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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. The suprachiasmatic nucleus (SCN) synchronises daily rhythms of behaviour and physiology to the light-dark cycle. Vasoactive intestinal peptide (VIP) is important for mediating SCN entrainment; however, the underlying mechanisms are incompletely understood. Here, the authors show that the effects of VIP on the SCN are mediated by ERK1/2 and DUSP4.

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock geneBmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input.We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAAsignaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

Socially contagious itch is ubiquitous in human society, but whether it exists in rodents is unclear. Using a behavioral paradigm that does not entail prior training or reward, we found that mice scratched after observing a conspecific scratching. Molecular mapping showed increased neuronal activity in the suprachiasmatic nucleus (SCN) of the hypothalamus of mice that displayed contagious scratching. Ablation of gastrin-releasing peptide receptor (GRPR) or GRPR neurons in the SCN abolished contagious scratching behavior, which was recapitulated by chemogenetic inhibition of SCN GRP neurons. Activation of SCN GRP/GRPR neurons evoked scratching behavior. These data demonstrate that GRP-GRPR signaling is necessary and sufficient for transmitting contagious itch information in the SCN. The findings may have implications for our understanding of neural circuits that control socially contagious behaviors.

The suprachiasmatic nucleus (SCN) serves as the body’s master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day–night cycle 1 – 4 . For example, the SCN opposes overnight adipsia by driving water intake before sleep 5 , 6 , and by driving the secretion of anti-diuretic hormone 7 , 8 and lowering body temperature 9 , 10 to reduce water loss during sleep 11 . These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase 12 – 16 . However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19—a time at which SCN VP (vasopressin) neurons are inactive—excited SCN VP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCN VP neurons and mimicked by optogenetic stimulation of SCN VP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLT GAD ) relay this information to SCN VP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLT GAD neuron axon terminals excited SCN VP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLT GAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLT GAD → SCN VP pathway and was prevented by chemogenetic inhibition of OVLT GAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks. The authors demonstrate that clock time can be regulated by non-photic physiologically relevant cues and that such cues can drive unscheduled homeostatic responses via clock-output networks.

Each day, over 50 billion synaptic signals, mediated by the neurotransmitter GABA, are sent between neurons in the central circadian pacemaker in the mammalian brain to time and coordinate daily events. Although GABA is the only signaling molecule sent and received by most, if not all of these neurons, its role is not well understood. Past studies have shown paradoxically that GABA can synchronize and desynchronize, as well as excite and inhibit, clock neurons. Through experiments and modeling characterizing the role of GABA in timekeeping, we propose the existence of two types of differentially regulated GABA signaling—fast signaling that regulates neuronal output, and slow signaling that modulates synchrony between neurons—a hypothesis that can explain many previous experimental results. The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by—and GABA-inhibited neurons repelled from—this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function.

Background: Signaling pathways like those depending on cAMP/PKA, calcium/calmodulin/CaMK, MEK-1/MAPK or PI3K/Akt have been described to modulate suprachiasmatic nucleus (SCN) neuronal signaling via influencing transcription factors like CREB. Here, we analyzed the effect of cyclic nucleotide phosphodiesterase inhibitors and structurally similar substances commonly used as autophagy modulators on a cell line stably expressing a cyclic nucleotide element-driven luciferase reporter. Methods: We used an SCN cell line stably transfected with a CRE-luciferase reporter (SCNCRE) to evaluate signaling and vitality responses to various isoform-selective PDE inhibitors and autophagy modulators to evaluate the mechanism of action of the latter. Results: In this study the different impacts of common PDE inhibitors and autophagy modulators on CRE-luciferase activity applied alone and in combination with known CRE-luciferase activating agents showed that (1) PDE3, 4 and 5 are present in SCNCRE cells, with (2) PDE3 being the most active and (3) the autophagy inhibitor 3-Methyladenin (3-MA) displaying PDE inhibitor-like behavior. Conclusions: Experiments provide evidence that, in addition to the extracellular signaling pathways components shown before to be involved in CRE-luciferase activity regulation like cAMP analogs, adenylate cyclase activators and beta-adrenoceptor agonists, cyclic nucleotide metabolism as realized by phosphodiesterase activity, or molecule/agents influencing processes like autophagy or inflammation, modulate transcriptional CRE-dependent activity in these cells. Specifically, we provide evidence that the autophagy inhibitor 3-MA, given that PDEs are expressed, may also act as a PDE inhibitor and inducer of CRE-mediated transcriptional activity.

Lithium salt has been widely used in treatment of Bipolar Disorder, a mental disturbance associated with circadian rhythm disruptions. Lithium mildly but consistently lengthens circadian period of behavioural rhythms in multiple organisms. To systematically address the impacts of lithium on circadian pacemaking and the underlying mechanisms, we measured locomotor activity in mice in vivo following chronic lithium treatment, and also tracked clock protein dynamics (PER2::Luciferase) in vitro in lithium-treated tissue slices/cells. Lithium lengthens period of both the locomotor activity rhythms, as well as the molecular oscillations in the suprachiasmatic nucleus, lung tissues and fibroblast cells. In addition, we also identified significantly elevated PER2::LUC expression and oscillation amplitude in both central and peripheral pacemakers. Elevation of PER2::LUC by lithium was not associated with changes in protein stabilities of PER2, but instead with increased transcription of Per2 gene. Although lithium and GSK3 inhibition showed opposing effects on clock period, they acted in a similar fashion to up-regulate PER2 expression and oscillation amplitude. Collectively, our data have identified a novel amplitude-enhancing effect of lithium on the PER2 protein rhythms in the central and peripheral circadian clockwork, which may involve a GSK3-mediated signalling pathway. These findings may advance our understanding of the therapeutic actions of lithium in Bipolar Disorder or other psychiatric diseases that involve circadian rhythm disruptions.

Although inhalational general anesthesia affects mammalian circadian rhythms, which are generated through oscillations in expression of clock genes in the suprachiasmatic nucleus (SCN), the impact of intravenous benzodiazepine (BDZ)-induced general anesthesia is not well understood. We investigated the effects of remimazolam (RMZ), an ultra-short-acting BDZ that has recently gained widespread use in clinical anesthesia, on circadian rhythms and clock gene expression in the SCN in male C57BL/6J mice. Phase shifts in behavioral rest-activity circadian rhythms were quantitatively measured under constant dark conditions. No significant differences were detected among the interventions tested: continuous RMZ injection, bolus RMZ injection, and saline injection at different circadian times. Analyses of day/night activity distribution and the percentage of mice in inactivity bouts showed no significant differences among these groups. To assess the effects of RMZ on clock gene expression, we used continuous RMZ injection, which better reflects clinical anesthesia conditions. Quantitative real-time polymerase chain reaction revealed that the mRNA expression levels of major clock genes Per2 , Bmal1 , Cry1 , and Npas2 in the SCN were decreased, whereas the neuronal activity marker Egr1 was increased. The period of circadian oscillations in clock gene Per2 expression–analyzed by bioluminescence rhythms of cultured SCN tissue from PER2::LUC mice–did not change with any concentration of RMZ administration. Our findings show that RMZ has minimal effects on circadian rhythms under our experimental protocols, suggesting that RMZ has the potential to reduce complications related to circadian rhythm disruption by general anesthesia.

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.

Ultradian (∼4 hr) rhythms in locomotor activity that do not depend on the master circadian pacemaker in the suprachiasmatic nucleus have been observed across mammalian species, however, the underlying mechanisms driving these rhythms are unknown. We show that disruption of the dopamine transporter gene lengthens the period of ultradian locomotor rhythms in mice. Period lengthening also results from chemogenetic activation of midbrain dopamine neurons and psychostimulant treatment, while the antipsychotic haloperidol has the opposite effect. We further reveal that striatal dopamine levels fluctuate in synchrony with ultradian activity cycles and that dopaminergic tone strongly predicts ultradian period. Our data indicate that an arousal regulating, dopaminergic ultradian oscillator (DUO) operates in the mammalian brain, which normally cycles in harmony with the circadian clock, but can desynchronize when dopamine tone is elevated, thereby producing aberrant patterns of arousal which are strikingly similar to perturbed sleep-wake cycles comorbid with psychopathology. The sleep-wake cycle of mammals is controlled by a ‘circadian clock’ within the brain, which is synchronized to the day–night cycle. However, other aspects of mammalian physiology including alertness and activity levels, as well as appetite and body temperature—fluctuate in cycles that repeat every few hours. These cycles are known as ultradian rhythms, and they may offer survival benefits by enabling potentially risky behaviors, such as foraging, to be coordinated between members of a group. Despite their widespread nature and the fact that they appear to be conserved in evolution, virtually nothing is known about the molecular basis of ultradian rhythms. Blum et al. have now identified a second internal clock within the brain, which they name ‘the DUO’, and shown that this clock normally works in concert with the circadian clock to regulate daily patterns of activity and alertness. Experiments in mice revealed that the DUO uses the brain chemical dopamine to generate bursts of activity roughly every four hours. Moreover, it continues to work when the circadian clock has been destroyed. Measurements of dopamine in freely moving mice showed that levels of the chemical fluctuate in synchrony with the animals' activity levels. Moreover, drugs that flood the brain with dopamine, such as methamphetamine, disrupt the 4-hour cycle by lengthening the period between bursts of activity, whereas drugs that block dopamine receptors have the opposite effect. As well as revealing a mechanism by which the brain coordinates processes that repeat several times per day, the identification of the DUO could also provide insights into the biological basis of psychiatric disorders. Conditions such as schizophrenia and bipolar disorder are often accompanied by disturbances in patterns of activity and rest. While these have previously been attributed to the disruption of circadian rhythms, there is little direct evidence for this, which raises the possibility that these changes might instead reflect the disruption of ultradian rhythms.