Journal of Biological Rhythms

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

Circadian clocks are evolutionarily conserved systems that orchestrate daily rhythms in human physiology, tissue behavior, and cellular function. In circadian research, the NanoString nCounter platform has emerged as a robust alternative to microarray and RNA sequencing. However, raw instrument output requires specialized preprocessing to handle technical noise. Furthermore, while harmonic regression is widely used to detect periodic signals in biological time-series data, evaluating rhythmicity remains highly challenging in clinical studies characterized by sparse sampling and small sample sizes. To address these limitations, we developed rhythmCI, an easy-to-use R package that integrates established normalization methods with a complete workflow for circadian data analysis. The package incorporates a bootstrapping procedure to estimate confidence intervals for harmonic regression amplitudes, offering a statistically transparent way to quantify rhythmicity in small-cohort studies. rhythmCI provides an end-to-end workflow for NanoString nCounter data, including automated preprocessing, normalization, and visualization. We evaluated the utility and accuracy of the package using synthetic time-series data generated with the Simphony framework, demonstrating that rhythmCI produces interpretable and reliable rhythmicity classifications under controlled simulation conditions. rhythmCI bridges a critical gap in circadian bioinformatics by offering a reliable method to characterize periodicity under challenging experimental designs typical of clinical research. By streamlining NanoString workflows and improving the statistical rigor of rhythm detection in small datasets, rhythmCI enhances the accessibility and reliability of circadian biology analysis.

Wearable light loggers are increasingly used to assess light exposure in everyday environments in studies of circadian physiology and sleep, yet their accuracy under diverse lighting conditions remains insufficiently understood. This study evaluated three commonly used wearable light loggers (ActLumus, Lido and OcuWEAR) by comparing measurements of photopic illuminance, melanopic equivalent daylight illuminance (m-EDI), and correlated colour temperature (CCT) against a reference spectroradiometer under controlled laboratory conditions and in the field. Measurements were conducted across fluorescent, incandescent, and multiple LED sources, as well as under dynamic daylight exposure. Device performance depended strongly on both light source characteristics and metric type. Illuminance exhibited lower variability, whereas m-EDI and CCT showed substantial device- and condition-specific biases, particularly under spectrally atypical illumination conditions. These differences were consistent with spectral mismatch effects arising from standard photometric calibration practices. Importantly, the impact of measurement bias varied by exposure context: under dim light conditions, small absolute deviations led to substantial misclassification relative to currently recommended thresholds, whereas brighter electric light and daylight conditions were more robust. These findings highlight that device- and context-dependent biases can influence the interpretation of light exposure in circadian and sleep research, underscoring the need for careful device selection and data interpretation.

Over the course of evolution, many species have adapted to the environmental conditions found at higher latitudes. This is likely also true of the circadian system, which regulates daily behavior and helps measure day length to facilitate adaptation to the seasons, though how the circadian clock adapts to latitude remains largely unknown. Here, we investigated daily activity patterns under long photoperiods and in constant darkness, as well as the underlying circadian clock network, in 52 Drosophila species from around the world. We found a strong, significant positive correlation between the clock's plasticity, measured as the phase difference between morning and evening activity, and the average geographical latitude from which the species originate. The mechanisms underpinning this increase in plasticity differed between species: in the subgenus Siphlodora, the clock weakened with increasing latitude, accompanied by a reduction of the neuropeptide Pigment-Dispersing Factor in the small ventrolateral clock neurons (s-LNv), whereas in the subgenus Drosophila the opposite was true. The subgenus Sophophora, by contrast, showed a negative correlation between circadian plasticity and PDF levels in the s-LNv axons. These results suggest that Drosophila species have evolved diverse, independent strategies to adapt their circadian clock to higher latitudes.

The circadian clock allows living systems to anticipate environmental daily variations by scheduling cellular mechanisms according to the time of the day. The reciprocal interplay between metabolism and circadian clock is essential for energy homeostasis, and the disruption of metabolic clock inputs contributes to dysfunction of clocks in peripheral tissues. However, the molecular mechanisms through which the feeding/fasting cycle entrains peripheral clocks remain incompletely understood. Here, we investigate experimentally and theoretically the role of AMP-dependent kinase (AMPK), a key fasting sensor, in metabolic regulation of the circadian clock. Using a luciferase reporter driven by the Bmal1 promoter to monitor molecular clock activity in human U2OS cells, and thanks to a signal processing method based on the Hilbert transform to retrieve the instantaneous phase and period of circadian signals, we show that pharmacological activation of AMPK by AICAR significantly elevates Bmal1 promoter activity and markedly lengthens the clock period in a dose-dependent manner. Conversely, the clock period is shortened by the pharmacological inhibition by SR18292 of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1-alpha). Importantly, the action of AICAR is abolished by FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, demonstrating that AMPK-dependent modulation of Bmal1 promoter activity requires NAD+ availability. These experimental results are recapitulated by numerical simulations of our previously published mathematical model, where PGC-1-alpha plays a key role in mediating AMPK activity to the clock. Moreover, the comparison of experimentally measured and theoretically predicted phase response curves, following AMPK activation at different circadian phases, provides an additional validation of the model. By integrating biological experiments with mathematical modeling, our results identify a key mechanism through which metabolic factors can entrain and regulate the circadian clock via NAD+-dependent AMPK-mediated regulation of Bmal1 promoter activity.

Chronic social isolation induces persistent behavioral and physiological changes across species. In humans, prolonged social isolation is associated with insomnia and increased risks of cognitive decline, cardiovascular disease, compromised immunity, and premature mortality. Yet whether these consequences are modified by the environment in which social isolation is experienced remains unclear. In particular, can access to social sensory information from conspecifics alleviate the effects of isolation and can various ambient environmental settings modify them? A related question arises during behavioral measurement because behavioral assays themselves impose a new environmental context. Whether these assays simply capture the enduring effects of prior social experience or further modify those effects remains unknown. Using social isolation-induced sleep loss in Drosophila as a model, we systematically examined how the social, spatial, and photoperiodic environments during isolation and behavioral monitoring influence the isolation phenotype. Providing isolated flies with visual, olfactory, or tactile social cues from conspecifics living behind a divider, either individually or in combination, was insufficient to alleviate sleep loss. Spatial confinement exacerbated isolation-induced sleep loss, while winter-like photoperiod in spatially confined Drosophila Activity Monitor assays revealed the clearest progressive decline in sleep during sleep monitoring. Together, these findings demonstrate that social isolation-induced sleep loss is both enduring and dynamic: it persists despite exposure to social cues yet remains sensitive to the environmental context in which isolation is experienced and behavior is measured. More broadly, our study establishes a tractable framework for investigating the environmental modulation of chronic social isolation.

Study Objectives: Glucagon-like peptide-1 (GLP-1) receptor agonists raise resting heart rate. We asked whether, after semaglutide initiation, the increase is larger during rapid eye movement (REM) sleep than during non-rapid eye movement (NREM) sleep. Methods: In the Human Phenotype Project, an Israeli cohort of generally healthy adults aged 40 to 70 years, 96 of 494 self-reported semaglutide initiators had a home sleep study (WatchPAT; sleep stages estimated from peripheral arterial tone and actigraphy) before and at least three months after starting. Each was propensity-matched 1:1 to a never-user of GLP-1 drugs measured at the same visits. We estimated the initiators' change minus their controls' change (difference-in-differences). The REM minus NREM contrast was designated primary after an exploratory analysis. Results: Relative to controls (90 to 94 pairs), heart rate rose during nocturnal wake (+3.89 beats per minute [bpm], 95% confidence interval 2.35 to 5.44), NREM sleep (+4.82 bpm, 3.12 to 6.52) and REM sleep (+6.25 bpm, 4.42 to 7.91); the REM increase exceeded the NREM increase by 1.42 bpm (0.98 to 1.89). The excess persisted across matching designs and on a relative scale; the proportion of REM sleep did not change. Never-users with similar weight loss showed no REM sleep heart-rate rise (+0.40 bpm, -0.63 to 1.50). Heart-rate variability decreases disappeared after adjustment for heart rate. Conclusions: Semaglutide initiation was associated with higher heart rate throughout sleep, most during REM sleep, unexplained by weight loss. Clinic measurements may understate this effect. Confirmation with polysomnographic staging is needed.

Seasonal daylight saving time (DST) transitions abruptly alter the alignment between clock time, environmental light, and sleep. In preregistered analyses, we used longitudinal Fitbit data from the All of Us Research Program to compare sleep around 13 seasonal transitions in Colorado, New Mexico, and Utah with sleep on the same dates in Arizona, where clocks do not change. The sample included 1,522 participants and 6,658 participant-transition windows. Following the spring transition, sleep duration changed more from one night to the next in Mountain states than in Arizona (14.6 minutes, 95% CI [7.2, 22.0]), even though sleep duration itself on day +1 showed little evidence of a DST-related reduction (-8.4 minutes, 95% CI [-17.3, 0.6]). Sleep timing shifted later after the spring transition and about 28 minutes earlier after the fall transition. Among Mountain-state observations with a large post-transition departure from usual sleep duration, the median return to the usual range was two nights. Nearly the same two-night pattern appeared in Arizona after similarly large sleep departures, indicating that this return pattern was not specific to DST. Greater baseline sleep-duration variability predicted a greater likelihood of remaining outside the usual range at days +7 and +14. Seasonal clock changes produced acute sleep disruption, while the Arizona comparison shows that a rapid return to baseline should not be interpreted as the duration of recovery from DST itself.

Consumer-grade wearables can continuously and longitudinally measure health signals such as heart rate (HR), activity, and sleep. While changes in these signals have been implicated in the early detection of infection (e.g., influenza, SARS-CoV-2) and real-time alerting systems have been developed, evidence from prospective deployment in pediatric transplant households remains limited. Here, we study the application of a resting heart rate (RHR)-based real-time alerting algorithm to detect respiratory virus infection in a cohort of solid organ transplant recipients and their household members (n=146) in a Phase IV clinical trial (NCT06161454). Reliable and efficient alerting for infection events may be particularly relevant for this population, enabling isolation, clinical management, and/or treatment. Of 31 antigen-test outcomes, nearly all (29/31, 93.5%) correspond to either influenza, COVID, or other patient-reported symptoms. We describe RHR, HR, activity, and sleep/circadian rhythm features around clinical alarm events, identifying candidate features for further investigation and algorithm improvement.

For sleep scoring in rodents, the use of electromyography (EMG) is considered the gold standard to assess muscle tone; however, EMG electrodes require invasive surgical implantation, are sensitive to muscular placement, can have extensive noise due to non-muscular signal contamination, and are at risk of failure due to mechanical stress. The use of an accelerometer (Acc) integrated on the recording headstage offers an alternative for movement detection. However, the degree of agreement between these two approaches has never been systematically evaluated. To understand if these tools allow similar scoring outcomes, seven trained scorers manually classified mouse sleep recordings using hippocampal local field potential (LFP) together with EMG or Acc. When comparing scoring between EMG and Acc, overall epoch-by-epoch accuracy exceeded 90% with a kappa score above 0.8, indicating excellent agreement. However, Acc was found to under classify Wake by 4.92% while overclassifying NREM sleep by 3.29%, possibly reflecting a decreased sensitivity of Acc to subtle changes in muscle tone used in distinguishing Wake from NREM sleep. Comparing EMG and Acc signal for agreed epochs vs disagreed epochs revealed a weaker correlation between the two signals for disagreed epochs, especially for Wake and NREM, suggesting that disagreements reflected genuine signal differences rather than scorer subjectivity. These findings support LFP/Acc as a valid approach to sleep scoring, however, it is noted that Acc based scoring slightly underestimated Wake epochs, favoring NREM sleep, relative to traditional EMG approach.

Sleep disturbances are a significant but underrecognized burden for individuals with Lennox-Gastaut Syndrome (LGS) and their caregivers. We conducted a large LGS caregiver survey to assess sleep quality, duration, specific sleep-related issues, and management strategies. Quantitative analysis revealed a significant association between nocturnal seizures and overall seizure frequency, but general sleep metrics in individuals with LGS (e.g., sleep hours, sleep quality) were not associated with seizure burden. In contrast, caregiver sleep was frequently disrupted - particularly in families who co-slept or reported frequent seizures. Nearly 40% of caregivers experienced nightly sleep disturbances. Respondents reported a range of strategies to manage sleep problems, including medication, environmental adjustments, and behavioral routines. Many also expressed frustrations over the limited effectiveness of strategies and inadequate support for sleep issues. These findings underscore the dual impact of LGS-related sleep disturbances on patients and caregivers, and highlight the need for comprehensive, family-centered approaches to sleep health.

Sleep disturbances compromise cognition, and overall physical and mental health; however, animal models of these effects remain limited, and typically impose more extreme disturbances than those experienced by humans. Here, we use behavioural interventions in mice to model two common types of sleep disruption: (1) sleep displacement associated with shiftwork and (2) sleep fragmentation associated with aging and disease. C57BL6/J mice were subjected to either a sleep deprivation protocol (n = 8, females = 4) or a fragmentation protocol (n = 8, females = 4), in which they were completely deprived of sleep or awoken every 2 min respectively, for 12 hours during their typical rest phase (lights-on) across 7 consecutive days. Age-matched control mice (n = 14, females = 7) remained undisturbed in their home cages. Activity during the 12-hour active phase was recorded via infrared motion sensors. After protocol completion, mice were injected with bromodeoxyuridine (BrdU, 200mg/kg) 2 hours prior to brain and blood collection; hippocampal neurogenesis and plasma corticosterone were then assessed. Results show that the sleep deprivation and fragmentation protocols had different, dynamic effects on behaviour; sleep deprivation was associated with an accumulating sleep need, operationalized as progressively reduced locomotor activity across the week, whereas fragmentation produced only an acute reduction of locomotor activity. Cell proliferation rates and corticosterone did not differ from controls. These protocols provide a foundation for future research into the mechanistic links between shiftwork and sleep fragmentation on health and cognition.

Synchrony between time-of-day and food intake is critical for maintaining homeostasis while desynchrony between these cues caused by repeated bouts of mistimed eating (ME) is profoundly disruptive to organismal health. However, it is unknown whether the same is true for a single bout of ME. While it is generally accepted that mammals recover quickly from acute alterations in feeding schedule and that chrono-disruption requires chronic, repeated changes, there have been no dedicated investigations of acute ME to suggest otherwise. Therefore, we exposed female and male C57Bl6/J mice to a single bout of ME or timed eating (TE) and continuously measured metabolic and behavioral responses for five days. A single bout of ME was sufficient to disrupt the daily rhythms of metabolic and behavioral outcomes for several days. Energy expenditure and wheel running activity were disrupted and light:dark variation in food intake and macronutrient utilization were altered for several days. Finally, ME changed the amount of time spent resting in for several days post-ME. The TE paradigm disrupted macronutrient utilization for two days but otherwise had no effect on metabolism or behavior. Our results demonstrate that one instance of ME alone has lasting impacts on daily rhythmicity and is an understudied source of circadian disruption.

Negative-feedback oscillators require a delay between the accumulation of a repressor's mRNA and the action of its protein. In the circadian clock, this delay has been attributed largely to post-translational control of PERIOD (PER) stability and nuclear entry. The RNA-binding proteins shown to regulate per translation, ATAXIN2 and its partners, promote it, leaving open whether any step holds clock transcripts back before they are translated. Here, using time-resolved miniTurbo proximity labeling of endogenous PER across four phases of the circadian cycle in Drosophila clock neurons, we define a 252-protein PER proximitome that partitions into a nuclear arm and a cytoplasmic RNA-metabolism arm. A behavioral RNAi screen identified two P-body components, the DEAD-box helicase Me31B (DDX6) and the 5'-3' exonuclease Pacman (Pcm; XRN1), as strong regulators of circadian rhythms. Using single-molecule RNA-FISH, proximity RNA editing and ribosome profiling, we show that as per and tim transcripts accumulate, they localize to Me31B-labeled P-bodies and are poorly translated, most prominently at ZT12. Me31B knockdown disrupts P-bodies and releases per mRNA from them, causing PER to accumulate earlier and to ~2-fold higher levels, whereas Me31B overexpression delays PER accumulation and lengthens the free-running period by ~2 h. Knockdown of Pcm, in contrast, impairs clearance of per mRNA, sustaining PER and TIM accumulation, prolonging the repression phase and abolishing cycling of ~89% of rhythmic transcripts. Together, these findings identify P-body sequestration as a repressive step that delays repressor synthesis, and Pcm-dependent decay as required to end repression on time. Given the deep conservation of DDX6 and XRN1, RNP compartments may provide a conserved means of generating delay in circadian and other negative-feedback circuits.

