The circadian clock in the brain: a structural and functional comparison between mammals and insects

The circadian master clocks in the brains of mammals and insects are compared in respect to location, organization and function. They show astonishing similarities. Both clocks are anatomically and functionally connected to the optic system and possess multiple output pathways allowing synchronization with the environmental light-dark cycles as well as the control of diverse endocrine, autonomic and behavioral functions. Both circadian master clocks are composed of multiple neurons, which are organized in populations with different morphology, physiology and neurotransmitter content and appear to subserve different functions. In the hamster and in the cockroach, the master clock consists of a core region that gets input from the eyes, and a shell region from which the majority of output projections originate. Communication between core and shell, between all other populations of clock neurons as well as between the master clocks of both brain hemispheres is a prerequisite of normal rhythmic function. Phenomena like rhythm splitting and internal desynchronization can be observed under constant light conditions and are caused by the uncoupling of the master clocks of both brain hemispheres.     

A partial hepatectomy results in altered expression of clock-related and cyclic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes

The liver is among the peripheral organs that display a clear circadian rhythmicity. To investigate whether specific pathological conditions affect circadian rhythms in the liver, we examined the expression profiles of the clock-related and glyceraldehyde 3-phosphate dehydrogenase (GADPH) genes following a partial hepatectomy in the mouse. This surgical procedure causes dynamic proliferation of residual hepatocytes and within one day of the operation the hepatectomized mice demonstrated higher expression of both mPer1 and mPer2 genes in the remaining liver tissue when compared to control mice that had undergone a Sham-operation. In contrast, the mCry1 gene in hepatectomized mice displayed a circadian gene expression profile that was similar to the control group. In addition, GAPDH levels, that demonstrated no oscillations in Sham-hepatectomized mice, underwent daily alterations following a partial hepatectomy. These findings suggest that the regenerative state of the liver affects the expression not only of clock-related genes but also of genes that are constitutively expressed under steady state conditions.     

Short-term propofol anaesthesia down-regulates clock genes expression in the master clock

ABSTRACT

Background: Propofol anesthesia triggers phase-advances of circadian rhythms controlled by the suprachiasmatic nuclei (SCN), the master clock. Besides, inhalational anesthesia has been associated with a subsequent reduction of Per2 mRNA levels in the whole brain of rodents. The acute effects of propofol anesthesia per se on the SCN molecular clockwork remain unclear. Here we aim to study the expression of Per1 and Per2 clock genes in the SCN of rats exposed to constant darkness after a single dose of propofol. Methods: Thirty 2-months old rats were randomly divided into 2 groups receiving a single dose of either 120 mg/kg propofol 1% (n=15), or intralipid® 10% (n=15) in late day (projected circadian time (CT) 10, i.e., 10h after the expected time of lights on). Thereafter, rat brains were sampled in darkness 1h, 2h or 3h after the treatment (projected CT11, CT12 or CT13). Expression of Per1 and Per2 mRNA was analyzed by in situ hybridization in SCN coronal sections. Results: Per1 expression was affected by time and treatment. Per1 expression in the SCN after propofol treatment decreased at CT11 and CT12 when compared to the vehicle group. For Per2 expression, we observed only a treatment effect. Observed in dark conditions without hypothermia or/and concomitant surgery, such down-regulation of clock genes Per is only correlated to propofol treatment. This may explain “jet-lag-like” symptoms described by patients after anesthesia. Conclusion: We show here for the first time that short-term propofol anesthesia leads to a transient down-regulation of Per1 and Per2 expression in the SCN.     

Timing is everything: regulation of Cdk1 and aneuploidy

The spindle checkpoint ensures the proper partition of the chromosomal content of dividing cells, by controlling the transition from metaphase to anaphase. In a recent issue of Cancer Cell, Vecchione and coworkers report that the protein product of the tumor suppressor gene Lzts1 (Leucine zipper tumor suppressor-1) binds the Cdk1 phosphatase Cdc25C and stabilizes it by protecting it from proteasomal degradation (Vecchione et al., 2007). Partial or complete loss of Lzts1 downregulates Cdc25C and inhibits Cdk1 activity during mitosis, leading to premature transition from metaphase to anaphase.     

Timing is everything

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.     

