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

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN., (© 2021 MRC Laboratory of Molecular Biology Published under the terms of the CC BY 4.0 license.)

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2 -null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2 -null controls expressing EGFP (SCN Con ) was arrhythmic, whereas Cry1-complemented mice (SCN Cry1 ) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCN Con mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCN Cry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS δ power and expression of REMS within total sleep (TS). Wakefulness in SCN Con mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCN Cry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS δ power, although the SCN Con mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCN Cry1 mice were comparable to WTs. Finally, SCN Con mice exhibited poor sleep-dependent memory but this was corrected in SCN Cry1 mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep. SIGNIFICANCE STATEMENT The circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but the presence of multiple local brain and peripheral clocks mean the respective roles of SCN and other clocks in regulating sleep are unclear. We therefore used virally mediated genetic complementation to restore molecular circadian functions in the suprachiasmatic hypothalamus, focusing on the SCN, in otherwise genetically clockless, arrhythmic mice. This initiated circadian activity-rest cycles, and circadian sleep-wake cycles, circadian patterning to the intensity of non-rapid eye movement sleep (NREMS) and circadian control of REMS as a proportion of total sleep (TS). Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the suprachiasmatic hypothalamus, alone, can direct circadian regulation of sleep-wake., (Copyright © 2021 Maywood et al.)

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling., (Copyright © 2017 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

Unlabelled: The suprachiasmatic nucleus (SCN) is the master circadian oscillator encoding time-of-day information. SCN timekeeping is sustained by a cell-autonomous transcriptional-translational feedback loop, whereby expression of the Period and Cryptochrome genes is negatively regulated by their protein products. This loop in turn drives circadian oscillations in gene expression that direct SCN electrical activity and thence behavior. The robustness of SCN timekeeping is further enhanced by interneuronal, circuit-level coupling. The aim of this study was to combine pharmacological and genetic manipulations to push the SCN clockwork toward its limits and, by doing so, probe cell-autonomous and emergent, circuit-level properties. Circadian oscillation of mouse SCN organotypic slice cultures was monitored as PER2::LUC bioluminescence. SCN of three genetic backgrounds-wild-type, short-period CK1ε(Tau/Tau) mutant, and long-period Fbxl3(Afh/Afh) mutant-all responded reversibly to pharmacological manipulation with period-altering compounds: picrotoxin, PF-670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride), and KNK437 (N-Formyl-3,4-methylenedioxy-benzylidine-gamma-butyrolactam). This revealed a remarkably wide operating range of sustained periods extending across 25 h, from ≤17 h to >42 h. Moreover, this range was maintained at network and single-cell levels. Development of a new technique for formal analysis of circadian waveform, first derivative analysis (FDA), revealed internal phase patterning to the circadian oscillation at these extreme periods and differential phase sensitivity of the SCN to genetic and pharmacological manipulations. For example, FDA of the CK1ε(Tau/Tau) mutant SCN treated with the CK1ε-specific inhibitor PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride) revealed that period acceleration in the mutant is due to inappropriately phased activity of the CK1ε isoform. In conclusion, extreme period manipulation reveals unprecedented elasticity and temporal structure of the SCN circadian oscillation., Significance Statement: The master circadian clock of the suprachiasmatic nucleus (SCN) encodes time-of-day information that allows mammals to predict and thereby adapt to daily environmental cycles. Using combined genetic and pharmacological interventions, we assessed the temporal elasticity of the SCN network. Despite having evolved to generate a 24 h circadian period, we show that the molecular clock is surprisingly elastic, able to reversibly sustain coherent periods between ≤17 and >42 h at the levels of individual cells and the overall circuit. Using quantitative techniques to analyze these extreme periodicities, we reveal that the oscillator progresses as a sequence of distinct stages. These findings reveal new properties of how the SCN functions as a network and should inform biological and mathematical analyses of circadian timekeeping., (Copyright © 2016 Patton et al.)

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells.

Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker in mammals, coordinating daily metabolic and physiological rhythms with the cycle of sleep and wakefulness. SCN neurons define circadian time via an auto-regulatory feedback loop in which the activation of Period (Per) and Cryptochrome genes is periodically suppressed by their own protein products. Casein kinase 1 (CK1) enzymes have a critical role in circadian pacemaking because they phosphorylate PER proteins and thereby direct their proteasomal degradation. In human pedigrees, individual mutations in either hCK1 or hPER2 lead to advanced sleep phase disorders, whereas in rodents, the Tau mutation of CK1 epsilon (CK1ε (Tau)) accelerates rest-activity cycles and shortens the period of the SCN molecular pacemaker. Biochemical analyses of recombinant PER proteins in cultured cells and endogenous proteins in peripheral tissues have identified PER1 and PER2, but not PER3, as direct substrates of CK1ε. The purpose of this study, therefore, was to determine the relative contributions of endogenous PER proteins to the period-accelerating effects of CK1ε (Tau), both in vivo and in vitro. CK1ε (Tau) mice were mated onto Per1-, Per2-, and Per1-Per2 (Per1/2) double-null backgrounds, in all cases carrying the Per1-luciferase bioluminescent circadian reporter gene. Mice lacking both PER1 and PER2 were behaviorally arrhythmic, confirming the inadequacy of PER3 as a circadian factor. Individual loss of either PER1 or PER2 had no significant effect on the circadian period or quality of wheel-running behavior, and CK1ε (Tau) accelerated behavioral rhythms in both Per1- and Per2-null mice. CK1ε (Tau) also accelerated in vitro molecular pacemaking in SCN lacking either PER1 or PER2, with a greater effect in PER2-dependent (i.e., Per1-null) SCN than in PER1-dependent slices. In double-null slices, some SCN were arrhythmic, whereas others exhibited transient rhythms, which trended nonsignificantly toward a shorter period. Both short-period and long-period rhythms could be identified in individual SCN neurons imaged by charge-coupled device camera. CK1ε (Tau) had no effect, however, on SCN-level or individual neuronal rhythms in the absence of PER1 and PER2. Thus, the CK1ε (Tau) allele has divergent actions, acting via both endogenous PER1 and PER2, but not PER3 protein, to mediate its circadian actions in vivo. Moreover, PER-independent cellular oscillations may contribute to pacemaking, but they are unstable and imprecise, and are not affected by the Tau mutation.

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.

The role of intracellular transcriptional/post-translational feedback loops (TTFL) within the circadian pacemaker of the suprachiasmatic nucleus (SCN) is well established. In contrast, contributions from G-coupled pathways and cytosolic rhythms to the intercellular control of SCN pacemaking are poorly understood. We therefore combined viral transduction of SCN slices with fluorescence/bioluminescence imaging to visualize GCaMP3-reported circadian oscillations of intracellular calcium [Ca2+]i alongside activation of Ca2+ /cAMP-responsive elements. We phase-mapped them to the TTFL, in time and SCN space, and demonstrated their dependence upon G-coupled vasoactive intestinal peptide (VIP) signaling. Pharmacogenetic manipulation revealed the individual contributions of Gq, Gs, and Gi to cytosolic and TTFL circadian rhythms. Importantly, activation of Gq-dependent (but not Gs or Gi) pathways in a minority of neurons reprogrammed [Ca2+]i and TTFL rhythms across the entire SCN. This reprogramming was mediated by intrinsic VIPergic signaling, thus revealing a Gq/[Ca2+]i-VIP leitmotif and unanticipated plasticity within network encoding of SCN circadian time., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The circadian clock of the suprachiasmatic nucleus (SCN) drives daily rhythms of behavior. Cryptochromes (CRYs) are powerful transcriptional repressors within the molecular negative feedback loops at the heart of the SCN clockwork, where they periodically suppress their own expression and that of clock-controlled genes. To determine the differential contributions of CRY1 and CRY2 within circadian timing in vivo, we exploited the N-ethyl-N-nitrosourea-induced afterhours mutant Fbxl3(Afh) to stabilize endogenous CRY. Importantly, this was conducted in CRY2- and CRY1-deficient mice to test each CRY in isolation. In both CRY-deficient backgrounds, circadian rhythms of wheel-running and SCN bioluminescence showed increased period length with increased Fbxl3(Afh) dosage. Although both CRY proteins slowed the clock, CRY1 was significantly more potent than CRY2, and in SCN slices, CRY1 but not CRY2 prolonged the interval of transcriptional suppression. Selective CRY-stabilization demonstrated that both CRYs are endogenous transcriptional repressors of clock-controlled genes, but again CRY1 was preeminent. Finally, although Cry1(-/-);Cry2(-/-) mice were behaviorally arrhythmic, their SCN expressed short period (~18 h) rhythms with variable stability. Fbxl3(Afh/Afh) had no effect on these CRY-independent rhythms, confirming its circadian action is mediated exclusively via CRYs. Thus, stabilization of both CRY1 and CRY2 are necessary and sufficient to explain circadian period lengthening by Fbxl3(Afh/Afh). Both CRY proteins dose-dependently lengthen the intrinsic, high-frequency SCN rhythm, and CRY2 also attenuates the more potent period-lengthening effects of CRY1. Incorporation of CRY-mediated transcriptional feedback thus confers stability to intrinsic SCN oscillations, establishing periods between 18 and 29 h, as determined by selective contributions of CRY1 and CRY2.

Sleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1] and often precedes related symptoms [2]. However, mechanistic basis for this association remains unknown. Therefore, we investigated the circadian phenotype of blind-drunk (Bdr), a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes modulated by prenatal factors and reversible by antipsychotic treatment [3, 4]. Notably, SNAP-25 has been implicated in schizophrenia from genetic [5-8], pathological [9-13], and functional studies [14-16]. We show here that the rest and activity rhythms of Bdr mice are phase advanced and fragmented under a light/dark cycle, reminiscent of the disturbed sleep patterns observed in schizophrenia. Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of mammals, coordinating daily rhythms of behavior and metabolism. Circadian timekeeping in SCN neurons revolves around transcriptional/posttranslational feedback loops, in which Period (Per) and Cryptochrome (Cry) genes are negatively regulated by their protein products. Recent studies have revealed, however, that these "core loops" also rely upon cytosolic and circuit-level properties for sustained oscillation. To characterize interneuronal signals responsible for robust pacemaking in SCN cells and circuits, we have developed a unique coculture technique using wild-type (WT) "graft" SCN to drive pacemaking (reported by PER2::LUCIFERASE bioluminescence) in "host" SCN deficient either in elements of neuropeptidergic signaling or in elements of the core feedback loop. We demonstrate that paracrine signaling is sufficient to restore cellular synchrony and amplitude of pacemaking in SCN circuits lacking vasoactive intestinal peptide (VIP). By using grafts with mutant circadian periods we show that pacemaking in the host SCN is specified by the genotype of the graft, confirming graft-derived factors as determinants of the host rhythm. By combining pharmacological with genetic manipulations, we show that a hierarchy of neuropeptidergic signals underpins this paracrine regulation, with a preeminent role for VIP augmented by contributions from arginine vasopressin (AVP) and gastrin-releasing peptide (GRP). Finally, we show that interneuronal signaling is sufficiently powerful to maintain circadian pacemaking in arrhythmic Cry-null SCN, deficient in essential elements of the transcriptional negative feedback loops. Thus, a hierarchy of paracrine neuropeptidergic signals determines cell- and circuit-level circadian pacemaking in the SCN.

Circadian pacemaking in suprachiasmatic nucleus (SCN) neurons revolves around transcriptional/posttranslational feedback loops, driven by protein products of "clock" genes. These loops are synchronized and sustained by intercellular signaling, involving vasoactive intestinal peptide (VIP) via its VPAC2 receptor, which positively regulates cAMP synthesis. In turn, SCN cells communicate circadian time to the brain via a daily rhythm in electrophysiological activity. To investigate the mechanisms whereby VIP/VPAC2/cAMP signaling controls SCN molecular and electrical pacemaking, we combined bioluminescent imaging of circadian gene expression and whole-cell electrophysiology in organotypic SCN slices. As a potential direct target of cAMP, we focused on hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Mutations of VIP-ergic signaling compromised the SCN molecular pacemaker, diminishing the amplitude and intercellular synchrony of circadian gene expression. These deficits were transiently reversed by elevation of cAMP. Similarly, cellular synchrony in electrical firing rates was lost in SCN slices lacking the VPAC2 receptor for VIP. Whole-cell current-clamp recordings in wild-type (WT) slices revealed voltage responses shaped by the conductance I(h), which is mediated by HCN channel activity. The influence of I(h) on voltage responses showed a modest peak in early circadian day, identifying HCN channels as a putative mediator of cAMP-dependent circadian effects on firing rate. I(h), however, was unaffected by loss of VIP-ergic signaling in VPAC2-null slices, and inhibition of cAMP synthesis had no discernible effect on I(h) but did suppress gene expression and SCN firing rates. Moreover, only sustained but not acute, pharmacological blockade of HCN channels reduced action potential (AP) firing. Thus, our evidence suggests that in the SCN, cAMP-mediated signaling is not a principal regulator of HCN channel function and that HCN is not a determinant of AP firing rate. VIP/cAMP-dependent signaling sustains the SCN molecular oscillator and action potential firing via mechanisms yet to be identified.

