Suprachiasmatic Nucleus Oscillators Research Articles

Modeling of Jet Lag and Searching for an Optimal Light Treatment.

The role of the hierarchical organization of the suprachiasmatic nucleus (SCN) in its functioning, jet lag, and the light treatment of jet lag remains poorly understood. Using the core-shell model, we mimic collective behavior of the core and shell populations of the SCN oscillators in transient states after rapid traveling east and west. The existence of a special region of slow dynamical states of the SCN oscillators can explain phenomena such as the east-west asymmetry of jet lag, instances when entrainment to an advance is via delay shifts, and the dynamics of jet lag recovery time. If jet lag brings the SCN state into this region, it will take a long time to leave it and restore synchronization among oscillators. We show that the population of oscillators in the core responds quickly to a rapid phase shift of the light-dark cycle, in contrast to the shell, which responds slowly. A slow recovery of the synchronization among the shell oscillators in transient states may strongly affect reentrainment in peripheral tissues and behavioral rhythms. We discuss the relationship between molecular, electrical, and behavioral rhythms. We also describe how light pulses affect the SCN and analyze the efficiency of the light treatment in facilitating the adaptation of the SCN to a new time zone. Light pulses of a moderate duration and intensity reduce the recovery time after traveling east, but not west. However, long duration and high intensity of light pulses are more detrimental than beneficial for speeding up reentrainment. The results of the core-shell model are compared with experimental data and other biologically motivated models of the SCN.

Photoperiod, food restriction and memory for objects and places in mice

The suprachiasmatic nucleus (SCN) contains a population of cell-autonomous circadian oscillators essential for entrainment to daily light–dark (LD) cycles. Synchrony among SCN oscillators is modified by photoperiod and determines functional properties of SCN clock cycling, including its amplitude, phase angle of entrainment, and free running periodicity (τ). For many species, encoding of daylength in SCN output is critical for seasonal regulation of metabolism and reproduction. C57BL/6 mice do not show seasonality in these functions, yet do show photoperiodic modulation of SCN clock output. The significance of this for brain systems and functions downstream from the SCN in these species is largely unexplored. C57BL/6 mice housed in a long-day photoperiod have been reported to perform better on tests of object, spatial and fear memory compared to mice in a standard 12 h photoperiod. We previously reported that encoding of photoperiod in SCN output, evident in τ in constant dark (DD), can be blocked by limiting food access to a 4 h mealtime in the light period. To determine whether this might also block the effect of long days on memory, mice entrained to 18 h:6 h (L18) or 6 h:18 h (L6) LD cycles were tested for 24 h object memory (novel object preference, NOP) and spatial working memory (Y-maze spontaneous alternation, SA), at 4 times of day, first with food available ad libitum and then during weeks 5–8 of daytime restricted feeding. Photoperiod modified τ as expected, but did not affect performance on the NOP and SA tests, either before or during restricted feeding. NOP performance did improve in the restricted feeding condition in both photoperiods, eliminating a weak time of day effect evident with food available ad-libitum. These results highlight benefits of restricted feeding on cognitive function, and suggest a dose–response relationship between photoperiod and memory, with no benefits at daylengths up to 18 h.

Circadian dysregulation and Alzheimer’s disease: A comprehensive review

Alzheimer’s disease (AD), the foremost variant of dementia, has been associated with a menagerie of risk factors, many of which are considered to be modifiable. Among these modifiable risk factors is circadian rhythm, the chronobiological system that regulates sleep‐wake cycles, food consumption timing, hydration timing, and immune responses amongst many other necessary physiological processes. Circadian rhythm at the level of the suprachiasmatic nucleus (SCN), is tightly regulated in the human body by a host of biomolecular substances, principally the hormones melatonin, cortisol, and serotonin. In addition, photic information projected along afferent pathways to the SCN and peripheral oscillators regulates the synthesis of these hormones and mediates the manner in which they act on the SCN and its substructures. Dysregulation of this cycle, whether induced by environmental changes involving irregular exposure to light, or through endogenous pathology, will have a negative impact on immune system optimization and will heighten the deposition of Aβ and the hyperphosphorylation of the tau protein. Given these correlations, it appears that there is a physiologic association between circadian rhythm dysregulation and AD. This review will explore the physiology of circadian dysregulation in the AD brain, and will propose a basic model for its role in AD‐typical pathology, derived from the literature compiled and referenced throughout.

Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal clock driving circadian rhythms of physiology and behavior that adapt mammals to environmental cycles. Disruption of SCN-dependent rhythms compromises health, and so understanding SCN time keeping will inform management of diseases associated with modern lifestyles. SCN time keeping is a self-sustaining transcriptional/translational delayed feedback loop (TTFL), whereby negative regulators inhibit their own transcription. Formally, the SCN clock is viewed as a limit-cycle oscillator, the simplest being a trajectory of successive phases that progresses through two-dimensional space defined by two state variables mapped along their respective axes. The TTFL motif is readily compatible with limit-cycle models, and in Neurospora and Drosophila the negative regulators Frequency (FRQ) and Period (Per) have been identified as state variables of their respective TTFLs. The identity of state variables of the SCN oscillator is, however, less clear. Experimental identification of state variables requires reversible and temporally specific control over their abundance. Translational switching (ts) provides this, the expression of a protein of interest relying on the provision of a noncanonical amino acid. We show that the negative regulator Cryptochrome 1 (CRY1) fulfills criteria defining a state variable: ts-CRY1 dose-dependently and reversibly suppresses the baseline, amplitude, and period of SCN rhythms, and its acute withdrawal releases the TTFL to oscillate from a defined phase. Its effect also depends on its temporal pattern of expression, although constitutive ts-CRY1 sustained (albeit less stable) oscillations. We conclude that CRY1 has properties of a state variable, but may operate among several state variables within a multidimensional limit cycle.

SynGAP is expressed in the murine suprachiasmatic nucleus and regulates circadian-gated locomotor activity and light-entrainment capacity.

The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master circadian clock. The phasing of the SCN oscillator is locked to the daily solar cycle, and an intracellular signaling cassette from the small GTPase Ras to the p44/42 mitogen-activated protein kinase (ERK/MAPK) pathway is central to thisentrainmentprocess. Here, we analyzed the expression and function of SynGAP-a GTPase-activating protein that serves as a negative regulator of Ras signaling-within the murine SCN. Using a combination of immunohistochemical and Western blotting approaches, we show that SynGAP is broadly expressed throughout the SCN. In addition, temporal profiling assays revealed that SynGAP expression is regulated over the circadian cycle, with peak expression occurring during the circadian night. Further, time-of-day-gated expression of SynGAP was not observed in clock arrhythmicBMAL1null mice, indicating that the daily oscillation in SynGAP is driven by the inherent circadian timing mechanism. We also show that SynGAP phosphorylation at serine 1138-an event that has been found to modulate its functional efficacy-is regulated by clock time and is responsive to photic input. Finally, circadian phenotypic analysis ofSyngap1heterozygous mice revealed enhanced locomotor activity, increased sensitivity to light-evoked clock entrainment, and elevated levels of light-evoked MAPK activity, which is consistent with the role of SynGAP as a negative regulator of MAPK signaling. These findings reveal that SynGAP functions as a modulator of SCN clock entrainment, an effect that may contribute to sleep and circadian abnormalities observed in patients withSYNGAP1gene mutations.

Ontogeny of Circadian Rhythms and Synchrony in the Suprachiasmatic Nucleus.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism.SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN.

Restricted Feeding Schedules Modulate in a Different Manner the Expression of Clock Genes in Rat Hypothalamic Nuclei.

