Mammalian Circadian Timing System Research Articles

Circadian Gene Expression in Individual Fibroblasts: Cell-Autonomous and Self-Sustained Oscillators Pass Time to Daughter Cells

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.

The mammalian circadian timing system: from gene expression to physiology.

Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues. Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms. In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism. The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus. However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons. This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology. In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation.

The brain's calendar: neural mechanisms of seasonal timing

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal component of the mammalian biological clock, the neural timing system that generates and coordinates a broad spectrum of physiological, endocrine and behavioural circadian rhythms. The pacemaker of the SCN oscillates with a near 24 h period and is entrained to the diurnal light-dark cycle. Consistent with its role in circadian timing, investigations in rodents and non-human primates furthermore suggest that the SCN is the locus of the brain's endogenous calendar, enabling organisms to anticipate seasonal environmental changes. The present review focuses on the neuronal organization and dynamic properties of the biological clock and the means by which it is synchronized with the environmental lighting conditions. It is shown that the functional activity of the biological clock is entrained to the seasonal photic cycle and that photoperiod (day length) may act as an effective zeitgeber. Furthermore, new insights are presented, based on electrophysiological and molecular studies, that the mammalian circadian timing system consists of coupled oscillators and that the clock genes of these oscillators may also function as calendar genes. In summary, there are now strong indications that the neuronal changes and adaptations in mammals that occur in response to a seasonally changing environment are driven by an endogenous circadian clock located in the SCN, and that this neural calendar is reset by the seasonal fluctuations in photoperiod.

The metabolism of phospholipids oscillates rhythmically in cultures of fibroblasts and is regulated by the clock protein PERIOD 1.

The mammalian circadian timing system is composed of countless cell oscillators distributed throughout the body and central pacemakers regulating temporal physiology and behavior. Peripheral clocks display circadian rhythms in gene expression both in vivo and in culture. We examined the biosynthesis of phospholipids as well as the expression of the clock gene period 1 (Per1) and its potential involvement in the regulation of the phospholipid metabolism in cultured quiescent NIH 3T3 cells synchronized by a 2 h serum shock. A 30 min pulse of radiolabeled precursor was given at phases ranging from 0.5 to 62 h after serum treatment. We observed a daily rhythm in the phospholipid labeling that persisted at least for two cycles, with levels significantly decreasing 29 and 58 h after treatment. Per1 expression exhibited a rapid and transient induction and a daily rhythmicity in antiphase to the lipid labeling. After Per1 expression knockdown, the rhythm of phospholipid labeling was lost. Furthermore, in cultures of CLOCK mutant fibroblasts--cells with a clock mechanism impairment--PER1 was equally expressed at all times examined and the phospholipid labeling did not oscillate. The results demonstrate that the biosynthesis of phospholipids oscillates daily in cultured fibroblasts by an endogenous clock mechanism involving Per1 expression.

Effect of Single Low Intensity Light Pulse Exposure and Melatonin Treatment on the Circadian Variation of Melatonin in Indian Palm Squirrel Funambulus pennanti

The endogenous circadian rhythm of melatonin in mammals provides information regarding the resetting response of the mammalian circadian timing system in response to the changes in light dark cycle. Photoperiodic changes are reported to have acute and chronic effect on melatonin rhythm. Our aim in present experiment was to study the effect of single light pulse of low intensity on the circadian variation of melatonin in Indian palm squirrel. A short pulse of 5min was given to the animals at 22:55 h on day 16th in natural photoperiodic condition of long day length (LD ~ 13.55:10.05) and melatonin levels were estimated at every 4-h interval on ZT scale on day 17th (DD). Observations suggest that the light pulse given on day 16th suppressed the melatonin level on day 17th (DD). Besides this, it was also found that there was phase delay in the peak value of melatonin. Further, we tested the ability of single melatonin injection on the light pulse induced phase shift of acrophase of melatonin in this species F. pennanti. We injected the single physiological dose of melatonin (25 microgram/100 g body wt.) just 5 min prior to the commencement of light pulse (22:50 h) on day 16 and melatonin levels were estimated on day 17th as above. Injection of melatonin prior to light pulse altered the suppressing and phase shifting effect of light in terms of peak concentration of melatonin in squirrels. Above data may lead us to conclude that the biological clock mechanism controlling circadian rhythm of melatonin in this rodent is in response to the phase shifting effect of light and acute melatonin treatment. Further, we may suggest that single melatonin injection has the capability to entrain melatonin rhythm but a dose dependent study is required to facilitate the suggestion.

