Electroacupuncture alleviates acute gouty arthritis by inhibiting NLRP3 inflammasome activation via modulation of the circadian-inflammation axis.

The hypothalamus is a key structure in the human brain, comprising numerous functionally distinct subnuclei that regulate critical physiological processes such as energy balance, stress response, and circadian rhythms. Due to the complexity and functional diversity of its subregions, precise segmentation is essential for elucidating its operational mechanisms. This review systematically summarizes hypothalamic segmentation methods and their applications in physiological and clinical research. Current approaches are categorized into two complementary types: anatomy-based manual segmentation and deep learning-based fully automated segmentation. The former provides a gold standard for algorithm validation through expert knowledge, enabling accurate identification of key functional subregions; the latter offers an efficient solution for large-scale studies, facilitating in-depth exploration of the hypothalamus's heterogeneous functional architecture. The review also highlights major challenges in the field, including the lack of unified segmentation protocols-which hinders cross-study comparability-and a significant methodological gap in pediatric population studies. Moving forward, it is crucial to establish standardized segmentation workflows, reduce subjective bias, improve reproducibility, and address the technical shortcomings in hypothalamic segmentation for children, thereby laying a foundation for comprehensively understanding the structure and function of this critical brain region.ABBREVIATIONS: ARC＝ arcuate nucleus; PVN＝ paraventricular nucleus; VM＝ ventromedial nucleus; DM＝ dorsomedial hypothalamic nucleus; SCh＝ suprachiasmatic nucleus; LH＝ lateral hypothalamus.

Dexamethasone affects the circadian clock in the fetal mouse suprachiasmatic nucleus at the transcriptional level.

Sleep amount and quality are important to maintain health, light exposure transported to the pineal gland via suprachiasmatic nucleus pineal pathway, and this affect melatonin synthesis. Melatonin exerts its action by receptors called MT1, MT2 that found in peripheral tissues including pancreas, which affect its endocrine function. In this study we investigate the effect of type of sleep disturbance on the histological structure and the expression of melatonin receptor (MTNR1B) in the pancreas. A sample of 45 adult healthy male rats had free access to water and feeding, divided into 3 groups (15 in each group): the control group had normal 24 h diurnal variation, group A subjected to interruption of sleep by light exposure for 2 h at three intervals, and group B subjected to a reduction in sleep duration by 7 h. This experiment continued for 14 days, animals were scarified by euthanasia by cervical dislocation, pancreatic tissue prepared for paraffin blocks and stained by H&E, and IHC for MTNR1B. Histomorhometric analysis done by image J (V 1.54), immunohistochemical assessment done by Aprioscope image analysis software 12.4.6.5003) mean total positivity of expression was selected, statistical analysis done by SSPS (25 V) and Tukeyꞌs test. Histological evaluation revealed changes in sleep disturbance groups including fat deposition, vascular dilatation, and apoptotic changes for group of cells within islets of Langerhans which showed significant reduction in their count in sleep disturbance groups compared to control, and non-significant change in their area across the groups, but significant reduction in area in sleep deprived group compared to sleep interruption. Immunohistochemical expression of MTNR1B showed it only expressed within the area of islets of Langerhans, with non-significant increase in expression in sleep disturbed groups, being more in sleep deprived group. This study showed that the pancreas is affected by sleep disturbance patterns, mainly its endocrine part (islets of Langerhans), with study expression of melatonin receptor within them. This may suggest a role of melatonin in maintaining pancreatic ꞵ cells.

Sleep disturbance is a prevalent yet poorly understood comorbidity in autism spectrum disorders (ASD). Here, we uncover a bidirectional regulatory axis connecting the ASD risk gene POGZ to core circadian mechanisms. We demonstrate that Pogz is widely expressed in the suprachiasmatic nucleus (SCN), the central pacemaker of the circadian rhythms, and exhibits circadian oscillations in both the hypothalamus and liver, with its transcription directly regulated by the circadian molecule DBP through a D-box element in its proximal enhancer. Pogz-deficient mice exhibited prolonged circadian periodicity, impaired light-induced phase shift, delayed adaption to an 8-hour advance jet-lag, and reduced SCN c-Fos activation in response to light pulses. Mechanistically, POGZ interacts with and enhances the transcription activity of CREB, a key regulator of light-induced phase resetting. Notably, Pogz deletion leads to ASD-related deficits in social novelty and cognition, with cognitive impairments influenced by both photoperiod and behavioral paradigm. Our findings, thus, reveal a critical, previously unrecognized intersection between an ASD risk gene and circadian clock, offering insights into the pathogenesis of core ASD symptoms and comorbid sleep disturbances.

