Aging does not compromise in vitro oscillation of the suprachiasmatic nuclei but makes it more vulnerable to constant light

Circadian regulation of behavior worsens with age, however, the mechanism behind this phenomenon is still poorly understood. Specifically, it is not clear to what extend the ability of the circadian clock in the suprachiasmatic nuclei (SCN) to generate the rhythm is affected by aging. This study aimed to ascertain the effect of aging on the functioning of the SCN of mPer2^Luciferase mice under unnatural lighting conditions, such as constant light (LL). Under LL, which worsened the age-induced effect on behavioral rhythms, a marginal age-dependent effect on in vitro rhythmicity in explants containing the middle, but not the rostral/caudal, regions of the SCN was apparent; the proportion of mice in which middle-region SCN explants were completely arrhythmic or had an extremely long period (>30 h) was 47% in aged mice and 27% in adults. The results suggest that in some of the aged animals, LL may weaken the coupling among oscillators in specific sub-regions of the SCN, leaving other sub-regions better synchronized. In the standard light/dark cycle and in constant darkness, the SCN ability to produce bioluminescence rhythms in vitro was not compromised in aged mice although aging significantly affected their SCN-driven locomotor activity rhythms. Therefore, our results demonstrate that although age worsened the SCN output rhythm, the SCN molecular core clock mechanism itself was relatively resilient to aging in these same animals. The results suggest the involvement of pathways downstream of the core clock mechanism which are responsible for this phenomenon.     

Daily injection of insulin attenuated impairment of liver circadian clock oscillation in the streptozotocin-treated diabetic mouse

Recent studies have shown a dampened amplitude of clock gene rhythm in the heart and liver of streptozotocin (STZ)-treated rats and mice, however it is unknown whether impairment is due to dysfunction of the suprachiasmatic nucleus (SCN) or not. Rhythmic expression of mPER2 was dampened in the STZ-treated mouse liver but not SCN and cerebral cortex. Injection of insulin could normalize an impairment of mPer2 and mPER2 expression rhythm in the liver, when it was injected at nighttime, but not at daytime. In the present study, we demonstrated that insulin-dependent diabetes impaired oscillation of the peripheral clock gene and its product. Insulin injection can recover dampened oscillation of the peripheral clock depending on its injection time.     

Age‐Related Effects on the Biological Clock and its Behavioral Output in a Primate

In humans, activity rhythms become fragmented and attenuated in the elderly. This suggests an alteration of the circadian system per se that could in turn affect the expression of biological rhythms. In primates, very few studies have analyzed the effect of aging on the circadian system. The mouse lemur provides a unique model of aging in non‐human primates. To assess the effect of aging on the circadian system of this primate, we recorded the circadian and daily rhythms of locomotor activity of mouse lemurs of various ages. We also examined age‐related changes in the daily rhythm of immunoreactivities for vasoactive intestinal polypeptide (VIP) and arginine‐vasopressin (AVP) in suprachiasmatic nucleus neurons (SCN), two major peptides of the biological clock. Compared to adult animals, aged mouse lemurs showed a significant increase in daytime activity and an advanced activity onset. Moreover, when maintained in constant dim red light, aged animals exhibited a shortening of the free‐running period compared to adult animals. In adults, AVP immunoreactivity (ir) peaked during the second part of the day, and VIP ir peaked during the night. In aged mouse lemurs, the peaks of AVP ir and VIP ir were significantly shifted with no change in amplitude. AVP ir was most intense at the beginning of the night; whereas, VIP ir peaked at the beginning of the daytime. A weakened oscillator could account for the rhythmic disorders often observed in the elderly. Changes in the daily rhythms of AVP ir and VIP ir may affect the ability of the SCN to transmit rhythmic information to other neural target sites, and thereby modify the expression of some biological rhythms.     

