Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes.

Circadian disruption as a result of shift work is associated with adverse metabolic consequences. Internal desynchrony between the phase of the suprachiasmatic nuclei (SCN) and peripheral clocks is widely believed to be a major factor contributing to these adverse consequences, but this hypothesis has never been tested directly. A GABAergic Cre driver combined with conditional casein kinase mutations (Vgat-Cre+CK1δfl/flεfl/+ ) was used to lengthen the endogenous circadian period in GABAergic neurons, including the SCN, but not in peripheral tissues, to create a Discordant mouse model. These mice had a long (27.4 h) behavioral period to which peripheral clocks entrained in vivo, albeit with an advanced phase (∼6 h). Thus, in the absence of environmental timing cues, these mice had internal desynchrony between the SCN and peripheral clocks. Surprisingly, internal desynchrony did not result in obesity in this model. Instead, Discordant mice had reduced body mass compared with Cre-negative controls on regular chow and even when challenged with a high-fat diet. Similarly, internal desynchrony failed to induce glucose intolerance or disrupt body temperature and energy expenditure rhythms. Subsequently, a lighting cycle of 2-h light/23.5-h dark was used to create a similar internal desynchrony state in both genotypes. Under these conditions, Discordant mice maintained their lower body mass relative to controls, suggesting that internal desynchrony did not cause the lowered body mass. Overall, our results indicate that internal desynchrony does not necessarily lead to metabolic derangements and suggest that additional mechanisms contribute to the adverse metabolic consequences observed in circadian disruption protocols.

Temperature-Sensitive Substrate and Product Binding Underlie Temperature-Compensated Phosphorylation in the Clock.

Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.

Glycogen synthase kinase 3 regulates photic signaling in the suprachiasmatic nucleus.

Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus (SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3ß levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 (GSK3-KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3-KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3-KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels.

Glycogen synthase kinase-3ß in the ventral tegmental area mediates diurnal variations in cocaine-induced conditioned place preference in rats.

Cocaine sensitization and reward are reported to be under the influence of diurnal rhythm. However, no previous studies have reported brain areas that play a role as modulators and underlie the mechanism of diurnal variations in cocaine reward. We examined (1) the diurnal rhythm of glycogen synthase kinase-3ß (GSK-3ß) activity in the suprachiasmatic nucleus (SCN) and reward-related brain areas in naive rats; (2) the effect of day and night on the acquisition of cocaine-induced conditioned place preference (CPP); (3) the influence of cocaine-induced CPP on GSK-3ß activity in the SCN and reward-related brain areas; and (4) the effect of the GSK-3ß inhibitor SB216763 microinjected bilaterally into the ventral tegmental area (VTA) on cocaine-induced CPP. A significant diurnal rhythm of GSK-3ß activity was found in the SCN and reward-related brain areas, with diurnal variations in cocaine-induced CPP. GSK-3ß activity in the SCN and reward-related brain areas exhibited marked diurnal variations in rats treated with saline. GSK-3ß activity in rats treated with cocaine exhibited distinct diurnal variations only in the prefrontal cortex and VTA. Cocaine decreased the expression of phosphorylated GSK-3ß (i.e. increased GSK-3ß activity) only in the VTA in rats trained and tested at ZT4 and ZT16. SB216763 microinjected into the VTA bilaterally eliminated the diurnal variations in cocaine-induced CPP, but did not affect the acquisition of cocaine-induced CPP. These findings suggest that the VTA may be a critical area involved in the diurnal variations in cocaine-induced CPP, and GSK-3ß may be a regulator of diurnal variations in cocaine-induced CPP.

Circadian expression and specific localization of a sialyltransferase gene in the suprachiasmatic nucleus.

