Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in 'arbitrary units', is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.     

Oscillatory Transpiration of Avena Plants: Perturbation Experiments Provide Evidence for a Stable Point of Singularity

Oscillatory plant water regulation of young Avena plants was studied. The period of the oscillations was around 40 min. Pulse perturbations were given to plants showing oscillations in the transpiration rate. Perturbations consisted in temporary irradiance changes of the leaf or in water potential changes around the root. The effect of the pulse perturbations on the amplitude of the oscillations was recorded. The oscillations could be permanently halted after a perturbation of suitable magnitude given at a suitable phase of The oscillations. A subsequent perturbation could restart the oscillations again. By means of simulations it was shown that a feedback model For the transpiration oscillations could explain the experimental outcome if a non finearity of a special kind was incorporated. The circadian eclosion rhythm of Drosophila pseudoobscura and the petal rhythm of Kalanchoe blossfeldiana show many features in common with the experiments reported. Biophysically the present results indicate that the transpiration oscillations of Avena plants have a stable point of equilibrium or a stable point of singularity.     

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.     

PPARalpha is a potential therapeutic target of drugs to treat circadian rhythm sleep disorders

Recent progress at the molecular level has revealed that nuclear receptors play an important role in the generation of mammalian circadian rhythms. To examine whether peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in the regulation of circadian behavioral rhythms in mammals, we evaluated the locomotor activity of mice administered with the hypolipidemic PPARalpha ligand, bezafibrate. Circadian locomotor activity was phase-advanced about 3h in mice given bezafibrate under light-dark (LD) conditions. Transfer from LD to constant darkness did not change the onset of activity in these mice, suggesting that bezafibrate advanced the phase of the endogenous clock. Surprisingly, bezafibrate also advanced the phase in mice with lesions of the suprachiasmatic nucleus (SCN; the central clock in mammals). The circadian expression of clock genes such as period2, BMAL1, and Rev-erbalpha was also phase-advanced in various tissues (cortex, liver, and fat) without affecting the SCN. Bezafibrate also phase-advanced the activity phase that is delayed in model mice with delayed sleep phase syndrome (DSPS) due to a Clock gene mutation. Our results indicated that PPARalpha is involved in circadian clock control independently of the SCN and that PPARalpha could be a potent target of drugs to treat circadian rhythm sleep disorders including DSPS.     

Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.     

Biological clocks in Arabidopsis thaliana

Biological rhythms are ubiquitous in eukaryotes, and the best understood of these occur with a period of approximately a day – circadian rhythms. Such rhythms persist even when the organism is placed under constant conditions, with a period that is close, but not exactly equal, to 24 h, and are driven by an endogenous timer – one of the many 'biological clocks'. In plants, research into circadian rhythms has been driven forward by genetic experiments using Arabidopsis . Higher plant genomes include a particularly large number of genes involved in metabolism, and circadian rhythms may well provide the necessary coordination for the control of these – for example, around the diurnal rhythm of photosynthesis – to suit changing developmental or environmental conditions. The endogenous timer must be flexible enough to support these requirements. Current research supports this notion most strongly for the input pathway, in which multiple photoreceptors have been shown to mediate light input to the clock. Both input and output components are now related to putative circadian oscillator mechanisms by sequence homology or by experimental observation. It appears that the pathways linking some domains of the basic clock model may be very short indeed, or the mechanisms of these domains may overlap. Components of the first plant circadian output pathway to be identified unequivocally will help to determine exactly how many output pathways control the various phases of overt rhythms in plants.     

Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Short-term and circadian rhythms in the behaviour of the vole,Microtus agrestis (L.)

