphasis has been distinctly time-oriented, and recent efforts have made use of powerful new strategies to develop temporal maps of gene expression and macromolecular assembly. Microarray analyses have exposed deeply rooted, cell-autonomous programs of timed gene activity, and in both areas sequential control is extensive. Circadian rhythms are associated with the ordered expression of 10% or more (see below) of the genome in Arabidopsis, Drosophila , and the mouse, and a comparable fraction of the genome appears to be regulated by the cell cycle in yeast (1–12). An even more pervasive system of temporal organization is now the subject of a study in this issue of PNAS by Klevecz et al. (13), who follow the yeast Saccharomyces cerevisiae . An initial search for clusters of gene expression that might support a well known ≈40-min respiratory cycle has instead revealed that ≈40-min rhythms are a genomewide phenomenon. Truly constitutive expression may not apply to any gene's activity in continuously grown, aerobic cultures of yeast. Respiratory oscillations have been observed in budding yeast for at least 30 years and are readily detected at higher cell densities in continuous culture. It has been proposed that a stage of ethanol metabolism may supply diffusible factors that effect synchrony (14). In addition to periodic fluctuations in the concentration of dissolved oxygen, NAD(P)H and reduced glutathione concentrations are rhythmic. Perturbations of the reduced glutathione concentration reset the phase …

Effects of uncoupling of mitochondrial energy conservation on the ultradian clock-driven oscillations in Saccharomyces cerevisiae continuous culture

Protonophores have several different perturbative effects on dissolved O 2 concentrations in continuous cultures of Saccharomyces cerevisiae. As well as uncoupling energy conservation from mitochondrial electron transport in vivo, they reset ultradian clock-driven respiratory oscillations and produce cell cycle effects. Thus, additions at low concentration (1.25 μM) of either m-chlorocarbonyl-cyanide phenylhydrazone (CCCP) or 5-chloro-3- t-butyl-2-chloro-4 1-nitrosalicylanilide (S 13) led to phase resetting of the 48 min ultradian clock-driven respiratory oscillations. At 2.5 μM CCCP or 4 μM S 13, transient inhibition of oscillatory respiration (for 5 h) preceded synchronisation of the cell division cycle seen as a slow (9 h period) wave that enveloped the 48 min oscillation. At still higher concentrations of CCCP (5 μM), the cell division cycle was prolonged by about 7 h, and during this phase, the respiratory oscillation became undetectable. The significance of these observations with respect to the time-keeping functions of the ultradian clock is discussed.