Sleep and circadian disruption adversely impacts mental health and physiology. Such disruption varies in degree and emerges from numerous causes, ranging from severe disruption arising from rotating shift work or frequent jetlag to more mild circadian stress experienced by people with circadian rhythm sleep disorders (CRSD). While the effects of extreme circadian disruption such as jetlag are established through animal studies, less is understood about more subtle mismatches between external zeitgeber cycles and internal circadian periods as is observed with CRSDs. The present study employed a slightly mismatched ~1-h disruption paradigm, whereby Black and Tan BRachyury (BTBR) and C57BL/6 mice were housed from conception onwards in a conventional 24-h light/dark (LD) cycle or a 22.75-h LD cycle closer to the endogenous period of BTBR mice. Starting on postnatal day 90-110, mice underwent a battery of anxiety screens (open field, LD box, elevated plus maze) or a spatial navigation task (Morris water maze) to assess how mild circadian disruption impacts learning and anxiety-like behavior in these strains. Mice raised in a LD cycle that was mismatched from their endogenous rhythm (LD22.75 for C57BL/6 and LD24 for BTBR) displayed more anxiety-like behavior during the open field test and the LD box test and more impulsive behavior during the elevated plus maze. While both disrupted mice and controls showed evidence of learning during the Morris water maze during training, disrupted female mice did not show significant improvements between day 1 and 5 of training, and disrupted mice spent less time in the target quadrant on the probe day. This shows that even a mild form of circadian disruption throughout the lifespan is sufficient to elicit deficits in learning and memory and increased anxiety-like behavior in mice. While circadian disruption in humans may be confounded with other factors, such as diet and socioeconomic status, this study demonstrates that mild circadian disruption alone can alter cognitive and psychological functioning in mice.
It has been hypothesized that most mass shootings are suicidal acts, necessitating investigation of potentially common elements. We investigated whether US mass shootings and deaths by suicide during 1 January 2014 through 31 October 2021 exhibited significant seasonal variations. Outcomes included temporal patterns of mass shooting events and deaths by suicide and their relationship, as characterized by multivariable linear regression models detrended to isolate seasonality and estimate peak phases and amplitudes. Overall, 3,305 mass shootings and 360,827 deaths by suicide were analyzed. Detrended models revealed significant seasonal variation in mass shootings and deaths by suicide. Mass shootings and deaths by suicide displayed concurrent estimated annual peaks occurring on 3 July and 23 June, respectively. Detrended models estimated that at peak-versus-nadir, seasonality accounted for 613% increased mass shootings and 12% increased deaths by suicide. US mass shooting events and deaths by suicide exhibited strikingly synchronous seasonality, with concurrent mid-summer peaks and mid-winter nadirs. This finding calls for expanded implementation of summertime gun violence and suicide-prevention programs and for equipping treatment centers to respond to increased summertime demands. Further research is needed to investigate potential mechanisms underlying the shared seasonality and the hypothesis that many mass shootings are both homicidal and suicidal, intended as final acts.
Drosophila littoralis and Drosophila montana are two widespread species of the virilis group that occur at latitudes between approximately 40°N and 70°N. To survive the winter, they undergo a photoperiodically controlled diapause triggered by the decreasing photoperiod in late summer to autumn. Previous studies have shown that the critical photoperiod at which diapause is triggered depends on latitude. Fly populations living at high latitudes have a longer critical photoperiod than populations living at low latitudes, meaning that they enter diapause earlier. It is not yet fully understood how fly species measure photoperiod, but there is increasing evidence that the circadian clock is involved in this process. A recent study on D. littoralis has shown that the properties of the circadian clock change with latitude in parallel with the latitudinal changes in critical photoperiod, supporting this hypothesis. In D. montana , latitudinal clines have been observed in several diapause-related traits that have not yet been studied in D. littoralis . However, it is not known whether D. montana populations exhibit similar latitudinal clines in circadian clock traits as D. littoralis . Here, we compare the circadian clock and diapause traits of D. littoralis strains from different latitudes in Europe and D. montana populations from different latitudes in North America, using previously published data and determining missing parameters. We show that the two species exhibit similar latitudinal clines in circadian clock traits such as rhythm strength and timing of evening activity, as well as in diapause-related traits such as critical photoperiod, critical temperature, post-reproductive diapause and stability of photoperiodic time-measurement, with minor differences in the correlation between latitude and the expression pattern of the neuropeptide pigment-dispersing factor. Overall, our findings give new insight into the role of the circadian clock in measuring photoperiods that are essential for diapause induction in both Drosophila species.
Single-component cosinor models are commonly used to assess circadian dysregulation in 24 h ambulatory blood pressure (BP) measurements, but they cannot capture more complex or asymmetric circadian patterns. This study evaluated 4 mixed-effects cosinor models by applying them to systolic and diastolic blood pressure (SBP and DBP) data collected from children with obstructive sleep apnea (OSA) and healthy controls (non-OSA) and compared the estimated circadian parameters between groups. The analysis included 24-h BP monitoring data from 219 age- and gender-matched children (117 controls, 52 with mild OSA, and 50 with moderate-to-severe OSA [MS-OSA]). Mixed-effects cosinor models with 1 to 4 components estimated various circadian parameters: acrophase, amplitude, and time arrived at peak velocity (TAPV). The 3-component mixed-effects cosinor model provided the best fit for SBP and DBP data. The estimated MESOR (midline estimating statistic of rhythm) was 106 mmHg for SBP and 64 mmHg for DBP in the control group; the MS-OSA group had a higher DBP MESOR (66 mmHg). Children with OSA had a dampened early afternoon BP peak and an increased late-evening BP peak. The timing differences for the first BP peak were more pronounced in the morning for SBP, and from morning to mid-day for DBP, particularly in MS-OSA. TAPV occurred in the morning in all models, with slight timing differences in SBP for mild OSA, and in DBP for MS-OSA, compared to controls. Although single-component cosinor models are traditionally used for 24-h BP rhythms, the multi-component mixed-effects models provided a better fit and captured disease-related differences.
Immunological homeostasis relies on a sophisticated bidirectional dialogue between the immune and nervous systems, primarily coordinated by cytokine signaling. These peripheral signals access the central nervous system (CNS) via circumventricular organs and are integrated within the hypothalamus to mount appropriate homeostatic responses. Given that the immune system is under rigorous circadian control, circadian desynchrony-such as constant light exposure-often compromises this coordination, thereby increasing disease vulnerability. This study investigates the hypothesis that interleukin-6 (IL-6) is a pivotal mediator in this neuroimmune dialogue, specifically examining how astrocytic IL-6 trans-signaling modulates hypothalamic activity following an immune challenge. Utilizing male wild-type (WT) and GFAP-sgp130Fc (TG) mice-the latter genetically engineered to express a soluble gp130 fusion protein that selectively binds the IL-6/sIL-6R complex, thereby acting as a specific decoy receptor to inhibit astrocytic IL-6 trans-signaling without altering classical membrane-bound signaling-we assessed physiological and molecular responses under standard LD cycles and constant light (LL) conditions, the latter serving as a model for circadian desynchronization. Our results reveal that TG mice exhibit compromised circadian patterns and significantly altered thermoregulatory responses to lipopolysaccharide (LPS) compared to WT controls. Notably, TG mice under LL conditions showed a complete abolition of the thermoregulatory response to LPS, identifying a critical failure in homeostatic integration when both astrocytic signaling and circadian organization are impaired. Furthermore, c-Fos immunoreactivity within the suprachiasmatic nucleus and paraventricular nucleus indicated a profound suppression of hypothalamic activation in TG animals. Peripheral analysis of hepatic mRNA (IL-1β, IL-6, TLR4) confirmed genotype- and photoperiod-dependent variations, with TG mice displaying a markedly blunted inflammatory profile. These findings underscore the essential role of astrocytic IL-6 trans-signaling in neuroimmune communication and demonstrate that its absence, coupled with circadian disruption, induces a state of "neuronal deafness" that severely compromises the ability of the CNS to respond to inflammatory challenges.
The circadian system coordinates daily physiology across nearly all tissues to temporally organize metabolism and maintain homeostasis. In the brain, circadian timing regulates neural activity, cellular function, and neuroimmune signaling, which is especially important during development. Yet, the ontogeny of circadian regulation during neurodevelopment remains poorly defined. Here, we characterized time-of-day variation in core clock and neuroimmune genes across multiple brain structures during early postnatal development, alongside circulating corticosterone concentrations. Using male and female C57BL mice housed in a standard light-dark cycle [12:12 light (150 lux)/dark (0 lux)], we measured the expression of Per1, Per2, and Rev-erbα in the suprachiasmatic nucleus, hippocampus, and medial prefrontal cortex, as well as in neuroimmune tissues (choroid plexus, meninges, and isolated microglia) across postnatal days (PND) 1-24. Across development, rhythms were seen in corticosterone concentrations and all brain regions, with increased amplitudes and gene-specific phase maturation toward adult-like timing by PND 24. Notably, the choroid plexus and meninges exhibited time-of-day differences in clock gene expression by PND 10-24. In contrast, isolated microglia did not display detectable time-of-day differences in clock gene expression; however, microglial phagocytic activity varied by time of day. Together, these findings demonstrate that circadian regulation of the brain emerges during the neonatal period, and the parameters of time-of-day differences are tissue- and gene-specific during development. In addition, functional rhythms may precede or occur independent of detectable transcriptional differences. This work establishes a developmental framework for circadian-neuroimmune interactions, with important implications for neuroimmune development and vulnerability. Given the neuroimmune system's role in shaping brain development, disruptions in these temporal processes may contribute to neurodevelopmental or mood disorders.
Night shift work is linked to more severe coronavirus pneumonia, suggesting that host resilience to these pathogens may depend on the timing of exposure. Here, we examined how the time of day influences the severity of coronavirus pneumonia in mice, using mouse hepatitis virus-1 (MHV-1) as a natural infection model. We found that the timing of infection influenced MHV-1 severity, with the highest mortality, peak viral load, and lung inflammation occurring with midday infection, which corresponds to the mid-rest phase in mice and is comparable with nighttime in humans. The time-of-day dependence in disease severity occurred prominently in males and was sensitive to global disruption of the clock gene Bmal1. Midday infection correlated with increased MHV-1 binding to and replication within alveolar macrophages (AM). Depleting AMs with clodronate or loading them with neutral liposomes before MHV-1 infection eliminated differences in pneumonia survival and peak viral load related to the timing of infection. These data suggest an immunologic rhythm underlying coronavirus outcomes, in which oscillations in "first contact" interactions between AMs and the virus shape subsequent pneumonia severity.
The mammalian circadian system has traditionally been viewed as a hierarchical network organized around a master pacemaker in the suprachiasmatic nucleus (SCN), which synchronizes peripheral clocks to coordinate daily rhythms in physiology and behavior. While this framework has provided a foundational model for circadian regulation, recent advances in molecular profiling, circuit tracing, and neuronal manipulation reveal a more complex architecture. Multiple brain regions outside the SCN contain neurons with intrinsic circadian properties that actively participate in processing temporal information, challenging the classical central-peripheral model. These findings raise a fundamental conceptual question: what defines a circadian clock neuron? Here, we synthesize recent molecular, cellular, and systems-level studies to examine the defining features of neurons embedded within brain circadian clocks. By integrating high-resolution molecular profiling with functional circuit analysis, we propose an operational framework for identifying clock neurons across brain circuits. Specifically, we outline four criteria that characterize circadian clock neurons: molecular oscillation, autonomy, physiological rhythmicity, and circuit influence. This framework reflects current experimental capabilities and highlights how multiscale approaches-from single-cell transcriptomics to causal circuit manipulation-are reshaping our understanding of circadian organization in the brain. Clarifying the identity of clock neurons will be essential for mapping distributed circadian circuits and for developing targeted interventions for neurological and neurodegenerative disorders associated with circadian and sleep disruption.
The Period (Per) gene was first identified in Drosophila as a key regulator of circadian rhythms, with mutations that altered or abolished behavioral rhythmicity. Mammals possess 3 homologs, Per1, Per2, and Per3. Here, we systematically compared the circadian properties of mice deficient in each Per gene, singly and in combination, under constant darkness (DD). We analyzed their free-running periods (τ) and phase response curves (PRCs) to 6-h light pulses (6-h LPs). Each Per-deficient line exhibited distinct circadian characteristics. Per1-deficient mice (including Per1-/- Per3-/- double mutants) showed high-amplitude PRCs with large phase shifts near CT18, consistent with Type 0 resetting. Per2-deficient mice displayed shorter τ and Type 1 PRCs with crossover points near CT17. Remarkably, some Per2-deficient mice lost circadian rhythmicity after a single 6-h LP delivered near the crossover but regained rhythmicity following a second pulse 12 days later. In contrast, Per1-/- Per2-/- mice, which retain Per3, failed to maintain stable rhythms in DD yet transiently reestablished a short-period (~19.5 h) rhythm in response to a 6-h LP. These findings indicate that rhythm loss in Per2-deficient mice does not represent oscillation stop but rather light-induced desynchrony among multiple oscillators that constitute the circadian pacemaker. Collectively, our results demonstrate that Per1 sustains oscillator strength, Per2 maintains inter-oscillator coupling, and together they ensure the robustness of the mammalian circadian system against strong photic perturbation.
During dark adaptation, pupil size changes in association with visual adaptation processes; however, the influence of diurnal phase (daytime vs nighttime) and aging on these changes remains unclear. In this study, we examined pupil size changes during dark adaptation across the daytime and nighttime in young, middle-aged, and aged C57BL/6N mice. In young mice, pupil size reached a maximum shortly after the onset of dark adaptation and then gradually decreased during the daytime, whereas it remained dilated during the nighttime. Furthermore, dopamine depletion induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly attenuated the gradual pupillary constriction following pupil dilation during dark adaptation in the daytime, suggesting a possible involvement of dopaminergic signaling in the later phase of the response. In middle-aged mice, pupillary light reflex-evoked constriction was preserved; however, the early (rapid) phase of constriction following the initial dilation during daytime dark adaptation was impaired, whereas the later (delayed) phase remained largely comparable to that in young mice, suggesting age-related alterations in regulatory mechanisms rather than a loss of constriction capacity. Pharmacological analyses further suggested that distinct neural mechanisms may differentially contribute to these temporal phases. In aged mice, maximal pupil dilation during dark adaptation was significantly reduced during both the daytime and nighttime. In addition, aged mice exhibited distinct abnormalities in pupil dilation immediately after the onset of dark adaptation during the nighttime, indicating mechanisms that differ from those observed in middle-aged mice. Together, these results demonstrate that pupillary dynamics during dark adaptation are modulated by diurnal phase and progressively altered with aging. Thus, analysis of pupil dynamics during dark adaptation may provide a useful approach for detecting age-related changes in neural function.
To characterize the presence and relationships between the rhythms of axillary temperature, heart rate, respiratory rate, blood pressure, and peripheral oxygen saturation, from the first (D1) to the third day of life (D3) in newborns (NBs) hospitalized in the neonatal intensive care unit (NICU). Primary, descriptive, nested clinical study within a primary, observational, and prospective clinical study, conducted in the NICU of a public hospital in the city of São Paulo, Brazil. Demographic data and vital signs (axillary temperature, heart rate, respiratory rate, blood pressure, and peripheral oxygen saturation) recorded in clinical charts were collected between the first and third day of life (D1-D3) at 4-h intervals (8 h; 12 h; 16 h; 20 h; 24 h; 4 h). The biological rhythms of the vital signs were analyzed using the Cosinor algorithm. Eighty-eight NBs participated (44 preterm and 44 term). Incipient variability of vital signs was observed in the first days, with reduced amplitudes and unstable phases. There was a correlation between the amplitude of axillary temperature and length of hospital stay in preterm infants, and between the amplitude of diastolic blood pressure and length of stay in term infants, suggesting the clinical potential of variability as a marker of neonatal maturation and clinical vulnerability. Altogether, the findings indicate the presence of early physiological rhythmicity and reinforce the importance of chronosensitive care and the incorporation of chronobiological parameters in clinical management, aiming to optimize the development and outcomes of hospitalized newborns.
The Moon's motion around the Earth is complex, governed by interactions among several types of cycles that arise from different aspects of its orbit. The 29.53-day synodic cycle occurs as the Moon passes through new-moon and full-moon alignments (syzygies) with the Earth and the Sun. The 27.32-day tropical cycle occurs as the Moon moves back and forth between its northernmost and southernmost positions (standstills) relative to the plane of the Earth's equator. The 27.55-day anomalistic cycle occurs as the Moon moves back and forth from its nearest and farthest distances from the Earth (perigees and apogees). The Moon's effects on luminance and gravity at the Earth's surface are greatest at times of syzygies, standstills, and perigees. Here, based on periodogram analyses of 14.5 patient-year records of a circular, rapid cycling type of bipolar disorder, we show that onsets of mania in a given individual can recur in association with 2 or more lunar cycles simultaneously, and with conjunctions of syzygies of the synodic cycle with standstills of the tropical cycle or with perigees of the anomalistic cycle. The results are consistent with the fact that the Moon's effects at the Earth's surface are the result of interactions among all of its constituent cycles, and they highlight the potential importance of long-term longitudinal designs in studies of lunar influence.
Chronotype reflects an individual's preferred timing of sleep/daily activities and is typically regarded as a stable trait, though it varies across the lifespan and may fluctuate also over shorter periods of time. Here, we investigate the temporal stability of self-assessed chronotype and its modulation by behavioral advice in a large cohort of University students participating in a 2-arm circadian hygiene education initiative (either encouraging consistency or advancement of sleep, meals, and exercise timing). Students provided demographic and sleep quality information, and repeated chronotype assessments (2-22 months) using the Self-Morningness/Eveningness (Self-ME) question, which offers a choice among 4 categories (definitely morning, morning, evening, definitely evening) and the Ultrashort Munich ChronoType Questionnaire (μMCTQ), which has midsleep and social jetlag as outcomes. Two main samples were analyzed: students completing baseline/2-month follow-up assessments (n = 1902) and students completing baseline/one later assessment, at any time (n = 2820); a subgroup with 3 available assessments (n = 1257) was also examined. Agreement between subsequent Self-ME assessments was good in all samples (0.6 < Cohen's k < 0.8), with approximately 70% of students confirming their first assessment at 2 months, with variations (62%-73%) depending on time between assessments and behavioral advice; transitions mostly occurred between adjacent Self-ME categories. Nonetheless, approximately 30% of participants changed category between 2 assessments, and those who moved once were more likely to move again. "Non movers" exhibited more extreme sleep-wake habits. Furthermore, midsleep and social jetlag changed over the calendar year in ways that seemed to reflect the time-course/constraints of the academic year. The 2 circadian hygiene interventions influenced chronotype stability slightly and differently, particularly in morning types, who probably found it easier to comply with the suggestion of advancing sleep-wake timing. In conclusion, chronotype in young adults shows relative stability but is plastic within limits, also in relation to the way it is measured.