Polysialic acids are implicated in various biological processes such as neural cell migration, axonal growth, synaptogenesis and resetting of the circadian rhythm. Recently, polysialation has been reported to be involved in the formation and resetting of the circadian clock. However, the genes that control the circadian rhythm of polysialation have not been elucidated. In the present study, we investigated the expression profile of ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 (ST8Sia VI) in the suprachiasmatic nucleus (SCN), which is one of the modification transferases that add sialic acids to type O carbohydrate chains. ST8Sia VI mRNA showed strong expression in the SCN with dynamic circadian rhythm. Further, the amount of ST8Sia VI mRNA in the SCN was increased by brief light exposure. Interestingly, the localization of ST8Sia VI mRNA in the SCN differs from those of arginine vasopressin and vasoactive intestinal peptide mRNAs, which are typical SCN subregion markers showing shell and core, dorsomedial and ventrolateral, or light-responsive and unresponsive regions, respectively. The present findings suggest that ST8siVI is involved in rhythmic polysialation in the SCN and that ST8siVI expression provides a novel compartmentation of the mammalian circadian center.

Regulation of MAPK/ERK signaling and photic entrainment of the suprachiasmatic nucleus circadian clock by Raf kinase inhibitor protein.

Activation of the MAPK/ERK signaling cascade in the suprachiasmatic nucleus (SCN) is a key event that couples light to circadian clock entrainment. However, we do not fully understand the mechanisms that shape the properties of MAPK/ERK signaling in the SCN, and how these mechanisms may influence overt circadian rhythms. Here we show that Raf kinase inhibitor protein (RKIP) controls the kinetics of light-induced MAPK/ERK activity in the SCN and photic entrainment of behavioral rhythms. Light triggers robust phosphorylation of RKIP in the murine SCN and dissociation of RKIP and c-Raf. Overexpression of a nonphosphorylatable form of RKIP in the SCN of transgenic mice blocks light-induced ERK1/2 activation in the SCN and severely dampens light-induced phase delays in behavioral rhythms. Conversely, in RKIP knock-out (RKIP(-/-)) mice, light-induced ERK1/2 activity in the SCN is prolonged in the early and late subjective night, resulting in augmentation of the phase-delaying and -advancing effects of light. Reentrainment to an advancing light cycle was also accelerated in RKIP(-/-) mice. In relation to the molecular clockwork, genetic deletion of RKIP potentiated light-evoked PER1 and PER2 protein expression in the SCN in the early night. Additionally, RKIP(-/-) mice displayed enhanced transcriptional activation of mPeriod1 and the immediate early gene c-Fos in the SCN in response to a phase-delaying light pulse. Collectively, our data reveal an important role of RKIP in the regulation of MAPK/ERK signaling in the SCN and photic entrainment of the SCN clock.

Impaired sodium levels in the suprachiasmatic nucleus are associated with the formation of cardiovascular deficiency in sleep-deprived rats.

Biological rhythms are a ubiquitous feature of all higher organisms. The rhythmic center of mammals is located in the suprachiasmatic nucleus (SCN), which projects to a number of brainstem centers to exert diurnal control over many physiological processes, including cardiovascular regulation. Total sleep deprivation (TSD) is a harmful condition known to impair cardiovascular activity, but the molecular mechanisms are unknown. As the inward sodium current has long been suggested as playing an important role in driving the spontaneous firing of the SCN, the present study aimed to determine if changes in sodium expression, together with its molecular machinery (Na-K ATPase) and rhythmic activity within the SCN, would occur during TSD. Adult rats subjected to different periods of TSD were processed for time-of-flight secondary ion mass spectrometry, Na-K ATPase assay, and cytochrome oxidase (COX) (an endogenous bioenergetic marker for neuronal activity) histochemistry. Cardiovascular dysfunction was determined through analysis of heart rate and changes in mean arterial pressure. Results indicated that, in normal rats, strong sodium signals were expressed throughout the entire SCN. Enzymatic data corresponded well with spectrometric findings in which high levels of Na-K ATPase and COX were observed in this nucleus. However, following TSD, all parameters including sodium imaging, sodium intensity as well as COX activities were drastically decreased. Na-K ATPase showed an increase in responsiveness following TSD. Both heart rate and mean arterial pressure measurements indicated an exaggerated pressor effect following TSD treatment. As proper sodium levels are essential for SCN activation, reduced SCN sodium levels may interrupt the oscillatory control, which could serve as the underlying mechanism for the initiation or development of TSD-related cardiovascular deficiency.

Behavioral rhythmicity of mice lacking AhR and attenuation of light-induced phase shift by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 microg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Mitogen-activated protein kinase is a functional component of the autonomous circadian system in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving behavioral and physiological rhythms in mammals. Circadian activation of mitogen-activated protein kinase [MAPK; also known as ERK (extracellular signal-regulated kinase)] is observed in vivo in the SCN under constant darkness, although the biological significance of this remains unclear. To elucidate this question, we first examined whether MAPK was autonomously activated in ex vivo SCN slices. Moreover, we investigated the effect of MAPK inhibition on circadian clock gene expression and neuronal firing rhythms using SCN-slice culture systems. We show herein that MAPK is autonomously activated in the SCN, and our data demonstrate that inhibition of the MAPK activity results in dampened rhythms and reduced basal levels in circadian clock gene expression at the SCN single-neuron level. Furthermore, MAPK inhibition attenuates autonomous circadian neuronal firing rhythms in the SCN. Thus, our data suggest that light-independent MAPK activity contributes to the robustness of the SCN autonomous circadian system.

Circadian and pharmacological regulation of casein kinase I in the hamster suprachiasmatic nucleus.

In mammals, the mechanism for the generation of circadian rhythms and entrainment by light-dark (LD) cycles resides in the hypothalamic suprachiasmatic nuclei (SCN), and the principal signal that adjusts this biological clock with environmental timing is the light:dark cycle. Within the SCN, rhythms are generated by a complex of molecular feedback loops that regulate the transcription of clock genes, including per and cry. Posttranslational modification plays an essential role in the regulation of biological rhythms; in particular, clock gene phosphorylation by casein kinase I , both epsilon (CKIepsilon) and delta (CKIdelta), regulates key molecular mechanisms in the circadian clock. In this paper, we report for the first time that CKI activity undergoes a significant circadian rhythm in the SCN (peaking at circadian time 12, the start of the subjective night), and its pharmacological inhibition alters photic entrainment of the clock, indicating that CKI may be a key element in this pathway.

Photic regulation of map kinase phosphatases MKP1/2 and MKP3 in the hamster suprachiasmatic nuclei.

MAP kinases (MAPKs) play a key role in photic entrainment signaling in the suprachiasmatic nuclei (SCN), the mammalian circadian clock. The control of MAPKs is a fine balance between specific kinases (MEKs) and phosphatases (MKPs), whose orchestration in the SCN is still unresolved. We have found MKP1/2 and MKP3 immunoreactive-cells in the hamster SCN, whose levels are rapidly increased in response to transient light stimulation in the subjective night (CT 18), when light is able to entrain the clock. Moreover, the expression level of MKP3 varies under light-dark cycles and constant darkness, peaking at noon, when MAPKs are in their activated state and begin their inactivation. These results show a different perspective on MAPKs in the SCN, which includes its regulation by a complex net of phosphatases.

Daily oscillation of phospholipase C beta4 in the mouse suprachiasmatic nucleus.

An endogenous biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) regulates the timing of an organism's physiology and behavior. A variety of receptors are found on SCN pacemaker cells which permit the clock mechanism to respond to extra- and intra-SCN chemical messengers. A subset of these receptors is coupled to G-proteins, which when bound, lead to the activation of a variety of intracellular signaling cascades. One common signaling pathway employs the phosphotidylinositol-specific phospholipase C enzyme to increase intracellular calcium levels. A specific isoform of this enzyme, phospholipase C beta4, is of particular interest to circadian biologists because in its absence, mice display a circadian phenotype. Moreover, it has been shown to be associated with receptor types that are involved in clock resetting. Despite compelling data that this enzyme could be a critical component of an intracellular signaling pathway in the SCN, no study to date has investigated the possible oscillation of phospholipase C in any mammalian tissue. In the present study, we analyzed the temporal variation in the number of phospholipase C beta4 immunoreactive cells in the SCN. Herein, we show that PLCbeta4 levels oscillate in the SCN of mice housed in a light:dark photoperiod. Protein levels reached a significant peak during the early night and a trough during the day. The oscillation was considerably damped in the SCN of mice housed in constant dark conditions indicating the cycle is photoperiod-dependent. These data are critical to understanding the temporal regulation of a variety of inputs to the mammalian central circadian clock.

Insight into the circadian clock within rat colonic epithelial cells.

BACKGROUND & AIMS: The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation, suggesting the presence of endogenous circadian rhythmicity. The aim of the study was to provide insight into the circadian core clock mechanism within the rat colon. Moreover, the potency of a restricted feeding regime to shift the circadian clock in the colon was tested. The question of whether the colonic clock drives circadian expression in NHE3, an electroneutral Na(+)/H(+) exchanger, was also addressed. METHODS: Daily profiles in expression of clock genes Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbalpha, and the NHE3 transporter were examined by reverse transcriptase-polymerase chain reaction and their mRNA levels, as well as PER1 and BMAL1 protein levels, were localized in the colonic epithelium by in situ hybridization and immunocytochemistry, respectively. RESULTS: Expression of Per1, Per2, Cry1, Bmal1, Clock, Rev-erbalpha, and NHE3, as well as PER1 and BMAL1 protein levels, exhibited circadian rhythmicity in the colon. The rhythms were in phase with those in the liver but phase-delayed relative to the master clock in the suprachiasmatic nucleus. Restricted feeding entrained the clock in the colon, because rhythms in clock genes as well as in NHE3 expression were phase-advanced similarly to the clock in the liver. CONCLUSIONS: The rat colon harbors a circadian clock. The colonic clock is likely to drive rhythmic NHE3 expression. Restricted feeding resets the colonic clock similarly to the clock in the liver.

Light-inducible and clock-controlled expression of MAP kinase phosphatase 1 in mouse central pacemaker neurons.

MAP kinase phosphatase 1 (MKP1) is a negative regulator for the mitogen-activated protein kinase (MAPK)-mediated signal transduction, a key pathway that leads to the regulated expression of circadian clock genes. Here the authors analyzed mkp1 expression by in situ hybridization and found that mkp1 is a light-inducible and clock-controlled gene expressed in the central pacemaker neurons of the hypothalamic SCN. Interestingly, mkp1 presents a marked similarity to the clock core gene per1 in terms of the gene expression profiles as well as the gene promoter organization. Both mkp1 and per1 are subject to bimodal regulation in the SCN: the external light-dependent acute up-regulation and the functional clock-dependent circadian oscillation. Consistent with this, the authors show that mkp1 gene has a per1-like promoter that contains 2 functionally distinct elements: cAMP-responsive element (CRE) and E-box. CRE sites present in the mkp1 promoter constitute the functional binding sites for the CRE binding protein (CREB), which serves as an important regulator that mediates the light-induced signaling cascades in the SCN neurons. Furthermore, the authors show that the E-box present in the mkp1 promoter is necessary and sufficient for transcriptional control exerted by circadian clock core regulators that include a positive complex CLOCK/BMAL1 and a negative factor CRY1. The authors' studies on mkp1 have identified for the first time a gene encoding a phosphatase that functions in light-dependent and time-of-day-dependent manners in the mammalian central clock structure SCN.

The molecular gatekeeper Dexras1 sculpts the photic responsiveness of the mammalian circadian clock.