The phenomenon of splitting was originally observed in hamsters which, after prolonged exposure to constant light, exhibit two rest/wake cycles within a subjective day. Splitting is a consequence of the left and right suprachiasmatic nuclei (SCN) falling out of synchrony. While it is known that split activity is characterized by an antiphase relationship between the left and right SCN and between the core and shell within each hemisphere, the role of the commissural projections that connect the right and left SCN is not known. In the present study, we investigate the impact of the inter-hemispheric connections on the split and unsplit dynamics of a computational model of the bilateral SCN. Our model has 4 nodes corresponding to each right and left core and shell. We simulated our bilateral model under different lighting conditions and measured its period and the phase relationships among the 4 nodes. To further characterize the dynamics of the system, we performed a bifurcation analysis. We found that the bilateral model automatically splits unless entrained by bright light/dark cycles, or unless it has excitatory inter-hemispheric connections. This suggests that excitatory cross-connections may be important for freerunning behavior. We found that constant light of varying intensities transitions the model between split and unsplit activity only in very limited conditions, but the strength and polarity of the contralateral connections play a much greater role in this dynamical transition. These findings suggest that splitting may involve plasticity of the inter-hemispheric connections of the SCN.

Disturbance of circadian rhythms is a hallmark of substance use disorders, with depressant drugs often causing soporific effects such as reduced sleep latency. The suprachiasmatic nucleus (SCN) of the hypothalamus is the central circadian pacemaker in mammals, regulating daily rhythms in physiology and behavior. However, the cellular mechanisms through which depressants alter SCN function remain poorly defined. We used whole-cell patch clamp electrophysiology in acute brain slices to examine how alcohol and opioids modulate excitatory glutamatergic transmission onto SCN neurons. Ethanol effects were examined both acutely and following chronic exposure paradigms. Optogenetic stimulation was used to activate either RHT input or μ-opioid receptor-expressing (MOR⁺) terminals, and MOR agonists were used to assess opioid-mediated effects on synaptic transmission. We show that acute application of ethanol paradoxically enhances SCN firing rates. In contrast, chronic alcohol exposure reduces glutamatergic drive. We also found that activating MOR + terminals produced bidirectional modulation of SCN firing and that MOR+ inputs formed functional glutamatergic synapses onto SCN neurons. Notably, this transmission could be suppressed by the MOR agonists DAMGO and fentanyl. Together, these findings reveal that both alcohol and opioids modulate glutamatergic input to the SCN. This work establishes the SCN as a novel target of depressant substances and highlights glutamatergic transmission as a key point of vulnerability in circadian dysregulation associated with substance use.

When nocturnal rodents are subjected to daytime restricted feeding, in which food is only available for a few hours per day, they typically become active a few hours before the onset of the scheduled mealtime. This so-called food-anticipatory activity (FAA) is controlled by an autonomous circadian pacemaker, which is independent from the central circadian pacemaker in the suprachiasmatic nucleus (SCN). Fred Stephan named this pacemaker the food-entrainable oscillator (FEO) because FAA re-entrains to a shifted feeding schedule. We recently developed a method to measure food-seeking nose-poking behavior by an operant feeding device and found that anticipatory food-seeking nose-poking for scheduled daily food availability shifts in parallel with phase-shifted environmental light-dark cycles, raising the possibility that anticipatory food-seeking behavior is controlled by an oscillator entrained to the environmental light-dark cycle. With this possible light-entrainability of the FEO, we revisited Stephan's historical experiment-testing whether the FEO entrains to feeding cycle in the absence of a light-dark cycle without functional SCN-using Period 1/2/3 triple knockout (KO) mice, in which the canonical circadian oscillators in the SCN and peripheral tissues are disabled. KO mice were subjected to restricted feeding under constant darkness. The food-seeking nose-poking activity of a subset of the KO mice indeed occasionally entrained to the feeding cycle and re-entrained to a shifted feeding cycle. Despite our previous study showing that anticipatory food-seeking behavior shifted with the environmental light-dark cycle, these data demonstrate that it can also entrain to the feeding cycle in the absence of an environmental light-dark cycle, supporting Stephan's observation that the FEO is indeed food-entrainable.

Circadian function in multicellular organisms arises from coordinated interactions amongst diverse cellular tissue populations. Existing approaches for long-term imaging of within-tissue circadian regulation remain low-throughput, highly specialized, and largely inaccessible. Here, we developed ClockCyte, a high-content fluorescent live-imaging platform that enables continuous monitoring of circadian rhythms in up to 144 brain tissue samples. Using the mouse suprachiasmatic nucleus as a model, ClockCyte captures the differential circadian tissue regulation of neurons and astrocytes. We further identified a previously uncharacterized oscillatory circadian compartment in axonal calcium, showing highly homogeneous activity, opposed to waves of intracellular neuronal calcium. By deleting Bmal1 in neurons, we reveal the network underpinnings connecting clock gene expression to network-wide axonal regulation. The discovery of distinct circadian properties of axonal calcium and their disruption by Bmal1 ablation highlights the potential to reveal new principles of intra-tissue network-level circadian organization. More broadly, this approach will enable systematic explorations of how cell-type-specific and compartmentalized subcellular rhythms contribute to brain physiology.

The suprachiasmatic nucleus regulates circadian rhythms and influences physiological and behavioural functions. Clock genes not only play a critical role in orchestrating circadian rhythms, but also regulate a variety of bodily functions. While Bmal1, a clock gene, is vital for maintaining optimal circadian rhythms, its therapeutic potential in type 2 diabetes remains unexplored. In this study, db/db mice, a well-established model of type 2 diabetes exhibiting arrhythmic behaviour and complications, were injected stereotaxically with AAV-Bmal1 or a control virus into the suprachiasmatic nucleus to evaluate the protective effects of Bmal1 overexpression on neurovascular deficits of type 2 diabetes. Given the complex neurovascular network and the eye's unique accessibility as a transparent system, ocular complications were selected as a model to examine the neuronal functional, behavioural and vascular benefits of central overexpression of Bmal1. Bmal1 overexpression decreased the free-running period, which otherwise is lengthened in db/db mice. Retinal neuronal function was restored on the electroretinogram, along with optomotor behaviour and visual acuity enhancements. Retinal vascular deficits were also significantly reduced. Notably, Bmal1 overexpression decreased fat content in genetically predisposed obese db/db mice compared with the untreated db/db group. As the suprachiasmatic nucleus is known to regulate hepatic glucose production via sympathetic mechanisms, glycaemic control and pyruvate tolerance tests were evaluated. Glucose homeostasis was improved in Bmal1-overexpressing mice, accompanied by a significant reduction in hepatic gluconeogenesis. Plasma noradrenaline (norepinephrine) and liver tyrosine hydroxylase levels were reduced, indicating a protective regulation of adrenergic signalling. Our study highlights the therapeutic potential of central overexpression of a clock gene, Bmal1, to mitigate metabolic and neurovascular deficits of type 2 diabetes, offering a compelling framework for incorporating circadian rhythms into managing diabetes and its complications.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) influence visual system development via melanopsin before photoreceptor-mediated vision, but how melanopsin signaling contributes to ipRGC circuit assembly remains unknown. Here we show that melanopsin coordinates retinohypothalamic tract development by regulating local translation in developing ipRGC axons. Loss of melanopsin selectively disrupted local translation in axons without affecting somatic translation. The affected transcripts encoded cytoskeletal regulators, adhesion molecules, and trafficking proteins, and activity-dependent changes in translation were restricted to the period before eye-opening. Consistent with impaired axonal growth and synaptogenesis, Opn4 knockout mice showed reduced ipsilateral suprachiasmatic nucleus innervation and fewer retinohypothalamic synapses, while nanoscale synaptic molecular organization and microglial engulfment were unaffected. Reduced visual drive in Opn4 knockouts further altered developmental gene expression programs across the retina, suprachiasmatic nucleus, and lateral geniculate nucleus, with region-specific differences in expression timing. These findings identify melanopsin as a regulator of local axonal translation during early circuit development, linking sensory phototransduction to translational control mechanisms that guide retinohypothalamic tract assembly and postsynaptic target maturation.

Sundowning, a common yet poorly understood neuropsychiatric syndrome in Alzheimer's disease (AD), manifests as evening-specific increases in agitation, confusion, and anxiety. Despite its prevalence and contribution to patient distress, its neural mechanisms remain elusive. Here, we establish a preclinical model of sundowning by characterizing sleep-wake, behavioral, and network-level alterations in an AD mouse model. Aged AD mice exhibit disrupted sleep-wake patterns and reduced slow-wave sleep. Behavioral and pose-tracking analyses revealed motor agitation and a distinct sundowning-like behavioral fingerprint selectively at Sundown. The suprachiasmatic nucleus (SCN) showed disruptions in time-of-day-dependent activation of vasopressin-expressing cells and brain-wide activity-dependent tagging identified hyperconnectivity amongst sensorimotor regions in AD mice at Sundown. Resting-state fMRI data from the Alzheimer's Disease Neuroimaging Initiative revealed analogous Salience Network alterations in AD subjects. Overall, these cross-species findings define a systems-level framework for sundowning and highlight regions that may be targeted to alleviate a debilitating symptom of AD.

The human circadian rhythm is controlled by central and peripheral clocks, primarily by the central clock in the suprachiasmatic nucleus (SCN). We investigated the diurnal variation of basic metabolism in the human cerebrum by measuring human baseline cerebral activity at rest contrasted with the SCN baseline activity. To this end, we utilized magnetic resonance imaging perfusion data of cerebral blood flow (CBF; N = 27, including both sexes), where each participant was scanned four times a day at 6 h intervals (18:00, 24:00, 6:00, and 12:00 local time). Similarly to the SCN exhibiting higher CBF activity at noon, we observed a consistent temporal activity pattern in the brain regions, including the limbic (cingulate, insular, and temporopolar) and sensorimotor (visual and somatosensory/motor) areas. In contrast, the hippocampus showed higher activity at midnight and lower activity at noon. To examine the functional interaction between the SCN and the cerebral regions showing diurnal variation, we calculated the resting-state functional connectivity using the database of the Human Connectome Project (N = 164, including both sexes). Notably, the hippocampus demonstrated greater functional connectivity with the SCN than the other regions. These results suggest that cerebral regions exhibit differential patterns of diurnal variation associated with their functional connectivity with the SCN.

Spatial and single-cell transcriptomic atlas of human suprachiasmatic nucleus.

The circadian clock is an internal timekeeping system that enables organisms to adapt to daily environmental changes. A defining property of this clock is temperature compensation, whereby the circadian period remains relatively constant despite fluctuations in temperature. Although this phenomenon has been extensively studied in cultured cells and tissues, how the mammalian circadian clock responds to hypothermia in vivo remains largely unknown. Here, we examined circadian dynamics in a hibernation-like state in mice, termed Q neuron-induced hypometabolic and hypothermic state (QIH), which lowers core and brain temperatures to approximately 25 °C for extended periods. We found that free-running behavioral and body temperature rhythms were preserved after QIH, exhibiting only minor phase changes. In vivo recordings further revealed that neuronal firing rhythms in the suprachiasmatic nucleus (SCN) and molecular rhythms of PER2::Luc bioluminescence in peripheral tissues persisted during QIH with dampened amplitudes but largely unaltered circadian periods. In contrast, SCN and kidney slice cultures maintained at the same temperature displayed strongly attenuated or reset PER2::Luc oscillations. Together, these findings demonstrate that the circadian period is robustly temperature compensated in vivo, likely supported by systemic regulatory mechanisms beyond cell-autonomous clockwork. Our results provide new insight into the fundamental biology of circadian robustness and establish a framework for understanding clock function during hibernation and potential medical hypothermia.

The circadian clock system has been demonstrated to modulate immune function, thereby resulting in a pronounced diurnal rhythm in the host's response to infection. However, the role of the circadian clock throughout the entire course of sepsis remains unclear. This study aims to investigate whether the endogenous circadian clock mediates differential effects on sepsis at different stages of the disease. We induced sepsis in mice by intraperitoneal injection of lipopolysaccharide (LPS) during the rest phase (Zeitgeber Time 6, ZT6) and the active phase (ZT18). We found a significant time-dependent dual effect: Mice in the ZT18 group exhibited a more intense inflammatory response in the acute phase within 24 h, characterized by more severe hypothermia and elevated serum and tissue levels of tumor necrosis factor (TNF-α), leading to higher acute-phase mortality. However, upon extending the observation period to 5 days, it was observed that ZT18 group had a higher survival rate. The long-term survival advantage of the ZT18 group was associated with stronger metabolic resilience, characterized by faster recovery of the respiratory exchange ratio (RER) and energy expenditure, as well as earlier restoration of daily activity capacity. Lesioning the central clock, the suprachiasmatic nucleus (SCN), eliminated the diurnal differences in LPS-induced mortality and metabolic disturbances, indicating that the phenomenon is driven by the central clock. These findings provide a new theoretical framework for developing stage-specific chronotherapies for sepsis.

