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.     

Photoperiodic regulation of PER1 and PER2 protein expression in rat peripheral tissues

Circadian oscillations in biological variables in mammals are controlled by a central pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus which coordinates circadian oscillators in peripheral tissues. The molecular clockwork responsible for this rhythmicity consists of several clock genes and their corresponding proteins that compose interactive feedback loops. In the SCN, two of the genes, Per1 and Per2, show circadian rhythmicity in their expression and protein production. This SCN rhythmicity is modified by the length of daylight, i.e. the photoperiod. The aim of the present study was to find out whether profiles of PER1 and PER2 proteins in peripheral organs are also affected by the photoperiod. Rats were maintained under a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) and under a short, LD 8:16, photoperiod. The PER1 and PER2 daily profiles were measured in peripheral organs by Western blotting. The photoperiod affected significantly the PER1 profile in livers and the PER2 profile in lungs and hearts. In lungs, PER2 in the cytoplasmic, but not in the nuclear fraction, was affected significantly. The effect of the photoperiod on PER1 profiles in peripheral organs appears to differ from that in the SCN.     

The tau mutation in the Syrian hamster differentially reprograms the circadian clock in the SCN and peripheral tissues

The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are synchronized to the solar day by the light-dark cycle, and in turn, they coordinate circadian oscillations in peripheral tissues. The tau mutation in the Syrian hamster is caused by a point mutation leading to a deficiency in the ability of Casein Kinase 1epsilon to phosphorylate its targets, including circadian PER proteins. How this accelerates circadian period in neural tissues is not known, nor is its impact on peripheral circadian oscillators established. We show that this mutation has no effect on per mRNA expression nor the nuclear accumulation of PER proteins in the SCN. It does, however, accelerate the clearance of PER proteins from the nucleus to an extent sufficient to explain the shortened circadian period of behavioral rhythms. The mutation also has novel, unanticipated consequences for circadian timing in the periphery, including tissue-specific phase advances and/or reduced amplitude of circadian gene expression. The results suggest that the tau mutation accelerates a specific phase, during mid-late subjective night of the SCN circadian feedback loop, rather than cause a global compression of the entire cycle. This reprogrammed output from the clock is associated with peripheral desynchrony, which in turn could account for impaired growth and metabolic efficiency of the mutant.     

Estrogen directly modulates circadian rhythms of PER2 expression in the uterus

Fluctuations in circulating estrogen and progesterone levels associated with the estrous cycle alter circadian rhythms of physiology and behavior in female rodents. Endogenously applied estrogen shortens the period of the locomotor activity rhythm in rodents. We recently found that estrogen implants affect Period (Per) gene expression in the suprachiasmatic nucleus (SCN; central clock) and uterus of rats in vivo. To explore whether estrogen directly influences the circadian clock in the SCN and/or tissues of the reproductive system, we examined the effects of 17beta-estradiol (E(2)) on PER2::LUCIFERASE (PER2::LUC) expression in tissue explant cultures from ovariectomized PER2::LUC knockin mice. E(2) applied to explanted cultures shortened the period of rhythmic PER2::LUC expression in the uterus but did not change the period of PER2::LUC expression in the SCN. Raloxifene, a selective estrogen receptor modulator and known E(2) antagonist in uterine tissues, attenuated the effect of E(2) on the period of the PER2::LUC rhythm in the uterus. These data indicate that estrogen directly affects the timing of the molecular clock in the uterus via an estrogen receptor-mediated response.     

