SummaryThe 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.

By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

The mammalian circadian clockwork is modelled as transcriptional and post-translational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3′5′ monophosphate (cAMP) signalling constitutes an additional, bona fide component of the oscillatory network. cAMP signalling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase and period. This role is general, being evident in peripheral mammalian tissues and cell lines, thus revealing an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signalling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles. ReportThe suprachiasmatic nuclei (SCN) of the hypothalamus are the principal circadian pacemaker in mammals, driving the sleep-wake cycle and co-ordinating subordinate clocks in other tissues(1). Disturbed circadian timing can have a major negative impact on human health(2). The molecular clockwork within the SCN has been modelled as a combination of transcriptional and post-translational negative feedback loops(3), whereby protein products of Period and Cryptochrome genes periodically suppress their own expression(4). It is unclear how long-term, high amplitude oscillations with a daily period are maintained, not least because transcriptional feedback loops are typically less precise than the oscillation of the circadian clock and oscillate at a higher frequency than one cycle per day(5). Moreover, recombinant cyanobacterial proteins can sustain circadian cycles of auto-phosphorylation in vitro, in the absence of transcription(6) and intracellular signalling molecules cADPR and Ca 2+ are essential regulators of circadian oscillation in Arabidopsis and Drosophila (7,8), indicating that transcriptional mechanisms may not be the sole, nor principal, arbiter of circadian pacemaking(9, 10). We show that the transcriptional feedback loops of the SCN are sustained by cytoplasmic adenosine 3′5′ monophosphate (cAMP) signalling, which determines their canonical properties of amplitude, phase and period. This extends the

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::LUCIFER-ASE 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.T he circadian pacemaker of the suprachiasmatic nucleus (SCN) directs, on a daily basis, the most fundamental processes of physiology and metabolism, sleep and wakefulness (1). The established molecular model of the SCN oscillator involves interlocked transcriptional/posttranslational negative feedback loops, centered on rhythmic expression of the transcriptional inhibitors Period (Per) and Cryptochrome (Cry), driven by the positive activators Clock and Bmal1. The period of the daily oscillation is determined by the transcriptional activity of Clock (1) and by the stability of Per and Cry proteins (2, 3). More recently the model of SCN neurons as autonomous transcriptional/translational feedback oscillators has been reappraised. Rhythmic cytosolic signaling pathways, particularly cAMP and Ca 2+ , not only are driven by the core feedback loops, but also support them in a mutually dependent fashion (4, 5). Furthermore, within SCN circuits, loss of signaling by the neuropeptide vasoactive intestinal peptide (VIP) through its VPAC2 receptor (a positive regulator of cAMP) both desynchronizes and damps cellular transcriptional pacemaking (6, 7). Conversely, intercellular coupling maintains robustness within SCN cells deficie...

Circadian (~24h) rhythms depend on intra-cellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multi-cellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this gene-to-cell-to-circuit-to-behavior pathway 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. These results provide a first demonstration that astrocytes can autonomously initiate and sustain complex mammalian behavior.

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2 −/− mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2 −/− mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.circadian clock | suprachiasmatic nucleus | period protein | Tau mutation | pacemaking

Summary In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal ∼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.