Genetic disorders affecting glucose transport and fasting as a trigger suggest that disruptions in brain glucose metabolism may contribute to migraine susceptibility. The activation of the peripheral sympathetic nervous system plays a critical role in responding to declines in blood sugar. Beyond its role in glucose regulation, sympathetic activity driven by locus coeruleus also influences astrocytes, the glial cells responsible for supporting neuronal function. During periods of fasting, stress, and sleep deprivation, sustained sympathetic activation from locus coeruleus may contribute to reduced availability of glycosyl units derived from glycogen in peri-synaptic astrocyte processes. This is attributed to transcriptional shifts favoring glycogen synthesis over utilization, in contrast to the transient low-level locus coeruleus activity that typically promotes glycogen breakdown. The metabolism of glycogen within astrocyte processes is tightly coupled to excitatory synaptic activity, helping to meet the high energetic demands of synaptic transmission, as well as supporting the uptake of glutamate and potassium released during neuronal firing. Disruptions in glycogen metabolism, which impair these processes, may contribute to migraine pathophysiology by compromising the uptake of glutamate and potassium. This environment reduces the cortical spreading depolarization threshold and facilitates the activation of parenchymal inflammatory signaling, both of which increase susceptibility to migraine headaches. The locus coeruleus is closely connected to several hypothalamic nuclei, including the suprachiasmatic nucleus, which helps synchronize circadian rhythms. The hypothalamus can also be activated by nociceptive input as well as external triggers. These reciprocal interactions may give rise to loop activity that fluctuates throughout migraine attacks and interictal periods, potentially influencing glycogen and glucose metabolism in cortical astrocytes. In conclusion, disruptions in astrocyte energy supply to synaptic activity, which is modulated by the extensive projections of locus coeruleus, may contribute to the initiation and progress of certain migraine forms.

Dysregulation of the serotonergic system is consistently noted in cases of psychiatric pathology. Circadian rhythm dysregulation is also a common comorbidity in psychiatric populations, and the circadian and serotonergic systems have a long history of coregulation. Despite this, it is not yet known whether serotonergic neurons house circadian molecular clocks, the transcription and translation feedback loops that drive circadian rhythms at the cellular level. To investigate this, brain tissue was extracted from adult male rats every 4 h throughout the light-dark cycle. Radiolabeled in situ hybridization was used to quantify clock gene expression in the dorsal and median raphe, the two nuclei responsible for providing the majority of serotonin to the brain. We discovered oscillatory rhythms in the expression of clock genes Bmal1, Per1, and Per2 with a period of approximately 24 h, and confirmed via fluorescent in situ hybridization that serotonergic (positive for Tph2, the rate-limiting enzyme in serotonin synthesis) neurons do express clock genes. The roughly antiphasic relationship between Bmal1 and the Per genes supports the existence of a circadian molecular clock in these cells. We next measured clock gene expression in neighboring brainstem regions that were not serotonergic, and found that although they all had similar daily clock gene expression profiles, the dorsal and median raphe had higher amplitude Bmal1 expression, and trending higher amplitude Per1 expression. This study adds to the growing list of extra-SCN (suprachiasmatic nucleus) molecular clocks reported in the brain. The prevalence of this circadian machinery, especially in regions of the brain so relevant to psychiatric health, underscores the importance of circadian rhythms to well-being. A greater understanding of the unique nature of circadian rhythms in discrete brain regions is a fruitful frontier for improving psychiatric treatment outcomes and overall health.

ABSTRACTCircadian rhythms are ~24‐h rhythms generated by the suprachiasmatic nucleus (SCN) in the mammalian hypothalamus. The regulation of circadian rhythms and downstream processes is highly dependent on the proper development and function of the SCN. Six3 and Six6 are homologous homeodomain transcription factors that have been shown to be required for SCN development; intriguingly, both Six3 and Six6 remain expressed in the adult SCN. To determine the role of Six3 and Six6 in the SCN after neurogenesis, we used Cre‐lox to conditionally knockdown either Six3 or Six6 from cells that express neuromedin‐S (NMS), a neuropeptide expressed in approximately half of SCN neurons. We found that the Nmscre allele turns on in the SCN after embryonic Day 16.5, limiting Cre‐lox‐mediated loss of Six3 or Six6 to the period after SCN neurogenesis. Using this approach, we hypothesized that Six3 and Six6 in NMS neurons regulate SCN circadian output and resulting reproductive function in males and females. Loss of Six6 from NMS neurons had no impact on puberty and reproduction. While loss of Six3 from NMS neurons had no effect in females, we found significantly decreased sperm motility in males, potentially through direct effects of Six3 in the testis. Loss of Six3, but not Six6, in NMS neurons resulted in shortened wheel‐running periods in constant darkness, indicating a shortening of the endogenous rhythm within the SCN. Together, these data indicate a role of Six3 in determining the circadian period, suggesting differing functions of Six3 and Six6 in the adult SCN.

Cellular- and systems-level profiling of amyloid-beta effects on circadian timing.