Summary The activity behaviour of the vole, Microtus agrestis, has been recorded in order to investigate the relationship between short-term rhythm and circadian rhythm. A simple device was developed, allowing separate monitoring of the time spent in or outside the nest, wheel-running, eating and drinking. Under natural light conditions during summer, a distinct differentiation between a short term rhythm of eating and drinking during the day-time and a circadian rhythm of wheel-running during the night was observed. The short-term rhythm depends closely on metabolic demands (hunger, thirst, excretion). Control of these demands by an endogenous oscillation could not be substantiated. The circadian rhythm of wheel-running activity is, however, controlled by an endogenous oscillation, synchronized by light conditions. It is subjected to seasonal variations. a) The threshold of light intensity below which wheel-running occurs is lowest during summer (<0.5 lx) and is higher during spring and autum (> 5 lx). b) Wheel-running is controlled by a circadian oscillation during summer only whereas it is an integrated part of the short-term rhythm during spring and autumn (experiments during the winter have not yet been performed). Experiments gave evidence that the properties of the cage can deeply influence the amount and pattern of wheel-running activity. It is concluded that wheel-running reflects a certain level of excitation, which may be caused by different behavioural intentions. The seasonal changes of the control of wheel-running activity are discussed with respect to this assumption. The relevancy of locomotor activity patterns as usually recorded in the laboratory to reveal the physiological and ecological significance of endogenously controlled behavioural patterns is discussed.Supported by the Deutsche Forschungsgemeinschaft     

ELF4 is required for oscillatory properties of the circadian clock

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 (ELF4) locus of Arabidopsis (Arabidopsis thaliana) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LONG ELONGATED HYPOCOTYL (LHY)-TIMING OF CAB EXPRESSION1 (TOC1) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4, therefore, can be considered a component of the central CCA1/LHY-TOC1 feedback loop in the plant circadian clock.     

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.     

The Circadian Rhythm of Stomatal Opening: Evidence for the Involvement of Potassium and Chloride Fluxes

Epidermal strips were taken from plants of Commelina communisat opposite phases of an entrained circadian rhythm, at timeswhich corresponded to the middle of the day phase and the middleof the night phase. Earlier observations of much reduced openingin response to external stimuli in the night phase were confirmed. Evidence is now presented that uptake or retention of specificions by the guard cells is affected by the phase of the rhythm.The overt expression of the rhythm was reduced if the epidermiswas presented with potassium iminodiacetate instead of potassiumchloride. Iminodiacetate is a non-absorbable anion, and thisresult suggested that chloride uptake is important for the fullexpression of the rhythm. The use of an anion channel inhibitor(DIDS) gave strong support for this conclusion. Reduced uptakeof chloride would explain an earlier observation that formationof malate is greater during the night phase. When the guard cells were presented with sodium chloride insteadof potassium chloride there was still an overt expression ofthe rhythm, but this was of reduced magnitude. This suggeststhat potassium movements might contribute to the rhythm, andstudies of ionic fluxes using ⁸⁶Rb as a tracer showed greatereffluxes during the night phase. It is concluded that the circadian rhythm in stomatal openingmay be the result of a varying ability of the guard cells toaccumulate or retain both chloride and potassium ions. Key words: Circadian rhythm, stomatal opening, Commelina communis, potassium and chloride fluxes     

The effects of intraventricular carbachol injections on the free-running activity rhythm of the hamster

The effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN) are mediated by the retinohypothalamic tract (RHT) and by the retinogeniculosuprachiasmatic tract (RGST). The neurotransmitter of the RGST is neuropeptide Y. The RHT may contain glutamate and aspartate. Recent evidence indicates that acetylcholine could also be involved in phase shifting by light. We determined that intraventricular injections with an acetylcholine agonist, carbachol, induces phase advances during the subjective day and phase delays during the early subjective night. No differences were observed between phase shifts induced in constant darkness and those induced in continuous light. A dose-response curve for carbachol was described at circadian time 6 (CT6). Injections at CT14 with various dosages of carbachol indicated the same dose dependency for this circadian time. Finally, carbachol injections in split animals resulted in similar responses of the two components of the split activity rhythm.