Effect of orexin-A on discharge rate of rat suprachiasmatic nucleus neurons in vitro.

The suprachiasmatic nuclei (SCN) constitute the principal pacemaker of the circadian timing system in mammals. The generated rhythm is forwarded mostly through projections to various hypothalamic nuclei. On the other hand, the regulated processes feedback to the SCN. One of the possible feedback pathways is the orexinergic projection from the lateral hypothalamus. Orexins are recently identified neuropeptides with an overall facilitatory effect on waking behaviors. Orexinergic fibers are widely distributed throughout the brain and are also present in the SCN. In this study we examined the effect of orexin-A on the spontaneous activity of rat SCN cell in vitro. Neurons showed 2 different firing pattern (continuous-regular, intermittent-irregular). Orexin-A increased firing rate in both cell types at 10(-8) M concentration, but caused a clear suppression of neuronal activity at 10(-7) M. Continuously firing neurons were less responsive than those firing intermittently. These results show that orexin-A may play a role in the modulation of the circadian pacemaker function. The neuropeptide might exert both direct, postsynaptic effects on SCN neurons and indirect, presynaptic effects on excitatory and inhibitory terminals. The dose-dependent modification of the firing rate indicate that the weight of these factors changes with the concentration of orexin-A.

Dorsal raphe nucleus modulates neuronal activity in rat intergeniculate leaflet

Serotonergic input from midbrain raphe nuclei is believed to have a significant effect on mammalian circadian timing system. The suprachiasmatic nucleus (SCN) receives its serotonergic input from the median raphe nucleus, while the intergeniculate leaflet (IGL) receives serotonergic innervation from the dorsal raphe nucleus (DRN). The present paper was aimed at determining whether projection from the DRN affected rhythmic neuronal oscillations in the IGL of rats. We investigated the impact of electrolytic lesions and electric stimulation of the DRN on spontaneous isoperiodic (i.e. burst firing with a constant interburst interval) neuronal activity recorded in the IGL. In all our experiments a complete lesion of the DRN always caused a significant increase (ca. 100%) of spontaneous activity of IGL neurons, their oscillatory character having been maintained, though. On the other hand, electric stimulation of the DRN produced a transient decrease in firing rate oscillations of the IGL neurons. The obtained results indicate that the neuronal projection from the DRN has a substantial modulating effect on IGL activity-an important element of the mechanism of the circadian time-keeping system that mediates the transfer of non-photic information to the SCN by modulating its activity. The observed increase of isoperiodic activity in the IGL after DRN lesion and a transient decrease in this activity after electric stimulation indicate an inhibitory character of this effect. The present findings corroborate the hypothesis that the DRN is a one of the major and extremely important source of the modulatory inputs to the mammalian circadian time-keeping system.

Functional CLOCK is not involved in the entrainment of peripheral clocks to the restricted feeding: entrainable expression of mPer2 and BMAL1 mRNAs in the heart of Clock mutant mice on Jcl:ICR background

The mammalian circadian timing system consists of a central pacemaker in brain hypothalamus and damping oscillators in most peripheral tissues. To investigate the mechanism that controls circadian rhythms in the mammalian peripheral tissues, we examined the expression rhythm of mPer2, BMAL1, albumin D-site binding protein ( DBP), and Rev-erbα mRNAs in the heart of homozygous Clock mutant mice on Jcl:ICR background under the temporal feeding restriction. Unexpectedly, the restricted feeding (RF) shifted the circadian phase of both mPer2 and BMAL1 mRNA expressions in the heart not only of wild-type mice but also of Clock mutant mice. Furthermore, in the Clock mutant mice, the amplitude of the circadian expression of mPer2 and BMAL1 mRNAs was dramatically increased by the RF. These data indicate that functional CLOCK is not required for an entrainment of peripheral clocks to RF. On the other hand, the expression levels of DBP and Rev-erbα mRNAs were blunted in Clock mutant mice not only under ad libitum but also under RF conditions. Thus, it seems that the rhythmic expression of Rev-erbα is not involved in the RF-induced circadian expression of BMAL1 mRNA, although REV-ERBα has been identified as a major regulator of BMAL1 transcription. Thus, the entraining mechanism of peripheral tissues to the RF seems to be different from that to the central clock in the suprachiasmatic nucleus.

Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of the mammalian circadian timing system. The SCN is composed of two anatomically and functionally distinct subdivisions, designated core and shell, which can be distinguished on the basis of their chemoarchitecture and connections in the rat. In the present study, we examine the intrinsic organization and the afferent and efferent connections of the mouse SCN using immunocytochemistry and ocular injections of cholera toxin. Neurons of the SCN shell contain GABA, calbindin (CALB), arginine vasopressin (AVP), angiotensin II (AII) and met-enkephalin (mENK), and receive input from galanin (GAL) and vasoactive intestinal polypeptide (VIP) immunoreactive fibers. Neurons of the SCN core synthesize GABA, CALB, VIP, calretinin (CALR), gastrin releasing peptide (GRP), and neurotensin (NT), and receive input from the retina and from fibers that contain neuropeptide Y (NPY) and 5-hydroxytryptamine (5HT). Fibers projecting from SCN neurons that are immunoreactive for AVP and VIP exhibit a characteristic morphology, and project to the lateral septum, a series of medial hypothalamic areas extending from the preoptic to the posterior hypothalamic area and to the paraventricular thalamic nucleus. The organization of the mouse SCN, and its connections, are similar to that in other mammalian species.

Alterations in arginine vasopressin neurons in the suprachiasmatic nucleus in depression.

Circadian rhythm disturbances are frequently found in depressed subjects. Although it has been presumed that these disturbances may reflect a disorder of the circadian pacemaker, this has never been established. The suprachiasmatic nucleus (SCN) is the pacemaker of the circadian timing system in mammals, and arginine vasopressin (AVP) is one of its major neuropeptides. As peptide content is often taken as a measure for activity, we hypothesized that a decreased number of AVP-immunoreactive (AVP-IR) neurons and amount of AVP-messenger RNA (mRNA) would be present in the SCN of depressed subjects. Brains of 11 subjects suffering from major depression (8 cases) and bipolar disorder (3 cases), and of 11 controls, matched for sex, age, and clock time at death, were collected. The number of AVP-IR neurons in the SCN was determined by means of a digitizer (CalComp Inc, Reading, England). The amount of AVP-mRNA expression in the SCN was quantified with the Interaktive Bild Analyse System image analysis system (Kontron, Munich, Germany). In depressed subjects, the number of AVP-IR neurons in the SCN was more than one and a half times higher than in controls, while the total masked area of silver grains, as an estimate of the amount of AVP-mRNA, was about one half that of controls. Contrary to our hypothesis, an increase in the number of AVP-IR neurons in the SCN in depression was found, together with an expected decrease in AVP-mRNA. These findings suggest that, in depressed patients, both the synthesis and release of AVP in the SCN is reduced, resulting in an impaired functional ability. A disbalance between AVP production and transport needs further investigation in future studies.

Effects of hypoxia and hypercapnia on circadian rhythms in the golden hamster (Mesocricetus auratus).

This study was designed to determine whether respiratory stimuli can influence the mammalian circadian timing system. Three-hour pulses of hypoxia (inspired O(2) concentration = 8%) or hypercapnia (inspired CO(2) concentration = 11%) were presented for 7 days at mid-subjective day (circadian time 6-9) under constant darkness. Hypoxic and hypercapnic pulses caused cumulative phase delays of 46. 4 +/- 6.9 and 25.9 +/- 12.3 min, respectively. Distance run per day was significantly reduced on hypoxic and hypercapnic pulse days, compared with nonpulsed days. Phase shifts were correlated with the reduction in daily running activity (multiple r(2) = 0.521, P = 0.036), metabolic depression (multiple r(2) = 0.772, P < 0.001), and reduction in body temperature (multiple r(2) = 0.539, P = 0.027), but not lung ventilation (multiple r(2) = 0.306, P = 0.414) during pulses. We conclude that hypoxia and hypercapnia can influence the phase and quantity of activity in free-running hamsters.