The protective effect of cycloastragenol on aging mouse circadian rhythmic disorder induced by d-galactose

Aging process in mammals is associated with a decline in amplitude and a long period of circadian behaviors which are regulated by a central circadian regulator in the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. It is unclear whether enhancing clock function can retard aging. Using fibroblasts expressing per2::lucSV and senescent cells, we revealed cycloastragenol (CAG), a natural aglycone derivative from astragaloside IV, as a clock amplitude enhancing small molecule. CAG could activate telomerase to antiaging, but no reports focused on its effects on circadian rhythm disorders in aging mice. Here we analyze the potential effects of CAG on d -galactose-induced aging mice on the circadian behavior and expression of clock genes. For this purpose, CAG (20 mg/kg orally), was administered daily to d -galactose (150 mg/kg, subcutaneous) mice model of aging for 6 weeks. An actogram analysis of free-running activity of these mice showed that CAG significantly enhances the locomotor activity. We further found that CAG increase expressions of per2 and bmal1 genes in liver and kidney of aging mouse. Furthermore, CAG enhanced clock protein BMAL1 and PER2 levels in aging mouse liver and SCN. Our results indicated that the CAG could restore the behavior of circadian rhythm in aging mice induced by d -galactose. These data of present study suggested that CAG could be used as a novel therapeutic strategy for the treatment of age-related circadian rhythm disruption.     

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.     

The role of {beta}-TrCP1 and {beta}-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2

The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein beta-TrCP1, a component of ubiquitin E3 ligase. Here, we show that beta-TrCP2 as well as beta-TrCP1 target PER2 protein in vitro. We also identified beta-TrCP binding site (m2) of PER2 being recognized by both beta-TrCP1 and beta-TrCP2. Luciferase-PER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of beta-TrCP1 and/or beta-TrCP2 attenuate circadian oscillations in NIH3T3 cell. beta-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that beta-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that beta-TrCP1 and beta-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of beta-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

Entrainment of the master circadian clock by scheduled feeding

The master circadian clock, located in the mammalian suprachiasmatic nuclei (SCN), generates and coordinates circadian rhythmicity, i.e., internal organization of physiological and behavioral rhythms that cycle with a near 24-h period. Light is the most powerful synchronizer of the SCN. Although other nonphotic cues also have the potential to influence the circadian clock, their effects can be masked by photic cues. The purpose of this study was to investigate the ability of scheduled feeding to entrain the SCN in the absence of photic cues in four lines of house mouse (Mus domesticus). Mice were initially housed in 12:12-h light/dark cycle with ad libitum access to food for 6 h during the light period followed by 4-6 mo of constant dark under the same feeding schedule. Wheel running behavior suggested and circadian PER2 protein expression profiles in the SCN confirmed entrainment of the master circadian clock to the onset of food availability in 100% (49/49) of the line 2 mice in contrast to only 4% (1/24) in line 3 mice. Mice from line 1 and line 4 showed intermediate levels of entrainment, 57% (8/14) and 39% (7/18), respectively. The predictability of entrainment vs. nonentrainment in line 2 and line 3 and the novel entrainment process provide a powerful tool with which to further elucidate mechanisms involved in entrainment of the SCN by scheduled feeding.     

Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day

In mammals, a light-entrainable clock located in the suprachiasmatic nucleus (SCN) regulates circadian rhythms by synchronizing oscillators throughout the brain and body. Notably, the nature of the relation between the SCN clock and subordinate oscillators in the rest of the brain is not well defined. We performed a high temporal resolution analysis of the expression of the circadian clock protein PERIOD2 (PER2) in the rat forebrain to characterize the distribution, amplitude and phase of PER2 rhythms across different regions. Eighty-four LEW/Crl male rats were entrained to a 12-h: 12-h light/dark cycle, and subsequently perfused every 30 min across the 24-h day for a total of 48 time-points. PER2 expression was assessed with immunohistochemistry and analyzed using automated cell counts. We report the presence of PER2 expression in 20 forebrain areas important for a wide range of motivated and appetitive behaviors including the SCN, bed nucleus, and several regions of the amygdala, hippocampus, striatum, and cortex. Eighteen areas displayed significant PER2 rhythms, which peaked at different times of day. Our data demonstrate a previously uncharacterized regional distribution of rhythms of a clock protein expression in the brain that provides a sound basis for future studies of circadian clock function in animal models of disease.