Caffeine lengthens circadian rhythms in mice

Although caffeine alters sleep in many animals, whether or not it affects mammalian circadian clocks remains unknown. Here, we found that incubating cultured mammalian cell lines, human osteosarcoma U2OS cells and mouse fibroblast NIH3T3 cells, with caffeine lengthened the period of circadian rhythms. Adding caffeine to ex vivo cultures also lengthened the circadian period in mouse liver explants from Per2::Luciferase reporter gene knockin mice, and caused a phase delay in brain slices containing the suprachiasmatic nucleus (SCN), where the central circadian clock in mammals is located. Furthermore, chronic caffeine consumption ad libitum for a week delayed the phase of the mouse liver clock in vivo under 12 h light–dark conditions and lengthened the period of circadian locomotor rhythms in mice under constant darkness. Our results showed that caffeine alters circadian clocks in mammalian cells in vitro and in the mouse ex vivo and in vivo.     

Embryonic development and maternal regulation of murine circadian clock function

The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene Per2::Luc and investigated rhythmicity and circadian traits of these tissues ex vivo. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of Clock-Δ19 mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, Clock gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.     

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.     

Early Chronotype and Tissue-Specific Alterations of Circadian Clock Function in Spontaneously Hypertensive Rats

Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.     

Age-related effects on the biological clock and its behavioral output in a primate

In humans, activity rhythms become fragmented and attenuated in the elderly. This suggests an alteration of the circadian system per se that could in turn affect the expression of biological rhythms. In primates, very few studies have analyzed the effect of aging on the circadian system. The mouse lemur provides a unique model of aging in non-human primates. To assess the effect of aging on the circadian system of this primate, we recorded the circadian and daily rhythms of locomotor activity of mouse lemurs of various ages. We also examined age-related changes in the daily rhythm of immunoreactivities for vasoactive intestinal polypeptide (VIP) and arginine-vasopressin (AVP) in suprachiasmatic nucleus neurons (SCN), two major peptides of the biological clock. Compared to adult animals, aged mouse lemurs showed a significant increase in daytime activity and an advanced activity onset. Moreover, when maintained in constant dim red light, aged animals exhibited a shortening of the free-running period compared to adult animals. In adults, AVP immunoreactivity (ir) peaked during the second part of the day, and VIP ir peaked during the night. In aged mouse lemurs, the peaks of AVP ir and VIP ir were significantly shifted with no change in amplitude. AVP ir was most intense at the beginning of the night; whereas, VIP ir peaked at the beginning of the daytime. A weakened oscillator could account for the rhythmic disorders often observed in the elderly. Changes in the daily rhythms of AVP ir and VIP ir may affect the ability of the SCN to transmit rhythmic information to other neural target sites, and thereby modify the expression of some biological rhythms.     

Ultradian feeding in mice not only affects the peripheral clock in the liver,but also the master clock in the brain

Restricted feeding during the resting period causes pronounced shifts in a number of peripheral clocks, but not the central clock in the suprachiasmatic nucleus (SCN). By contrast, daily caloric restriction impacts also the light-entrained SCN clock, as indicated by shifted oscillations of clock (PER1) and clock-controlled (vasopressin) proteins. To determine if these SCN changes are due to the metabolic or timing cues of the restricted feeding, mice were challenged with an ultradian 6-meals schedule (1 food access every 4 h) to abolish the daily periodicity of feeding. Mice fed with ultradian feeding that lost <10% body mass (i.e. isocaloric) displayed 1.5-h phase-advance of body temperature rhythm, but remained mostly nocturnal, together with up-regulated vasopressin and down-regulated PER1 and PER2 levels in the SCN. Hepatic expression of clock genes (Per2, Rev-erbα, and Clock) and Fgf21 was, respectively, phase-advanced and up-regulated by ultradian feeding. Mice fed with ultradian feeding that lost >10% body mass (i.e. hypocaloric) became more diurnal, hypothermic in late night, and displayed larger (3.5 h) advance of body temperature rhythm, more reduced PER1 expression in the SCN, and further modified gene expression in the liver (e.g. larger phase-advance of Per2 and up-regulated levels of Pgc-1α). While glucose rhythmicity was lost under ultradian feeding, the phase of daily rhythms in liver glycogen and plasma corticosterone (albeit increased in amplitude) remained unchanged. In conclusion, the additional impact of hypocaloric conditions on the SCN are mainly due to the metabolic and not the timing effects of restricted daytime feeding.     