Food access restriction is associated to changes in gene expression of the circadian clock system. However, there are only a few studies investigating the effects of non-photic synchronizers, such as food entrainment, on the expression of clock genes in the central oscillators. We hypothesized that different feeding restriction patterns could modulate the expression of clock genes in the suprachiasmatic nucleus (SCN) “master” clock and in extra-SCN oscillators such as the paraventricular (PVN) and arcuate (ARC) hypothalamic nuclei. Wistar rats were divided into four groups: Control group (CG; food available ad libitum), Restricted night-fed (RF-n; food access during 2 h at night), Restricted day-fed (RF-d; food access during 2 h at daytime), Day-fed (DF; food access during 12 h at daytime). After 21 days, rats were decapitated between ZT2-ZT3 (0800–0900 h); ZT11-ZT12 (1700–1800 h), or ZT17-18 (2300–2400 h). Plasma corticosterone was measured by radioimmunoassay (RIA). The expression of Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, and Rorα were assessed in SCN, PVN, and ARC hypothalamic nuclei by RT-PCR and calculated by the 2[−DeltaDeltaCT(Cyclethreshold)](2−ΔΔCT) method. Restricted food availability during few h led to decreased body weight in RF-n and RF-d groups compared to controls and DF group. We also observed an anticipatory corticosterone peak before food availability in RF-n and RF-d groups. Furthermore, the pattern of clock gene expression in response to RF-n, RF-d, and DF schedules was affected differently in the SCN, PVN, and ARC hypothalamic nuclei. In conclusion, the master oscillator in SCN as well as the oscillator in PVN and ARC, all brain areas involved in food intake, responds in a tissue-specific manner to feeding restriction.

Modulation of learning and memory by the targeted deletion of the circadian clock gene Bmal1 in forebrain circuits

A large body of literature has shown that the disruption of circadian clock timing has profound effects on mood, memory and complex thinking. Central to this time keeping process is the master circadian pacemaker located within the suprachiasmatic nucleus (SCN). Of note, within the central nervous system, clock timing is not exclusive to the SCN, but rather, ancillary oscillatory capacity has been detected in a wide range of cell types and brain regions, including forebrain circuits that underlie complex cognitive processes. These observations raise questions about the hierarchical and functional relationship between the SCN and forebrain oscillators, and, relatedly, about the underlying clock-gated synaptic circuitry that modulates cognition. Here, we utilized a clock knockout strategy in which the essential circadian timing gene Bmal1 was selectively deleted from excitatory forebrain neurons, whilst the SCN clock remained intact, to test the role of forebrain clock timing in learning, memory, anxiety, and behavioral despair. With this model system, we observed numerous effects on hippocampus-dependent measures of cognition. Mice lacking forebrain Bmal1 exhibited deficits in both acquisition and recall on the Barnes maze. Notably, loss of forebrain Bmal1 abrogated time-of-day dependent novel object location memory. However, the loss of Bmal1 did not alter performance on the elevated plus maze, open field assay, and tail suspension test, indicating that this phenotype specifically impairs cognition but not affect. Together, these data suggest that forebrain clock timing plays a critical role in shaping the efficiency of learning and memory retrieval over the circadian day.

The unfolded protein response, inflammation, oscillators, and disease: a systems biology approach

AbstractNon-communicable diseases (NCDs) such as cardiovascular disease, cancers, diabetes and obesity are responsible for about two thirds of mortality worldwide, and all of these ailments share a common low-intensity systemic chronic inflammation, endoplasmic reticulum stress (ER stress), and the ensuing Unfolded Protein Response (UPR). These adaptive mechanisms are also responsible for significant metabolic changes that feedback with the central clock of the suprachiasmatic nucleus (SCN) of the hypothalamus, as well as with oscillators of peripheral tissues. In this review we attempt to use a systems biology approach to explore such interactions as a whole; to answer two fundamental questions: (1) how dependent are these adaptive responses and subsequent events leading to NCD with their state of synchrony with the SCN and peripheral oscillators? And, (2) How could modifiers of the activity of SCN for instance, food intake, exercise, and drugs, be potentially used to modulate systemic inflammation and ER stress to ameliorate or even prevent NCDs?

Disrupted reproduction, estrous cycle, and circadian rhythms in female mice deficient in vasoactive intestinal peptide.

The female reproductive cycle is gated by the circadian timing system and may be vulnerable to disruptions in the circadian system. Prior work suggests that vasoactive intestinal peptide (VIP)-expressing neurons in the suprachiasmatic nucleus (SCN) are one pathway by which the circadian clock can influence the estrous cycle, but the impact of the loss of this peptide on reproduction has not been assessed. In the present study, we first examine the impact of the genetic loss of the neuropeptide VIP on the reproductive success of female mice. Significantly, mutant females produce about half the offspring of their wild-type sisters even when mated to the same males. We also find that VIP-deficient females exhibit a disrupted estrous cycle; that is, ovulation occurs less frequently and results in the release of fewer oocytes compared with controls. Circadian rhythms of wheel-running activity are disrupted in the female mutant mice, as is the spontaneous electrical activity of dorsal SCN neurons. On a molecular level, the VIP-deficient SCN tissue exhibits lower amplitude oscillations with altered phase relationships between the SCN and peripheral oscillators as measured by PER2-driven bioluminescence. The simplest explanation of our data is that the loss of VIP results in a weakened SCN oscillator, which reduces the synchronization of the female circadian system. These results clarify one of the mechanisms by which disruption of the circadian system reduces female reproductive success.

Differential modulation of clock gene expression in the suprachiasmatic nucleus, liver and heart of aged mice

Studies on the molecular clockwork during aging have been hitherto addressed to core clock genes. These previous investigations indicate that circadian profiles of core clock gene expression at an advanced age are relatively preserved in the master circadian pacemaker and the hypothalamic suprachiasmatic nucleus (SCN), and relatively impaired in peripheral tissues. It remains to be clarified whether the effects of aging are confined to the primary loop of core clock genes, or also involve secondary clock loop components, including Rev-erbα and the clock-controlled genes Dbp and Dec1. Using quantitative real-time RT-PCR, we here report a comparative analysis of the circadian expression of canonical core clock genes (Per1, Per2, Cry1, Cry2, Clock and Bmal1) and non-core clock genes (Rev-erbα, Dbp and Dec1) in the SCN, liver, and heart of 3month-old vs 22month-old mice. The results indicate that circadian clock gene expression is significantly modified in the SCN and peripheral oscillators of aged mice. These changes are not only highly tissue-specific, but also involve different clock gene loops. In particular, we here report changes of secondary clock loop components in the SCN, changes of the primary clock loop in the liver, and minor changes of clock gene expression in the heart of aged mice. The present findings outline a track to further understanding of the role of primary and secondary clock loop components and their crosstalk in the impairment of circadian output which characterizes aging.

PeriodGene Expression in the Brain of a Dual-Phasing Rodent, theOctodon degus

Clock gene expression is not only confined to the master circadian clock in the suprachiasmatic nucleus (SCN) but is also found in many other brain regions. The phase relationship between SCN and extra-SCN oscillators may contribute to known differences in chronotypes. The Octodon degus is a diurnal rodent that can shift its activity-phase preference from diurnal to nocturnal when running wheels become available. To understand better the relationship between brain clock gene activity and chronotype, we studied the day-night expression of the Period genes, Per1 and Per2, in the SCN and extra-SCN brain areas in diurnal and nocturnal degus. Since negative masking to light and entrainment to the dark phase are involved in the nocturnalism of this species, we also compare, for the first time, Per expression between entrained (EN) and masked nocturnal (MN) degus. The brains of diurnal, MN, and EN degus housed with wheels were collected during the light (ZT4) and dark (ZT16) phases. Per1 and Per2 mRNA levels were analyzed by in situ hybridization. Within the SCN, signals for Per1 and Per2 were higher at ZT4 irrespective of chronotype. However, outside of the SCN, Per1 expression in the hippocampus of EN degus was out of phase (higher values at ZT16) with SCN values. Although a similar trend was seen in MN animals, this day-night difference in Per1 expression was not significant. Interestingly, daily differences in Per1 expression were not seen in the hippocampus of diurnal degus. For other putative brain areas analyzed (cortices, striatum, arcuate, ventromedial hypothalamus), no differences in Per1 levels were found between chronotypes. Both in diurnal and nocturnal degus, Per2 levels in the hippocampus and in the cingulate and piriform cortices were in phase with their activity rhythms. Thus, diurnal degus showed higher Per2 levels at ZT4, whereas in both types of nocturnal degus, Per2 expression was reversed, peaking at ZT16. Together, the present study supports the hypothesis that the mechanisms underlying activity-phase preference in diurnal and nocturnal mammals reside downstream from the SCN, but our data also indicate that there are fundamental differences between nocturnal masked and entrained degus.

MicroRNA in the molecular mechanism of the circadian clock in mammals

The biochemical activity of mammals is controlled by an internal timekeeping mechanism driving a clock to run in approximate 24-hour (circadian) cycles. In mammals, this circadian clock is located both in the suprachiasmatic nuclei (SCN) and peripheral oscillators. Recently, microRNAs have emerged as significant players in circadian clock timing. The biological implications of miRNAs are extended further by recent studies that microRNAs are expressed in the SCN and peripheral circadian oscillators. In this study, we review recent work revealing the role of microRNAs in the molecular mechanism of circadian clock in mammals.

Heteroplasmy of Mouse mtDNA Is Genetically Unstable and Results in Altered Behavior and Cognition

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.

Period Coding of Bmal1 Oscillators in the Suprachiasmatic Nucleus

Circadian oscillators in the suprachiasmatic nucleus (SCN) collectively orchestrate 24 h rhythms in the body while also coding for seasonal rhythms. Although synchronization is required among SCN oscillators to provide robustness for regular timekeeping (Herzog et al., 2004), heterogeneity of period and phase distributions is needed to accommodate seasonal variations in light duration (Pittendrigh and Daan, 1976b). In the mouse SCN, the heterogeneous phase distribution has been recently found in the cycling of clock genes Period 1 and Period 2 (Per1, Per2) and has been shown to reorganize by relative day lengths (Inagaki et al., 2007). However, it is not yet clearly understood what underlies the spatial patterning of Per1 and Per2 expression (Yamaguchi et al., 2003; Foley et al., 2011) and its plasticity. We found that the period of the oscillation in Bmal1 expression, a positive-feedback component of the circadian clock, preserves the behavioral circadian period under culture and drives clustered oscillations in the mouse SCN. Pharmacological and physical isolations of SCN subregions indicate that the period of Bmal1 oscillation is subregion specific and is preserved during culture. Together with computer simulations, we show that either the intercellular coupling does not strongly influence the Bmal1 oscillation or the nature of the coupling is more complex than previously assumed. Furthermore, we have found that the region-specific periods are modulated by the light conditions that an animal is exposed to. Based on these, we suggest that the period forms the basis of seasonal coding in the SCN.

The Suprachiasmatic Nucleus Gets Split: Why Does Cortisol Respond But Not ACTH?