The mammalian master clock, located in the suprachiasmatic nucleus (SCN), is exquisitely sensitive to photic timing cues, but the key molecular events that sculpt both the phasing and magnitude of responsiveness are not understood. Here, we show that the Ras-like G-protein Dexras1 is a critical factor in these processes. Dexras1-deficient mice (dexras1-/-) exhibit a restructured nighttime phase response curve and a loss of gating to photic resetting during the day. Dexras1 affects the photic sensitivity by repressing or activating time-of-day-specific signaling pathways that regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). During the late night, Dexras1 limits the capacity of pituitary adenylate cyclase (PAC) activating peptide (PACAP)/PAC1 to affect ERK/MAPK, and in the early night, light-induced phase delays, which are mediated predominantly by NMDA receptors, are reduced as reported previously. Daytime photic phase advances are mediated by a novel signaling pathway that does not affect the SCN core but rather stimulates ERK/MAPK in the SCN shell and triggers downregulation of clock protein expression.

Constitutive activation of the ERK-MAPK pathway in the suprachiasmatic nuclei inhibits circadian resetting.

Circadian entrainment involves photic stimulation of the suprachiasmatic molecular oscillator, including activation of the ERK/MAP kinase, which is phosphorylated endogenously during the day and in response to light during the night. We aimed to disrupt the diurnal cycle of ERK phosphorylation by in vivo transfection of a constitutively active form of MEK, a MAPK kinase. This procedure did not affect normal circadian parameters, but completely inhibited light-induced phase advances. Therefore, circadian regulation of the ERK pathway is not essential for the normal mechanism of the biological clock, but it is fundamental as an interface with environmental entrainment by light.

Posttranslational regulation of the mammalian circadian clock by cryptochrome and protein phosphatase 5.

The molecular oscillator that drives circadian rhythmicity in mammals obtains its near 24-h periodicity from posttranslational regulation of clock proteins. Activity of the major clock kinase casein kinase I (CKI) epsilon is regulated by inhibitory autophosphorylation. Here we show that protein phosphatase (PP) 5 regulates the kinase activity of CKIepsilon. We demonstrate that cryptochrome regulates clock protein phosphorylation by modulating the effect of PP5 on CKIepsilon. Like CKIepsilon, PP5 is expressed both in the master circadian clock in the suprachiasmatic nuclei and in peripheral tissues independent of the clock. Expression of a dominant-negative PP5 mutant reduces PER phosphorylation by CKIepsilon in vivo, and down-regulation of PP5 significantly reduces the amplitude of circadian cycling in cultured human fibroblasts. Collectively, these findings indicate that PP5, CKIepsilon, and cryptochrome dynamically regulate the mammalian circadian clock.

Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats.

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.

Hypothalamic nitric oxide synthase in affective disorder: focus on the suprachiasmatic nucleus.

Depression is frequently associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to repeated episodes of hypercortisolemia. Hypothalamic paraventricular neurons are believed to trigger these processes by aberrant generation and/or release of corticotropin releasing hormone, oxytocin, vasopressin, and nitric oxide (NO). Recent findings from two independent laboratories have demonstrated that the suprachiasmatic nucleus, which in part controls the cellular activity of paraventricular neurons (PVN), is also involved in affective disorder. The aim of the present study was to elucidate by stereological analysis, whether suprachiasmatic nucleus (SCN) nitric oxide synthase and neurophysin generating neurons are affected in neuropsychiatric disorders. We show that compared to controls the number of nitric oxide synthase immunoreactive neurons is greatly reduced both in depression and in schizophrenia. In subjects with affective disorder there was a correlation between the number of NOS-expressing cells and duration of treatment with antidepressants. The number of neurophysin-expressing SCN neurons was also fewer in cases with mood disorder. It is concluded that SCN-derived NO may be a relevant pathophysiological factor in neuropsychiatric disorders.