An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433–451, 2000)     

Daily Expression of Six Clock Genes in Central and Peripheral Tissues of a Night-Migratory SongBird: Evidence for Tissue-Specific Circadian Timing

In birds, independent circadian clocks reside in the retina, pineal, and hypothalamus, which interact with each other and produce circadian time at the functional level. However, less is known of the molecular clockwork, and of the integration between central and peripheral clocks in birds. The present study investigated this, by monitoring the timed expression of five core clock genes (Per2. Cry1. Cry2. Bmal1, and Clock) and one clock-controlled gene (E4bp4) in a night-migratory songbird, the redheaded bunting (rb; Emberiza bruniceps). The authors first partially cloned these six genes, and then measured their 24-h profiles in central (retina, hypothalamus) and peripheral (liver, heart, stomach, gut, testes) tissues, collected at six times (zeitgeber time 2 [ZT2], ZT6, ZT11, ZT13, ZT18, and ZT23; ZT0?=?lights on) from birds (n?=?5 per ZT) on 12?h:12?h light-dark cycle. rbPer2. rbCry1. rbBmal1, and rbClock were expressed with a significant rhythm in all the tissues, except in the retina (only rbClock) and testes. rbCry2, however, had tissue-specific expression pattern: a significant rhythm in the hypothalamus, heart, and gut, but not in the retina, liver, stomach, and testes. rbE4bp4 had a significant mRNA rhythm in all the tissues, except retina. Further, rbPer2 mRNA peak was phase aligned with lights on, whereas rbCry1. rbBmal1, and rbE4bp4 mRNA peaks were phase aligned with lights off. rbCry2 and rbClock had tissue-specific scattered peaks. For example, both rbCry2 and rbClock peaks were close to rbCry1 and rbBmal1 peaks, respectively, in the hypothalamus, but not in other tissues. The results are consistent with the autoregulatory circadian feedback loop, and indicate a conserved tissue-level circadian time generation in buntings. Variable phase relationships between gene pairs forming positive and negative limbs of the feedback loop may suggest the tissue-specific contribution of individual core circadian genes in the circadian time generation.     

The circadian system and melatonin: lessons from rats and mice

The circadian system (CS) comprises three key components: (1) endogenous oscillators (clocks) generating a circadian rhythm; (2) input pathways entraining the circadian rhythm to the astrophysical day; and (3) output pathways distributing signals from the oscillator to the periphery. This contribution briefly reviews some general aspects of the organization of the rodent CS and pays particular attention to recent results obtained with various mouse strains, related to molecular mechanisms involved in entraining the endogenous clock and the role of the pineal hormone melatonin as a hand of the endogenous clock.     

The circadian regulation of extracellular ATP

Extracellular ATP is a potent signaling molecule released from various cells throughout the body and is intimately involved in the pathophysiological functions of the nervous system and immune system by activating P2 purinergic receptors. Recent increasingly studies showed that extracellular ATP exhibits circadian oscillation with an approximately 24-h periodicity, which participates in regulatory pathways of central oscillator suprachiasmatic nucleus and peripheral oscillator bladder, respectively. Oscillators modulate the protein expression of ATP release channels and ectonucleotidase activity through clock genes; indeed, real-time alterations of ATP release and degradation determine outcomes of temporal character on extracellular ATP rhythm. The regulatory pathways on extracellular ATP rhythm are different in central and peripheral systems. In this review, we summarize the circadian rhythm of extracellular ATP and discuss several circadian regulatory pathways in different organs via ATP release and degradation, to provide a new understanding for purinergic signaling in the regulatory mechanism of circadian rhythm and a potential target to research the circadian regulation of extracellular ATP in other circadian oscillators.     

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433-451, 2000)     

Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN

Proper completion of mitosis requires careful coordination of numerous cellular events. It is crucial, for example, that cells do not initiate spindle disassembly and cytokinesis until chromosomes have been properly segregated. Cells have developed numerous safeguards or checkpoints to delay exit from mitosis and initiation of the next cell cycle in response to defects in late mitosis. In this review, we discuss recent work on two homologous signaling pathways in budding and fission yeast, termed the mitotic exit network (MEN) and septation initiation network (SIN), respectively, that are essential for coordinating completion of mitosis and cytokinesis with other mitotic events.     

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

Scheduled Feeding Alters the Timing of the Suprachiasmatic Nucleus Circadian Clock in Dexras1-Deficient Mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1^?/?) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1^?/? mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1^?/? mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca)