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Circadian disruptions impact nearly all people with Alzheimer's disease (AD), emphasizing both their potential role in pathology and the critical need to investigate the therapeutic potential of circadian-modulating interventions. Here, we show that time-restricted feeding (TRF) without caloric restriction improved key disease components including behavioral timing, disease pathology, hippocampal transcription, and memory in two transgenic (TG) mouse models of AD. We found that TRF had the remarkable capability of simultaneously reducing amyloid deposition, increasing Aβ42 clearance, improving sleep and memory, and normalizing daily transcription patterns of multiple genes, including those associated with AD and neuroinflammation. Thus, our study unveils for the first time the pleiotropic nature of timed feeding on AD, which has far-reaching effects beyond metabolism, ameliorating neurodegeneration and the misalignment of circadian rhythmicity. Since TRF can substantially modify disease trajectory, this intervention has immediate translational potential, addressing the urgent demand for accessible approaches to reduce or halt AD progression.

ObjectiveThe increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function.MethodsBehavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincreallele.ResultsConditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization.ConclusionOur study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.

The homeodomain transcription factors sine oculis homeobox 3 (Six3) and ventral anterior homeobox 1 (Vax1) are required for brain development. Their expression in specific brain areas is maintained in adulthood, where their functions are poorly understood. To identify the roles of Six3 and Vax1 in neurons, we conditionally deleted each gene using Synapsincre , a promoter targeting maturing neurons, and generated Six3syn and Vax1syn mice. Six3syn and Vax1syn females, but not males, had reduced fertility, due to impairment of the luteinizing hormone (LH) surge driving ovulation. In nocturnal rodents, the LH surge requires a precise timing signal from the brain's circadian pacemaker, the suprachiasmatic nucleus (SCN), near the time of activity onset. Indeed, both Six3syn and Vax1syn females had impaired rhythmic SCN output, which was associated with weakened Period 2 molecular clock function in both Six3syn and Vax1syn mice. These impairments were associated with a reduction of the SCN neuropeptide vasoactive intestinal peptide in Vax1syn mice and a modest weakening of SCN timekeeping function in both Six3syn and Vax1syn mice. Changes in SCN function were associated with mistimed peak PER2::LUC expression in the SCN and pituitary in both Six3syn and Vax1syn females. Interestingly, Six3syn ovaries presented reduced sensitivity to LH, causing reduced ovulation during superovulation. In conclusion, we have identified novel roles of the homeodomain transcription factors SIX3 and VAX1 in neurons, where they are required for proper molecular circadian clock function, SCN rhythmic output, and female fertility.

Bipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30-40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of "clock genes" and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.

AbstractLight, circadian clocks, and rhythmic behaviors interact closely to produce a temporal order that is essential for the survival of most living organisms. In mammals, the principal circadian pacemaker in the brain is the suprachiasmatic nucleus (SCN), which receives direct retinal input and synchronizes itself and other brain regions to the external light-dark cycle. Altered day length (photoperiod) and disrupted circadian rhythms are associated with impaired memory and mood in both humans and animal models. Prior work demonstrated that altering photoperiod can change neurotransmitter (NT) expression in the periventricular nucleus (PeVN) of the hypothalamus in adult rat brain. Here we show that neuromedin S-(NMS-) and vasoactive intestinal polypeptide-(VIP-) expressing neurons in the SCN also display photoperiod-induced neurotransmitter switching. Such photoperiod-dependent NT plasticity is retained in Bmal1-KO mice, indicating that NT plasticity in the SCN does not require a functional circadian clock. Utilizing a conditional viral DO-DIO vector as an historical marker of NT expression in the SCN, we further reveal that short-day photoperiod induces a cluster of non-NMS-expressing neurons to undergo NT switching and acquire the NMS phenotype. Selective chemogenetic activation of NMS neurons, but not VIP neurons, during the dark phase induces a significant delay in the timing of locomotor activity onset and is sufficient to increase the number of dopaminergic neurons in the PeVN. Our findings provide novel insights into molecular adaptations of the SCN neuronal network in response to altered photoperiod that affect neuronal circuit function in the hypothalamus and lead to changes in circadian behavior.

The nucleus accumbens (NAc), a central component of the midbrain dopamine reward circuit, exhibits disturbed circadian rhythms in the postmortem brains of depressed patients. We hypothesized that normal mood regulation requires proper circadian timing in the NAc, and that mood disorders are associated with dysfunctions of the NAc cellular circadian clock. In mice exhibiting stress-induced depression-like behavior (helplessness), we found altered circadian clock function and high nighttime expression of the core circadian clock component CRYPTOCHROME (CRY) in the NAc. In the NAc of helpless mice, we found that higher expression of CRY is associated with decreased activation of dopamine 1 receptor-expressing medium spiny neurons (D1R-MSNs). Furthermore, D1R-MSN-specific CRY-knockdown in the NAc reduced susceptibility to stress-induced helplessness and increased NAc neuronal activation at night. Finally, we show that CRY inhibits D1R-induced G protein activation, likely by interacting with the Gs protein. Altered circadian rhythms and CRY expression were also observed in human fibroblasts from major depressive disorder patients. Our data reveal a causal role for CRY in regulating the midbrain dopamine reward system, and provide a mechanistic link between the NAc circadian clock and vulnerability to depression.

Shift-work and jet-lag-related disorders are caused by the limited flexibility of the suprachiasmatic nucleus (SCN), a master circadian clock in the hypothalamus, to adjust to new light-dark (LD) cycles. Recent findings confirmed here establish that behavioral jet lag after simulated time-zone travel is virtually eliminated following bifurcated circadian entrainment under a novel and atypical 24-h light:dark:light:dark (LDLD) cycle. To investigate the mechanisms of this fast resetting, we examined the oscillatory stability of the SCN and peripheral tissues in LDLD-bifurcated mice employing the dissection procedure as a perturbing resetting stimulus. SCN, lung, liver, and adrenal tissue were extracted at times throughout the day from female and male PER2::Luciferase knock-in mice entrained to either LDLD or a normal LD cycle. Except for adrenals, the phase of the cultured explants was more strongly set by dissection under LDLD than under normal LD. Acute bioluminescence levels of SCN explants indicate that the rhythm amplitude of PER2 is reduced and phase is altered in LDLD. Real-time quantitative PCR suggests that amplitude and rhythmicity of canonical clock genes in the lung, liver, and kidney are also significantly reduced in LDLD in vivo. Furthermore, spatiotemporal patterns of PER2 peak time in cultured SCN were altered in LDLD. These results suggest that altered gene expression patterns in the SCN caused by bifurcation likely result in fast resetting of behavior and cultured explants, consistent with previously reported mathematical models. Thus, non-invasive, simple light manipulations can make circadian rhythms more adaptable to abrupt shifts in the environmental LD cycle.

Bmal1 is the only single circadian clock gene that is essential for rhythmic gene expression in the mammalian circadian timing system. Genetic approaches targeting Bmal1 expression have been used to further assess its role in the circadian clock and to test for behavioral effects of clock disruption. In particular, disruptions in circadian clock function have been implicated in human mood disorders, and clock gene manipulation in mice may provide valuable models for studying depression-like behavior. In this review, we explore various approaches to manipulating Bmal1 in mouse models and review their effects on the brain's master circadian pacemaker, on circadian rhythmicity in other brain regions, and on circadian and mood-related behavior.

Bipolar disorder (BD) is a serious mood disorder associated with circadian rhythm abnormalities. Risk for BD is genetically encoded and overlaps with systems that maintain circadian rhythms. Lithium is an effective mood stabilizer treatment for BD, but only a minority of patients fully respond to monotherapy. Presently, we hypothesized that lithium-responsive BD patients (Li-R) would show characteristic differences in chronotype and cellular circadian rhythms compared to lithium non-responders (Li-NR). Selecting patients from a prospective, multi-center, clinical trial of lithium monotherapy, we examined morning vs. evening preference (chronotype) as a dimension of circadian rhythm function in 193 Li-R and Li-NR BD patients. From a subset of 59 patient donors, we measured circadian rhythms in skin fibroblasts longitudinally over 5 days using a bioluminescent reporter (Per2-luc). We then estimated circadian rhythm parameters (amplitude, period, phase) and the pharmacological effects of lithium on rhythms in cells from Li-R and Li-NR donors. Compared to Li-NRs, Li-Rs showed a difference in chronotype, with higher levels of morningness. Evening chronotype was associated with increased mood symptoms at baseline, including depression, mania, and insomnia. Cells from Li-Rs were more likely to exhibit a short circadian period, a linear relationship between period and phase, and period shortening effects of lithium. Common genetic variation in the IP3 signaling pathway may account for some of the individual differences in the effects of lithium on cellular rhythms. We conclude that circadian rhythms may influence response to lithium in maintenance treatment of BD.

Disruption of circadian clocks is strongly associated with mood disorders. Chronotherapies targeting circadian rhythms have been shown to be very effective treatments of mood disorders, but still are not widely used in clinical practice. The mechanisms by which circadian disruption leads to mood disorders are poorly characterized and, therefore, may not convince clinicians to apply chronotherapies. Hence, in this review, we describe specific potential mechanisms, in order to make this connection more credible to clinicians. We believe that four major features of disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. Discussing these attributes and giving plausible examples, we will discuss prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice. Key messages In this review, we describe specific potential mechanisms by which disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. We provide prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

Seasonal changes in light exposure have profound effects on behavioral and physiological functions in many species, including effects on mood and cognitive function in humans. The mammalian brain's master circadian clock, the suprachiasmatic nucleus (SCN), transmits information about external light conditions to other brain regions, including some implicated in mood and cognition. Although the detailed mechanisms are not yet known, the SCN undergoes highly plastic changes at the cellular and network levels under different light conditions. We therefore propose that the SCN may be an essential mediator of the effects of seasonal changes of day length on mental health. In this review, we explore various forms of neuroplasticity that occur in the SCN and other brain regions to facilitate seasonal adaptation, particularly altered phase distribution of cellular circadian oscillators in the SCN and changes in hypothalamic neurotransmitter expression.

BackgroundCircadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock.ResultsWe found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events.ConclusionsHere we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3-4weeks old) and adult (9-30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.

BackgroundMajor depressive disorder is associated with disturbed circadian rhythms. To investigate the causal relationship between mood disorders and circadian clock disruption, previous studies in animal models have employed light/dark manipulations, global mutations of clock genes, or brain area lesions. However, light can impact mood by noncircadian mechanisms; clock genes have pleiotropic, clock-independent functions; and brain lesions not only disrupt cellular circadian rhythms but also destroy cells and eliminate important neuronal connections, including light reception pathways. Thus, a definitive causal role for functioning circadian clocks in mood regulation has not been established.MethodsWe stereotactically injected viral vectors encoding short hairpin RNA to knock down expression of the essential clock gene Bmal1 into the brain's master circadian pacemaker, the suprachiasmatic nucleus (SCN).ResultsIn these SCN-specific Bmal1-knockdown (SCN-Bmal1-KD) mice, circadian rhythms were greatly attenuated in the SCN, while the mice were maintained in a standard light/dark cycle, SCN neurons remained intact, and neuronal connections were undisturbed, including photic inputs. In the learned helplessness paradigm, the SCN-Bmal1-KD mice were slower to escape, even before exposure to inescapable stress. They also spent more time immobile in the tail suspension test and less time in the lighted section of a light/dark box. The SCN-Bmal1-KD mice also showed greater weight gain, an abnormal circadian pattern of corticosterone, and an attenuated increase of corticosterone in response to stress.ConclusionsDisrupting SCN circadian rhythms is sufficient to cause helplessness, behavioral despair, and anxiety-like behavior in mice, establishing SCN-Bmal1-KD mice as a new animal model of depression.

UnlabelledBipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs.ControlsPharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

Endogenous circadian (∼24 h) clocks regulate key physiological and cognitive processes via rhythmic expression of clock genes. The main circadian pacemaker is the hypothalamic suprachiasmatic nucleus (SCN). Mood disorders, including bipolar disorder (BD), are commonly associated with disturbed circadian rhythms. Dopamine (DA) contributes to mania in BD and has direct impact on clock gene expression. Therefore, we hypothesized that high levels of DA during episodes of mania contribute to disturbed circadian rhythms in BD. The mood stabilizer valproic acid (VPA) also affects circadian rhythms. Thus, we further hypothesized that VPA normalizes circadian disturbances caused by elevated levels of DA. To test these hypotheses, we examined locomotor rhythms and circadian gene cycling in mice with reduced expression of the dopamine transporter (DAT-KD mice), which results in elevated DA levels and mania-like behavior. We found that elevated DA signaling lengthened the circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants. In contrast, we found that VPA shortened circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants, hippocampal cell lines, and human fibroblasts from BD patients. Thus, DA and VPA have opposing effects on circadian period. To test whether the impact of VPA on circadian rhythms contributes to its behavioral effects, we fed VPA to DAT-deficient Drosophila with and without functioning circadian clocks. Consistent with our hypothesis, we found that VPA had potent activity-suppressing effects in hyperactive DAT-deficient flies with intact circadian clocks. However, these effects were attenuated in DAT-deficient flies in which circadian clocks were disrupted, suggesting that VPA functions partly through the circadian clock to suppress activity. Here, we provide in vivo and in vitro evidence across species that elevated DA signaling lengthens the circadian period, an effect remediated by VPA treatment. Hence, VPA may exert beneficial effects on mood by normalizing lengthened circadian rhythm period in subjects with elevated DA resulting from reduced DAT.

Light is the primary signal that calibrates circadian neural circuits and thus coordinates daily physiological and behavioral rhythms with solar entrainment cues. Drosophila and mammalian circadian circuits consist of diverse populations of cellular oscillators that exhibit a wide range of dynamic light responses, periods, phases, and degrees of synchrony. How heterogeneous circadian circuits can generate robust physiological rhythms while remaining flexible enough to respond to synchronizing stimuli has long remained enigmatic. Cryptochrome is a short-wavelength photoreceptor that is endogenously expressed in approximately half of Drosophila circadian neurons. In a previous study, physiological light response was measured using real-time bioluminescence recordings in Drosophila whole-brain explants, which remain intrinsically light-sensitive. Here we apply analysis of real-time bioluminescence experimental data to show detailed dynamic ensemble representations of whole circadian circuit light entrainment at single neuron resolution. Organotypic whole-brain explants were either maintained in constant darkness (DD) for 6 days or exposed to a phase-advancing light pulse on the second day. We find that stronger circadian oscillators support robust overall circuit rhythmicity in DD, whereas weaker oscillators can be pushed toward transient desynchrony and damped amplitude to facilitate a new state of phase-shifted network synchrony. Additionally, we use mathematical modeling to examine how a network composed of distinct oscillator types can give rise to complex dynamic signatures in DD conditions and in response to simulated light pulses. Simulations suggest that complementary coupling mechanisms and a combination of strong and weak oscillators may enable a robust yet flexible circadian network that promotes both synchrony and entrainment. A more complete understanding of how the properties of oscillators and their signaling mechanisms facilitate their distinct roles in light entrainment may allow us to direct and augment the circadian system to speed recovery from jet lag, shift work, and seasonal affective disorder.

An association between circadian rhythms and mood regulation is well established, and disturbed circadian clocks are believed to contribute to the development of mood disorders, including major depressive disorder. The circadian system is coordinated by the suprachiasmatic nucleus (SCN), the master pacemaker in the hypothalamus that receives light input from the retina and synchronizes circadian oscillators in other brain regions and peripheral tissues. Lacking the tight neuronal network that couples single-cell oscillators in the SCN, circadian clocks outside the SCN may be less stable and more susceptible to disturbances, for example by clock gene mutations or uncontrollable stress. However, non-SCN circadian clocks have not been studied extensively in rodent models of mood disorders. In the present study, it was hypothesized that disturbances of local circadian clocks in mood-regulating brain areas are associated with depression-like behaviour in mice. Using the learned helplessness procedure, depression-like behaviour was evoked in mice bearing the PER2::LUC circadian reporter, and then circadian rhythms of PER2 expression were examined in brain slices from these mice using luminometry and bioluminescence imaging. It was found that helplessness is associated with absence of circadian rhythms in the nucleus accumbens and the periaqueductal grey, two of the most critical brain regions within the reward circuit. The current study provides evidence that susceptibility of mice to depression-like behaviour is associated with disturbed local circadian clocks in a subset of mood-regulating brain areas, but the direction of causality remains to be determined.

Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3β (GSK-3β), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3β. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.

UnlabelledBipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca(2+) changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2::luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 h in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca(2+) less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs.ControlsWe conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms.

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.

Psychiatric disorders such as schizophrenia, bipolar disorder, and major depressive disorder are often accompanied by metabolic dysfunction symptoms, including obesity and diabetes. Since the circadian system controls important brain systems that regulate affective, cognitive, and metabolic functions, and neuropsychiatric and metabolic diseases are often correlated with disturbances of circadian rhythms, we hypothesize that dysregulation of circadian clocks plays a central role in metabolic comorbidity in psychiatric disorders. In this review paper, we highlight the role of circadian clocks in glucocorticoid, dopamine, and orexin/melanin-concentrating hormone systems and describe how a dysfunction of these clocks may contribute to the simultaneous development of psychiatric and metabolic symptoms.

Circadian neural circuits generate near 24-hr physiological rhythms that can be entrained by light to coordinate animal physiology with daily solar cycles. To examine how a circadian circuit reorganizes its activity in response to light, we imaged period (per) clock gene cycling for up to 6 days at single-neuron resolution in whole-brain explant cultures prepared from per-luciferase transgenic flies. We compared cultures subjected to a phase-advancing light pulse (LP) to cultures maintained in darkness (DD). In DD, individual neuronal oscillators in all circadian subgroups are initially well synchronized but then show monotonic decrease in oscillator rhythm amplitude and synchrony with time. The small ventral lateral neurons (s-LNvs) and dorsal lateral neurons (LNds) exhibit this decrease at a slower relative rate. In contrast, the LP evokes a rapid loss of oscillator synchrony between and within most circadian neuronal subgroups, followed by gradual phase retuning of whole-circuit oscillator synchrony. The LNds maintain high rhythmic amplitude and synchrony following the LP along with the most rapid coherent phase advance. Immunocytochemical analysis of PER shows that these dynamics in DD and LP are recapitulated in vivo. Anatomically distinct circadian neuronal subgroups vary in their response to the LP, showing differences in the degree and kinetics of their loss, recovery and/or strengthening of synchrony, and rhythmicity. Transient desynchrony appears to be an integral feature of light response of the Drosophila multicellular circadian clock. Individual oscillators in different neuronal subgroups of the circadian circuit show distinct kinetic signatures of light response and phase retuning.

The state of being helpless is regarded as a central aspect of depression, and therefore the learned helplessness paradigm in rodents is commonly used as an animal model of depression. The term 'learned helplessness' refers to a deficit in escaping from an aversive situation after an animal is exposed to uncontrollable stress specifically, with a control/comparison group having been exposed to an equivalent amount of controllable stress. A key feature of learned helplessness is the transferability of helplessness to different situations, a phenomenon called 'trans-situationality'. However, most studies in mice use learned helplessness protocols in which training and testing occur in the same environment and with the same type of stressor. Consequently, failures to escape may reflect conditioned fear of a particular environment, not a general change of the helpless state of an animal. For mice, there is no established learned helplessness protocol that includes the trans-situationality feature. Here we describe a simple and reliable learned helplessness protocol for mice, in which training and testing are carried out in different environments and with different types of stressors. We show that with our protocol approximately 50% of mice develop learned helplessness that is not attributable to fear conditioning.

Seasonal effects on mood have been observed throughout much of human history.  Seasonal changes in animals and plants are largely mediated through the changing photoperiod (i.e., the photophase or duration of daylight).  We review that in mammals, daylight specifically regulates SCN (suprachiasmatic nucleus) circadian organization and its control of melatonin secretion.  The timing of melatonin secretion interacts with gene transcription in the pituitary pars tuberalis to modulate production of TSH (thyrotropin), hypothalamic T3 (triiodothyronine), and tuberalin peptides which modulate pituitary production of regulatory gonadotropins and other hormones.  Pituitary hormones largely mediate seasonal physiologic and behavioral variations.  As a result of long winter nights or inadequate illumination, we propose that delayed morning offset of nocturnal melatonin secretion, suppressing pars tuberalis function, could be the main cause for winter depression and even cause depressions at other times of year.  Irregularities of circadian sleep timing and thyroid homeostasis contribute to depression.  Bright light and sleep restriction are antidepressant and conversely, sometimes trigger mania.  We propose that internal desynchronization or bifurcation of SCN circadian rhythms may underlie rapid-cycling manic-depressive disorders and perhaps most mania.  Much further research will be needed to add substance to these theories.

Many psychiatric disorders are characterized by circadian rhythm abnormalities, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Animal models with mutations in circadian "clock genes" commonly show disturbances in reward processing, locomotor activity and novelty seeking behaviors, further supporting the idea of a connection between the circadian clock and psychiatric disorders. However, if circadian clock dysfunction is a common risk factor for multiple psychiatric disorders, it is unknown if and how these putative clock abnormalities could be expressed differently, and contribute to multiple, distinct phenotypes. One possible explanation is that the circadian clock modulates the biological responses to stressful environmental factors that vary with an individual's experience. It is known that the circadian clock and the stress response systems are closely related: Circadian clock genes regulate the physiological sensitivity to and rhythmic release of glucocorticoids (GC). In turn, GCs have reciprocal effects on the clock. Since stressful life events or increased vulnerability to stress are risk factors for multiple psychiatric disorders, including post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), bipolar disorder (BD), major depressive disorder (MDD), alcohol use disorder (AUD) and schizophrenia (SCZ), we propose that modulation of the stress response is a common mechanism by which circadian clock genes affect these illnesses. Presently, we review how molecular components of the circadian clock may contribute to these six psychiatric disorders, and present the hypothesis that modulation of the stress response may constitute a common mechanism by which the circadian clock affects multiple psychiatric disorders.

An association between circadian clock function and mood regulation is well established and has been proposed as a factor in the development of mood disorders. Patients with depression or mania suffer disturbed sleep-wake cycles and altered rhythms in daily activities. Environmentally disrupted circadian rhythms increase the risk of mood disorders in the general population. However, proof that a disturbance of circadian rhythms is causally involved in the development of psychiatric disorders remains elusive. Using clock gene mutants, manipulations of sleep-wake and light-dark cycles, and brain lesions affecting clock function, animal models have been developed to investigate whether circadian rhythm disruptions alter mood. In this review, selected animal models are examined to address the issue of causality between circadian rhythms and affective behavior.

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.

BackgroundClinical and genetic studies suggest circadian clock genes may contribute to biological mechanisms underlying alcohol use disorders (AUD). In particular, the Per2 gene regulates alcohol consumption in mutant animals, and in humans with AUD, the 10870 variant in PER2 has been associated with alcohol consumption. However, with respect to function, the molecular clock remains largely uncharacterized in AUD patients.MethodsIn skin fibroblast cultures from well-characterized human AUD patients (n = 19) and controls (n = 13), we used a bioluminescent reporter gene (Per2::luc) to measure circadian rhythms in gene expression at high sampling density for 5 days. Cells were genotyped for the PER2 10870 variant. The rhythm parameters period and amplitude were then analyzed using a case-control design and by genetic and clinical characteristics of the AUD subjects.ResultsThere were no differences between AUD cases and controls in rhythm parameters. However, period was inversely correlated with illness severity (defined as the number of alcohol dependence criteria met). The PER2 variant 10870 was not associated with differences in rhythm parameters.ConclusionsOur data suggest that differences in the cellular circadian clock are not pronounced in fibroblasts from AUD cases and controls. However, we found evidence that the circadian clock may be associated with an altered trajectory of AUD, possibly related to illness severity. Future work will be required to determine the mechanistic basis of this association.

Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1-/- cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2-/- cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1-/- cells. In previous work, we found that low K⁺ suppresses fibroblast rhythmicity, and we and others have found that either low K⁺ or low Ca²⁺ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K⁺ (21 mM), high Ca²⁺ (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K⁺ or Ca²⁺ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts.

In mammals, the suprachiasmatic nucleus (SCN) is the central pacemaker organizing circadian rhythms of behavior and physiology. At the cellular level, the mammalian clock consists of autoregulatory feedback loops involving a set of "clock genes," including the Cryptochrome (Cry) genes, Cry1 and Cry2. Experimental evidence suggests that Cry1 and Cry2 play distinct roles in circadian clock function. In mice, Cry1 is required for sustained circadian rhythms in dissociated SCN neurons or fibroblasts but not in organotypic SCN slices or at the behavioral level, whereas Cry2 is not required at any of these levels. It has been argued that coupling among SCN cellular oscillators compensates for clock gene defects to preserve oscillatory function. Here we test this hypothesis in Cry1(-/-) mice by first disrupting intercellular coupling in vivo using constant light (resulting in behavioral arrhythmicity) and then examining circadian clock gene expression in SCN slices at the single cell level. In this manner, we were able to test the role of intercellular coupling without drugs and while preserving tissue organization, avoiding the confounding influences of more invasive manipulations. Cry1(-/-) mice (as well as control Cry2(-/-) mice) bearing the PER2::LUC knock-in reporter were transferred from a standard light:dark cycle to constant bright light (~650 lux) to induce arrhythmic locomotor patterns. In SCN slices from these animals, we used bioluminescence imaging to monitor PER2::LUC expression in single cells. We show that SCN slices from rhythmic Cry1(-/-) and Cry2(-/-) mice had similarly high percentages of functional single-cell oscillators. In contrast, SCN slices from arrhythmic Cry1(-/-) mice had significantly fewer rhythmic cells than SCN slices from arrhythmic Cry2(-/-) mice. Thus, constant light in vivo disrupted intercellular SCN coupling to reveal a cell-autonomous circadian defect in Cry1(-/-) cells that is normally compensated by intercellular coupling in vivo.

Precise determination of a noisy biological oscillator's period from limited experimental data can be challenging. The common practice is to calculate a single number (a point estimate) for the period of a particular time course. Uncertainty is inherent in any statistical estimator applied to noisy data, so our confidence in such point estimates depends on the quality and quantity of the data. Ideally, a period estimation method should both produce an accurate point estimate of the period and measure the uncertainty in that point estimate. A variety of period estimation methods are known, but few assess the uncertainty of the estimates, and a measure of uncertainty is rarely reported in the experimental literature. We compare the accuracy of point estimates using six common methods, only one of which can also produce uncertainty measures. We then illustrate the advantages of a new Bayesian method for estimating period, which outperforms the other six methods in accuracy of point estimates for simulated data and also provides a measure of uncertainty. We apply this method to analyze circadian oscillations of gene expression in individual mouse fibroblast cells and compute the number of cells and sampling duration required to reduce the uncertainty in period estimates to a desired level. This analysis indicates that, due to the stochastic variability of noisy intracellular oscillators, achieving a narrow margin of error can require an impractically large number of cells. In addition, we use a hierarchical model to determine the distribution of intrinsic cell periods, thereby separating the variability due to stochastic gene expression within each cell from the variability in period across the population of cells.

Biological oscillators naturally exhibit stochastic fluctuations in period and amplitude due to the random nature of molecular reactions. Accurately measuring the precision of noisy oscillators and the heterogeneity in period and strength of rhythmicity across a population of cells requires single-cell recordings of sufficient length to fully represent the variability of oscillations. We found persistent, independent circadian oscillations of clock gene expression in 6-week-long bioluminescence recordings of 80 primary fibroblast cells dissociated from PER2::LUC mice and kept in vitro for 6 months. Due to the stochastic nature of rhythmicity, the proportion of cells appearing rhythmic increases with the length of interval examined, with 100% of cells found to be rhythmic when using 3-week windows. Mean period and amplitude are remarkably stable throughout the 6-week recordings, with precision improving over time. For individual cells, precision of period and amplitude are correlated with cell size and rhythm amplitude, but not with period, and period exhibits much less cycle-to-cycle variability (CV 7.3%) than does amplitude (CV 37%). The time series are long enough to distinguish stochastic fluctuations within each cell from differences among cells, and we conclude that the cells do exhibit significant heterogeneity in period and strength of rhythmicity, which we measure using a novel statistical metric. Furthermore, stochastic modeling suggests that these single-cell clocks operate near a Hopf bifurcation, such that intrinsic noise enhances the oscillations by minimizing period variability and sustaining amplitude.

Circadian rhythm abnormalities in bipolar disorder (BD) have led to a search for genetic abnormalities in circadian "clock genes" associated with BD. However, no significant clock gene findings have emerged from genome-wide association studies (GWAS). At least three factors could account for this discrepancy: complex traits are polygenic, the organization of the clock is more complex than previously recognized, and/or genetic risk for BD may be shared across multiple illnesses. To investigate these issues, we considered the clock gene network at three levels: essential "core" clock genes, upstream circadian clock modulators, and downstream clock controlled genes. Using relaxed thresholds for GWAS statistical significance, we determined the rates of clock vs. control genetic associations with BD, and four additional illnesses that share clinical features and/or genetic risk with BD (major depression, schizophrenia, attention deficit/hyperactivity). Then we compared the results to a set of lithium-responsive genes. Associations with BD-spectrum illnesses and lithium-responsiveness were both enriched among core clock genes but not among upstream clock modulators. Associations with BD-spectrum illnesses and lithium-responsiveness were also enriched among pervasively rhythmic clock-controlled genes but not among genes that were less pervasively rhythmic or non-rhythmic. Our analysis reveals previously unrecognized associations between clock genes and BD-spectrum illnesses, partly reconciling previously discordant results from past GWAS and candidate gene studies.

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.

Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber).

Genes responsible for generating circadian oscillations are expressed in a variety of brain regions not typically associated with circadian timing. The functions of this clock gene expression are largely unknown, and in the present study we sought to explore the role of the Per2 (Period 2) gene in hippocampal physiology and learned behaviour. We found that PER2 protein is highly expressed in hippocampal pyramidal cell layers and that the expression of both protein and mRNA varies with a circadian rhythm. The peaks of these rhythms occur in the late night or early morning and are almost 180° out-of-phase with the expression rhythms measured from the suprachiasmatic nucleus of the same animals. The rhythms in Per2 expression are autonomous as they are present in isolated hippocampal slices maintained in culture. Physiologically, Per2-mutant mice exhibit abnormal long-term potentiation. The underlying mechanism is suggested by the finding that levels of phosphorylated cAMP-response-element-binding protein, but not phosphorylated extracellular-signal-regulated kinase, are reduced in hippocampal tissue from mutant mice. Finally, Per2-mutant mice exhibit deficits in the recall of trace, but not cued, fear conditioning. Taken together, these results provide evidence that hippocampal cells contain an autonomous circadian clock. Furthermore, the clock gene Per2 may play a role in the regulation of long-term potentiation and in the recall of some forms of learned behaviour.

The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBalpha and beta are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbalpha. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBalpha and beta function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.

The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBalpha and beta are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbalpha. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBalpha and beta function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.