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The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca 2+ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca 2+ -dependent kinase regulates the clock gene transcription. However, the Ca 2+ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca 2+ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca 2+ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca 2+ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca 2+ rhythms, whereas blocking of Ca 2+ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP 3 ) receptors had a minor effect on either Ca 2+ rhythms. We conclude that the in-phasic circadian Ca 2+ rhythms in the cytosol and nucleus are mainly driven by Ca 2+ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca 2+ rhythms directly regulate transcription in situ.

Glucose is a significant energy resource for maintaining physiological activities, including body temperature homeostasis, and glucose homeostasis is tightly regulated in mammals. Although ambient temperature tunes glucose metabolism to maintain euthermia, the significance of body temperature in metabolic regulation remains unclear owing to strict thermoregulation. Activation of Qrfp neurons in the preoptic area induced a harmless hypothermic state known as Q-neuron–induced hypothermia and hypometabolism (QIH), which is suitable for studying glucose metabolism under hypothermia. In this study, we observed that QIH mice had hyperinsulinemia and insulin resistance. This glucose hypometabolic state was abolished by increasing the body temperature to euthermia. Moreover, QIH-mediated inappetence and locomotor inactivity were recovered in euthermia QIH mice. These results indicate that body temperature is considerably more powerful than ambient temperature in regulating glucose metabolism and behavior, and the glucose hypometabolism in QIH is secondary to hypothermia rather than modulated by Qrfp neurons. QIH is a hibernation-like state characterized by hypothermia and hypometabolism, yet its glucose metabolic profile remains unclear. In this study, the authors used the QIH model and discovered that a diabetes-like state is induced by hypothermia.

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABAA receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that causes cognitive decline. Uncovering the mechanisms of neurodegeneration in the early stages is essential to establish a treatment for AD. Recent research has proposed the hypothesis that amyloid-β (Aβ) oligomers elicit an excessive glutamate release from astrocytes toward synapses through intracellular free Ca 2+ ([Ca 2+ ] i ) elevations in astrocytes, finally resulting in neuronal dendritic spine loss. Under physiological conditions, astrocytic [Ca 2+ ] i elevations range spatially from microdomains to network-wide propagation and temporally from milliseconds to tens of seconds. Astrocytic localized and fast [Ca 2+ ] i elevations might correlate with glutamate release; however, the Aβ-induced alteration of localized, fast astrocytic [Ca 2+ ] i elevations remains unexplored. In this study, we quantitatively investigated the Aβ dimers-induced changes in the spatial and temporal patterns of [Ca 2+ ] i in a primary culture of astrocytes by two-photon excitation spinning-disk confocal microscopy. The frequency of fast [Ca 2+ ] i elevations occurring locally in astrocytes (≤ 0.5 s, ≤ 35 µm 2 ) and [Ca 2+ ] i event occupancy relative to cell area significantly increased after exposure to Aβ dimers. The effect of Aβ dimers appeared above 500 nM, and these Aβ dimers-induced [Ca 2+ ] i elevations were primarily mediated by a metabotropic purinergic receptor (P2Y1 receptor) and Ca 2+ release from the endoplasmic reticulum. Our findings suggest that the Aβ dimers-induced alterations and hyperactivation of astrocytic [Ca 2+ ] i is a candidate cellular mechanism in the early stages of AD.

In mammals, the master circadian clock is located in the suprachiasmatic nucleus (SCN), where most neurons show circadian rhythms of intracellular Ca 2+ levels. However, the origin of these Ca 2+ rhythms remains largely unknown. In this study, we successfully monitored the intracellular circadian Ca 2+ rhythms together with the circadian PER2 and firing rhythms in a single SCN slice ex vivo , which enabled us to explore the origins. The phase relation between the circadian PER2 and Ca 2+ rhythms, but not between the circadian PER2 and firing rhythms, was significantly altered in Cry1 / Cry2 double knockout mice, which display a loss of intercellular synchronization in the SCN. In addition, in Cry1 / Cry2 double knockout mice, circadian Ca 2+ rhythms were abolished in the dorsolateral SCN, but were maintained in the majority of the ventromedial SCN. These findings indicate that intracellular circadian Ca 2+ rhythms are composed of an exogenous and endogenous component involving PER2 expression.

The circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multioscillator system in which neuronal networks play crucial roles in expressing coherent rhythms in physiology and behavior. However, our understanding of the neuronal network is still incomplete. Intracellular calcium mediates the input signals, such as phase-resetting stimuli, to the core molecular loop involving clock genes for circadian rhythm generation and the output signals from the loop to various cellular functions, including changes in neurotransmitter release. Using a unique large-scale calcium imaging method with genetically encoded calcium sensors, we visualized intracellular calcium from the entire surface of SCN slice in culture including the regions where autonomous clock gene expression was undetectable. We found circadian calcium rhythms at a single-cell level in the SCN, which were topologically specific with a larger amplitude and more delayed phase in the ventral region than the dorsal. The robustness of the rhythm was reduced but persisted even after blocking the neuronal firing with tetrodotoxin (TTX). Notably, TTX dissociated the circadian calcium rhythms between the dorsal and ventral SCN. In contrast, a blocker of gap junctions, carbenoxolone, had only a minor effect on the calcium rhythms at both the single-cell and network levels. These results reveal the topological specificity of the circadian calcium rhythm in the SCN and the presence of coupled regional pacemakers in the dorsal and ventral regions. Neuronal firings are not necessary for the persistence of the calcium rhythms but indispensable for the hierarchical organization of rhythmicity in the SCN.

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 μm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 μm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Circadian rhythms in Per1 , PER2 expression and intracellular Ca 2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100–200 μm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca 2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca 2+ under these conditions.

The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca -dependent kinase regulates the clock gene transcription. However, the Ca regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca rhythms remain unclear. In the present study, we monitored circadian-timescale Ca dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca rhythms, whereas blocking of Ca release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP ) receptors had a minor effect on either Ca rhythms. We conclude that the in-phasic circadian Ca rhythms in the cytosol and nucleus are mainly driven by Ca influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca rhythms directly regulate transcription