Circadian timed wakefulness at dawn opposes compensatory sleep responses after sleep deprivation in Octodon degus.

The circadian timing system in mammals is thought to promote wakefulness and oppose sleep drive that accumulates across the activity phase in diurnal and nocturnal species. Whether the circadian system actively opposes compensatory sleep responses in mammals with episodes of alertness consolidated at dawn and dusk is unknown. In the present study, an interaction between circadian timed arousal at dawn and compensatory sleep responses after sleep deprivation (SD) was examined in Octodon degus, a hystricomorph rodent with crepuscular episodes of wakefulness. Recovery sleep was compared after 6 hours and 12 hours of SD ending at either CT 21 or 12, just before the dawn, and just after the dusk crepuscular episodes of consolidated wakefulness, respectively. Total sleep time and NREM sleep after SD increased proportionally to the amount of sleep loss; however, compensatory sleep responses after SD were attenuated at CT 23, a circadian time when a crepuscular event of wakefulness occurs in this species. EEG slow-wave activity (SWA) and body temperature levels in the first two hours after 6 and 12 hours of SD ending at CT 12 were similar. However, both were significantly higher than after 12 hours of SD ending at CT 21, suggesting factors other than the amount of prior wake duration can influence SWA levels. This study provides evidence that the circadian arousal system opposes compensatory sleep responses at dawn by actively promoting wakefulness in this species.

Vasoactive intestinal peptide neurons as synaptic targets for vasopressin neurons in the suprachiasmatic nucleus. Double-label immunocytochemical demonstration in the rat

Cellular relationships between neurons producing vasopressin or vasoactive intestinal peptide in the suprachiasmatic nucleus of the hypothalamus, the main component of the central circadian timing system in mammals, were investigated in the rat using double immunocytochemistry. Analysis of serial confocal images revealed that the vasopressin-synthesizing neurons not only are important targets for the vasoactive intestinal peptide-synthesizing neurons, as previously demonstrated, but also establish reciprocal axosomatic contacts with these neurons, which have never been reported. On average, 5.4 vasoactive intestinal peptide contacts per vasopressin perikaryon and 1.7 vasopressin contacts per vasoactive intestinal peptide perikaryon were counted. That both types of neurons are linked by reciprocal synapses was confirmed at the electron microscopic level using a combination of immunoperoxidase and immunogold–silver labeling. Existence of an anatomical substrate for a vasopressinergic control of the vasoactive intestinal peptide neurons may have important functional consequences. In view (i) of the presumed, direct or indirect, involvement of the vasopressin neurons in relaying pacemaker information within and outside the suprachiasmatic nucleus, and (2) of the established role of the vasoactive intestinal peptide neurons as the main light-sensitive cells, it provides support for a neuronal mechanism through which the circadian clock may regulate inputs related to environmental messages. Our electron-microscopic data also extended earlier observations, pointing to the involvement of vasopressin and vasoactive intestinal peptide terminals in so-called double synapses that, conceivably, could regulate neuronal synchronization in the suprachiasmatic nucleus. A morphological basis for non-synaptic interactions that could be involved in ephaptic and/or paracrine communication between both types of peptidergic neurons is also reported.

Effect of Ambient Temperature on the Orcadian Activity Rhythm in Common Marmosets, Callithrix J. Jacchus (Primates)