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.     

Repeated psychosocial stress at night,but not day,affects the central molecular clock

We have recently demonstrated that the outcome of repeated social defeat (SD) on behavior, physiology and immunology is more negative when applied during the dark/active phase as compared with the light/inactive phase of male C57BL/6 mice. Here, we investigated the effects of the same stress paradigm, which combines a psychosocial and novelty stressor, on the circadian clock in transgenic PERIOD2::LUCIFERASE (PER2::LUC) and wildtype (WT) mice by subjecting them to repeated SD, either in the early light phase (social defeat light?=?SDL) or in the early dark phase (social defeat dark?=?SDD) across 19 days. The PER2::LUC rhythms and clock gene mRNA expression were analyzed in the suprachiasmatic nucleus (SCN) and the adrenal gland, and PER2 protein expression in the SCN was assessed. SDD mice showed increased PER2::LUC rhythm amplitude in the SCN, reduced Per2 and Cryptochrome1 mRNA expression in the adrenal gland, and increased PER2 protein expression in the posterior part of the SCN compared with single-housed control (SHC) and SDL mice. In contrast, PER2::LUC rhythms in the SCN of SDL mice were not affected. However, SDL mice exhibited a 2-hour phase advance of the PER2::LUC rhythm in the adrenal gland compared to SHC mice. Furthermore, plasma levels of brain-derived neurotrophic factor (BDNF) and BDNF mRNA in the SCN were elevated in SDL mice. Taken together, these results show that the SCN molecular rhythmicity is affected by repeated SDD, but not SDL, while the adrenal peripheral clock is influenced mainly by SDL. The observed increase in BDNF in the SDL group may act to protect against the negative consequences of repeated psychosocial stress.     

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.     

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master–slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.     

Modulation of mammalian circadian rhythms by tumor necrosis factor-α

Systemic low doses of the endotoxin lipopolysaccharide (LPS, 100?µg/kg) administered during the early night induce phase-delays of locomotor activity rhythms in mice. Our aim was to evaluate the role of tumor necrosis factor (Tnf)-alpha and its receptor 1/p55 (Tnfr1) in the modulation of LPS-induced circadian effects on the suprachiasmatic nucleus (SCN). We observed that Tnfr1-defective mice (Tnfr1 KO), although exhibiting similar circadian behavior and light response to that of control mice, did not show LPS-induced phase-delays of locomotor activity rhythms, nor LPS-induced cFos and Per2 expression in the SCN and Per1 expression in the paraventricular hypothalamic nucleus (PVN) as compared to wild-type (WT) mice. We also analyzed Tnfr1 expression in the SCN of WT mice, peaking during the early night, when LPS has a circadian effect. Peripheral inoculation of LPS induced an increase in cytokine/chemokine levels (Tnf, Il-6 and Ccl2) in the SCN and in the PVN. In conclusion, in this study, we show that LPS-induced circadian responses are mediated by Tnf. Our results also suggest that this cytokine stimulates the SCN after LPS peripheral inoculation; and the time-related effect of LPS (i.e. phase shifts elicited only at early night) might depend on the increased levels of Tnfr1 expression. We also confirmed that LPS modulates clock gene expression in the SCN and PVN in WT but not in Tnfr1 KO mice.

Highlights: We demonstrate a fundamental role for Tnf and its receptor in circadian modulation by immune stimuli at the level of the SCN biological clock.