The central circadian pacemaker of the suprachiasmatic nucleus (SCN) is characterized as a series of transcriptional/posttranslational feedback loops. How this molecular mechanism coordinates daily rhythms in the SCN and hence the organism is poorly understood. We conducted the first systematic exploration of the "circadian intracellular proteome" of the SCN and revealed that approximately 13% of soluble proteins are subject to circadian regulation. Many of these proteins have underlying nonrhythmic mRNAs, so they have not previously been noted as circadian. Circadian proteins of the SCN include rate-limiting factors in metabolism, protein trafficking, and, intriguingly, synaptic vesicle recycling. We investigated the role of this clock-regulated pathway by treating organotypic cultures of SCN with botulinum toxin A or dynasore to block exocytosis and endocytosis. These manipulations of synaptic vesicle recycling compromised circadian gene expression, both across the SCN as a circuit and within individual SCN neurons. These findings reveal how basic cellular processes within the SCN are subject to circadian regulation and how disruption of these processes interferes with SCN cellular pacemaking. Specifically, we highlight synaptic vesicle cycling as a novel point of clock cell regulation in mammals.

The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3',5'-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

The secretion of hormones is temporally precise and periodic, oscillating over hours, days, and months. The circadian timekeeper within the suprachiasmatic nuclei (SCN) is central to this coordination, modulating the frequency of pulsatile release, maintaining daily cycles of secretion, and defining the time base for longer-term rhythms. This central clock is driven by cell-autonomous, transcriptional/posttranslational feedback loops incorporating Period (Per) and other clock genes. SCN neurons exist, however, within neural circuits, and an unresolved question is how SCN clock cells interact. By monitoring the SCN molecular clockwork using fluorescence and bioluminescence videomicroscopy of organotypic slices from mPer1::GFP and mPer1::luciferase transgenic mice, we show that interneuronal neuropeptidergic signaling via the vasoactive intestinal peptide (VIP)/PACAP2 (VPAC2) receptor for VIP (an abundant SCN neuropeptide) is necessary to maintain both the amplitude and the synchrony of clock cells in the SCN. Acute induction of mPer1 by light is, however, independent of VIP/VPAC2 signaling, demonstrating dissociation between cellular mechanisms mediating circadian control of the clockwork and those mediating its retinally dependent entrainment to the light/dark cycle. The latter likely involves the Ca(2+)/cAMP response elements of mPer genes, triggered by a MAPK cascade activated by retinal afferents to the SCN. In the absence of VPAC2 signaling, however, this cascade is inappropriately responsive to light during circadian daytime. Hence VPAC2-mediated signaling sustains the SCN cellular clockwork and is necessary both for interneuronal synchronization and appropriate entrainment to the light/dark cycle. In its absence, behavioral and endocrine rhythms are severely compromised.

The suprachiasmatic nucleus (SCN), the brain's principal circadian pacemaker, coordinates adaptive daily cycles of behavior and physiology, including the rhythm of sleep and wakefulness. The cellular mechanism sustaining SCN circadian timing is well characterized, but the neurochemical pathways by which SCN neurons coordinate circadian behaviors remain unknown. SCN transplant studies suggest a role for (unidentified) secreted factors, and one potential candidate is the SCN neuropeptide prokineticin 2 (Prok2). Prok2 and its cognate prokineticin receptor 2 (Prokr2/Gpcr73l1) are widely expressed in both the SCN and its neural targets, and Prok2 is light-regulated. Hence, they may contribute to cellular timing within the SCN, entrainment of the clock, and/or they may mediate circadian output. We show that a targeted null mutation of Prokr2 disrupts circadian coordination of the activity cycle and thermoregulation. Specifically, mice lacking Prokr2 lost precision in timing the onset of nocturnal locomotor activity; and under both a light/dark cycle and continuous darkness, there was a pronounced temporal redistribution of activity away from early to late circadian night. Moreover, the coherence of circadian behavior was significantly reduced, and nocturnal body temperature was depressed. Entrainment by light is not, however, dependent on Prokr2, and bioluminescence real-time imaging of organotypical SCN slices showed that the mutant SCN is fully competent as a circadian oscillator. We conclude that Prokr2 is not necessary for SCN cellular timekeeping or entrainment, but it is an essential link for coordination of circadian behavior and physiology by the SCN, especially in defining the onset and maintenance of circadian night.