Whereas the (zeitgeber) effect of ambient temperature Ta and temperature cycles TaC's on circadian rhythmicity has been well documented for heterofhermic mammals, inconsistent results have been obtained for strictly homeothermic species. Hence, it might be inferred that the susceptibility of the mammalian circadian timing system (CTS) to Ta and TaC's depends on the range of the animals' core and/or brain temperature rhythm. This hypothesis was tested in the common marmoset (Callithrix j. jacchus, n=12), a small diurnal primate with an amplitude in body temperature rhythm that is larger than for other homeothermic primates studied so far. Within the range 20-30°C, no systematic effects of constant Ta on most parameters of the marmosets' light-dark (LD)-entrained and free-running circadian activity rhythm (CAR) were found. Significant differences could be established in the average amount of activity per circadian cycle. It was highest at Ta 25°C (LD) and 20°C (light-light, LL) and most probably reflected a temperature-induced masking effect. A 24h trapezoidal TaC of 20:30°C entrained the free-running CAR in two of six marmosets and produced relative coordination in all others. Accordingly, in all animals tested, it had an effect on the CTS. In marmosets free running in LL at a Ta of 20°C or 30°C, 3h warm and cold pulses of 30°C and 20°C, respectively, produced neither systematic phase responses nor period responses of the CAR. So, there is no evidence of a phase-response mechanism underlying circadian entrainment. The results show that large-amplitude TaC's function as a weak zeitgeber for the marmosets' CTS. Since this zeitgeber effect is significantly larger than that found in owl monkeys, the results are consistent with the starting hypothesis that the zeitgeber effect of a given T,C on the mammalian CTS may be related to the amplitude of the species' core and/or brain temperature cycle.

An NK 1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK 1, NK 2, and NK 3. The NK 1 subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK 1 antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK 1 receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK 1 antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK 1 antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK 1 antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK 1 antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK 1 receptors, modulate photic responses of the SCN pacemaker.

Components and connections of the circadian timing system in mammals

The circadian timing system is a neural network consisting of the hypothalamic suprachiasmatic nucleus, aided by the retina, other hypothalamic nuclei, autonomic regions of the spinal cord, sympathetic ganglia and the pineal gland. Extensive studies conducted over the last two decades have unravelled the principal items of its functional neuroanatomy. The system is responsible for the generation of the circadian rhythm, its synchronization by environmental factors such as light, and its mediation with respect to morphological, physiological and biochemical parameters of mammals that exhibit distinct alterations throughout the 24-h cycle. This review characterizes the brain sites involved and the pathways responsible for the generation and maintenance of circadian rhythmicity.

Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation

Serotonergic innervation is believed to inhibit the effects of light on the mammalian circadian timing system. Two anatomical components of this system, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL), receive serotonergic input from midbrain raphe nuclei. The present studies use retrograde and anterograde tracing as well as neurotoxic lesion techniques to demonstrate that serotonergic cells in the median raphe nucleus (MR) project to the SCN and that serotonergic cells in the dorsal raphe nucleus (DR) project to the IGL. Neurotoxic lesions were also used to investigate the effects of selective serotonin (5-HT) neuron loss in the MR or DR on circadian rhythm parameters of animals entrained to a light/dark cycle or housed in constant light. 5-HT depletion in the MR, but not in the DR, induces an advance in onset, a delay in offset, and a longer duration of the nocturnal running-wheel activity phase. Circadian rhythm disruption in constant light is also more frequent in hamsters with MR lesions. A second experiment was designed to investigate the relationship between lesion location, 5-HT-immunoreactive (5-HT-IR) fiber loss, and behavioral changes. Destruction of 5-HT neurons in the MR causes 5-HT-IR fiber loss in the SCN, which may account for the observed changes in circadian parameters. DR lesions result in 5-HT-IR fiber depletion of the IGL, with no associated changes in the entrained rhythm. The anatomical and behavioral results support the view that a 5-HT projection from the MR mediates 5-HT effects on circadian rhythm regulation in hamsters.

Gravitational biology and the mammalian circadian timing system

Mammals have evolved under the influence of many selective pressures. Two of these pressures have been the static force of gravity and the daily variations in the environment due to the rotation of the earth. It is now clear that each of these pressures has led to specific adaptations which influence how organisms respond to changes in either gravity or daily time cues. However, several unpredicted responses to altered gravitational environments occur within the homeostatic and circadian control systems. These results may be particularly relevant to biological and medical issues related to spaceflight. This paper demonstrates that the homeostatic regulation of rat body temperature, heart rate, and activity become depressed following exposure to a 2 G hyperdynamic field, and recovers within 5–6 days. In addition, the circadian rhythms of these same variables exhibit a depression of rhythm amplitude; however, recovery required a minimum of 7 days.