Circadian rhythms are endogenous biological cycles with a period of approximately 24 h that integrate a wide range of physiological and behavioral processes in living organisms. In addition to circadian rhythms, many other biological processes also exhibit diurnal (24 h) rhythms driven by external environmental cues. With the significance of circadian and diurnal regulation becoming increasingly recognized, the field of chronobiology is exhibiting unprecedented growth in the life sciences and translational medicine. Over the past 2 decades, a variety of computational methods have been developed to detect and extract rhythmic signals from the time-series data of different species. However, existing rhythmic analysis tools are often fragmented, demand programming expertise, exhibit limited visualization capabilities, and impose inconsistent requirements on data types and sampling intervals. Therefore, there is an urgent need to establish a convenient and comprehensive tool for detecting and analyzing the circadian and diurnal rhythmicity. To meet this demand, RhythmInsight was developed as an open access, web-based platform for comprehensive analysis and visualization of circadian and diurnal rhythms across various species and data types, including physiological, omics, and experimental data. RhythmInsight includes 3 modules: Rhythmic Analysis, Differential Rhythmicity Analysis, and Rhythmic Visualization. The Rhythmic Analysis module incorporates 9 algorithms (JTK_CYCLE, Cosinor, CircaCompare, meta2d, Lomb-Scargle, RAIN, ARSER, Fisher's G-test, and Robust G-test) to detect and characterize rhythmic signals. The Differential Rhythmicity Analysis module, based on CircaCompare, detects and compares rhythmic parameters (amplitude, phase, and the Midline Estimating Statistic of Rhythm) between 2 experimental conditions. The Rhythmic Visualization module provides powerful graphical tools, including line plots, fitted curves, heatmaps, polar plots, and boxplots, for the intuitive visualization of time-dependent trends. By integrating rhythmic algorithmic analysis with interactive visualization, RhythmInsight simplifies the analysis process and enhances accessibility for researchers without programming backgrounds, particularly experimental biologists and early-career scientists. RhythmInsight is freely available at https://RhythmInsight.com.
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.
This letter follows up on an article recently published in the Journal of Biological Rhythms. The authors of the original article used actigraphy data from a sample of 720 participants to show that the mean chronotype of male participants is not significantly different from the mean chronotype of female participants but also that the mean chronotype of Black participants is about 20 min later than the mean chronotype of White participants. I reached the same conclusions using a larger data set of 7562 participants from National Health and Nutrition Examination Survey (NHANES) 2013-2014. This corroboration is important because the associations of race and gender with chronotype have been inconsistent in studies in which chronotype was measured with questionnaires rather than with objective rest-activity measures.
Circadian neuronal plasticity describes daily recurring changes at the level of neuronal morphology, connectivity and synaptic processes. Disturbance of these plastic changes could result in inflexibility of an organism to adapt behavior to changing environmental cues. The mitogen activated protein kinases (MAPK)/ERK signaling pathway is involved both in circadian processes and neuronal plasticity. Ribosomal S6 kinases (RSK) act as downstream mediators of ERK signaling with apparently pleiotropic-but sometimes poorly understood- functions in the nervous system. This is illustrated by some major gaps in our understanding of the pathophysiological processes caused by RSK2 mutations in humans that lead to intellectual disabilities. Previous studies described the role of Drosophila RSK as one regulator of the molecular circadian oscillator. Here we could show that RSK kinase activity is required to control another aspect of circadian rhythmicity, the daily remodeling of the dorsal branching pattern of the small ventral lateral neurons (s-LNv) as the central pacemaker cells. Loss of RSK function resulted in more fasciculated and less branched s-LNv's in the early morning, which could affect synaptic in- or output connectivity. Increased fasciculation correlated with a reduced number of Bruchpilot sites as a marker for presynapses. Analysis of the expression of the Pigment Dispersing Factor PDF in s-LNv's, the most important signaling factor between clock neurons, revealed no evidence of changes in RSK mutants. Consistent with unaffected PDF signaling as a major output from the s-LNv's, RSK mutant flies are rhythmic. Their free-running rhythms show even a significantly higher power than those of the wild-type controls. This robustness is at the expense of flexibility to adapt their activity to variations in light conditions. Together with the known role of RSK in olfactory learning and memory processes our results suggest that RSK is required to maintain experience dependent plasticity.