In mammals, circadian timing is dependent on a pacemaker or clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus (1). The clock is reset daily by environmental lighting transmitted to the SCN from the retina via the retinohypothalamic pathway (2). This mechanism guarantees that daily rhythms in behavior and physiology including hypothalamic-pituitary-adrenal (HPA) activity are coordinated with the daily light-dark cycle. The HPA axis is characterized by a prominent circadian rhythm that is essential for providing circulating glucocorticoids to support metabolic, cardiovascular, and neuronal processes at the onset of daily activity periods. Circadian rhythms in adrenal corticosterone are driven by in-phase rhythms in plasma ACTH and in adrenal sensitivity to ACTH (3). Adrenal sensitivity is modulated by splanchnic neural activity (4, 5). Taken together, these findings have led to the hypothesis that rhythms in adrenal secretion of glucocorticoids are regulated by not only pituitary ACTH but also sympathetic adrenal innervation (6, 7). In the present issue of Endocrinology, Lilley et al. (8) provide novel results supporting the existence of pathways from the SCN that could subserve dual regulation of glucocorticoid rhythmicity. In this study, Syrian golden hamsters were induced to split rhythms of locomotor activity by exposure to constant light. As shown previously, the split rhythm is manifested as two bouts of locomotor activity separated by approximately 12 h (9, 10). The episodes of locomotion are thought to result from the activity of two coupled circadian oscillators cycling in antiphase ( 12 h out of phase) corresponding to the left and right SCN nuclei (10). By collecting serial blood samples at 1-h intervals throughout a 24-h period in awake hamsters, the study by Lilley et al. (8) showed a clear difference in cortisol rhythms between split and unsplit animals. Whereas unsplit control hamsters showed a single peak of cortisol that preceded a single period of locomotor activity, the split hamsters showed two peaks of cortisol separated by approximately 12 h that were associated temporally with the split rhythm in locomotion. This unique observation is consistent with the hypothesis that dual SCN oscillators are capable of controlling glucocorticoid secretion. Nonetheless, these data do not answer the major question: are there pathways from the SCN that sustain glucocorticoid rhythmicity by activating both HPA and sympathoadrenal systems? To address this issue, the investigators completed another experiment to examine whether peaks in plasma cortisol are associated temporally with peaks in ACTH. To overcome the technical hurdle of obtaining a sufficient sample volume to measure ACTH without activating a hypovolemia-induced stress response, samples were collected at 3-h intervals, a sampling frequency still sufficient to characterize rhythmicity. Results showed that in unsplit control hamsters, a single peak in plasma ACTH was associated with the peak in plasma cortisol. The unexpected finding, however, was that split hamsters showing two peaks in cortisol showed no peaks in plasma ACTH. This finding supports the hypothesis that an extra-ACTH mechanism is responsible for regulating cortisol secretion in the split hamster. The investigators propose that sympathoadrenal control of glucocorticoid secretion is the extra-ACTH mechanism [Fig. 3 (8)]. Although experimental results are not presented, there is compelling evidence to support this conjecture. The adrenal cortex receives sympathetic in-

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.

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, VPAC2, play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC2 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 VPAC2 receptor expression; both VPAC2 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 VPAC2 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 VPAC2 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.

Circadian rhythm of metabolic oscillation in suprachiasmatic nucleus depends on the mitochondrial oxidation state, reflected by cytochrome C oxidase and lactate dehydrogenase

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the Clock/Bmal1 heterodimer binding to the E-box in target genes (e.g., Arg-vasopressin). Similar transcriptional regulation by Npas2/Bmal1 heterodimer formation operates in the brain, which is dependent on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state and glycolysis levels, we measured glucose, lactate dehydrogenase (LDH), LDH mRNA, and cytochrome C oxidase, energy-producing biochemical materials from mitochondria/cytosol, in rats kept under a light-dark cycle. Mitochondrial cytochrome C oxidase activity, measured by the changes in absorption at 550 nm, was higher during the light period than during the dark period. Glucose concentration was higher during the light period. In contrast, LDH and its coding mRNA were higher during the dark period. Mitochondrial aggregation, which is reflected by mitochondrial membrane potential, indexed by JC-1 fluorescence, was higher during the light period. The results indicate that the glycolysis energy pathway in the SCN, which exhits higher metabolic activity during the day than at night, might be involved in the generation of circadian rhythm.

Circadian rhythm of enolase in suprachiasmatic nucleus depends on mitochondrial function

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the CLOCK/BMAL1 heterodimer binding to the E-box in target genes (e.g., Arg- vasopressin). Similar transcriptional regulation by NPAS2/BMAL1 heterodimer formation operates in the brain, which depends on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state, two-dimensional electrophoresis was carried out on the mitochondrial fraction of SCN, obtained from rats kept under a light:dark cycle and constant under dim light. The electrophoretic pattern with TOF-mass spectrometry analysis revealed that enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. The enolase activity, coupled with lactate dehydrogenase, was higher during the light period than that in the dark. However, enolase mRNA, analyzed by RT-PCR, showed higher levels during the dark period than in the light. The clock gene products Per2, Bmal1, Rev-erbα, and AVP mRNA in the mitochondrial fraction of SCN developed a circadian rhythm showing almost the same peak time as that in whole SCN. These mRNA rhythms ran free except for that of Rev-erbα mRNA. The results indicate that, in the glycolysis-related energy pathway, enolase might be involved in higher metabolic activity during the day than at night, at least in part.