Background: Emerging evidence suggested the role of sleep and circadian rhythm in the development of mental resilience, but the association of an integrated analysis of objective sleep and circadian measures with multifaceted mental resilience in healthy adults has been underexplored. Objective: This study aims to explore the associations between multifaceted mental resilience and sleep and circadian rhythm in healthy adults. Methods: A total of 156 healthy adults (Age: Mean (SD): 29.54 (5.28) years; 60.9% female) underwent 14-day actigraphy, one night polysomnography (PSG) and dim light melatonin onset (DLMO) assessments. Resilience was operationalized as both a coping capacity and a positive mental health outcome in the context of accumulative adversity (life stressful events and childhood adversities). We first identified the covariance patterns between multiple sleep and circadian health domains (Sleep macro- and microstructures, dim light melatonin onset, rest activity pattern) and multifaceted mental resilience using partial least squares regression (PLSR), and followed by univariate analysis as complementary to illustrate the distribution trends of sleep and circadian rhythm parameters across integrative resilience score derived from principal component analysis (PCA). Findings: The PLSR model identified a modest but significant association of greater mental resilience with a sleep and circadian profile (Bootstrapping r = 0.15, 95%CI: 0.02-0.28), characterized by greater frontal-central slow waves activity in NREM sleep, greater frontal theta activity in REM sleep, lower occipital spectral power across fast and slow frequency bands in NREM and REM sleep, and probably earlier dim light melatonin onset timing. Conclusions: Sleep and circadian metrics, specifically frontal slow waves and REM theta activity, attenuated overall occipital spectral power during sleep, and earlier dim light melatonin onset time, represent quantifiable targets that may serve to improve resilience and stress-related mental health in adults. Longitudinal and interventional studies are needed to establish whether sleep interventions can be endorsed as evidence-based approaches for resilience enhancement. Clinical implications: Targeting sleep and circadian aspects may be an important approach to promote stress-related mental health.

Background: Commercial spaceflight now carries civilians on short missions, yet their stress-axis physiology is poorly described. Earlier shuttle data suggest that sympathetic responses predominate after short flights and glucocorticoid responses after long ones. The neutrophil-to-lymphocyte ratio (NLR) is a low-cost readout of both. Methods: Preregistered secondary analysis of open clinical laboratory data (NASA Open Science Data Repository OSD-569 and OSD-575) from the four Inspiration4 crew members, each sampled seven times: three draws before launch, one the day after return (R+1) and three during recovery (R+45 to R+194). Log-transformed NLR was modelled with a linear mixed model with a random intercept per crew member and Kenward-Roger inference. Lymphocyte and eosinophil counts and C-reactive protein were secondary outcomes with false discovery rate correction. Results: All 28 draws were analysed. NLR at R+1 did not differ from pre-flight (fold change 1.04; 95% CI 0.81 to 1.34; p = 0.73); two crew members increased (+25% and +46%) and two decreased (-27% and -11%). During recovery NLR was 23% lower than pre-flight (fold change 0.77; 95% CI 0.64 to 0.92; p = 0.005). No secondary outcome changed after correction; lymphocytes were 18% lower at R+1 (95% CI -35% to +4%; q = 0.31). Between-person differences explained 58% of the variance. An ordinary regression ignoring repeated measures missed the recovery effect (p = 0.08). Conclusions: A 3-day orbital flight produced no consistent leukocyte stress signature one day after return. The lower NLR during recovery suggests that pre-flight values, not recovery values, deviate from the resting state, which has implications for how baselines are defined in short-duration spaceflight studies.

Artificial light at night (ALAN) disrupts the natural light-dark cycle, which is the most important cue for synchronizing circadian clocks. If populations most exposed to ALAN adapt to get less disturbed by it, we expect forest and city populations to differ genetically. We tested this prediction in a common garden experiment using a songbird, the great tit (Parus major), originating from forest and city populations. We simulated ALAN exposure during the breeding season by keeping the birds in pairs under ALAN or dark nights for three months and then repeated the experiment in the following year with the same pairs under the opposite treatment. The advancement of daily onset under ALAN disappeared over time for city birds, but not for their forest counterparts. For these same birds, we measured their onset of daily activity under constant dim light and shift in activity in response to light pulses and showed that the city/forest difference in behavioural response was not reflected in any clock property measured. This indicates that city and forest birds differ genetically in the downstream behavioural response to ALAN, and not in the circadian clock. Thus, urbanization is unlikely to select for divergence in circadian clock properties in songbirds.

Objective. Temperature readings are routinely stratified by race and ethnicity, though significant group differences may reflect physiology, instrument noise, or categorization rather than biology. This study tests whether these categories partition facial infrared thermography measurements beyond individual variation and baseline device noise. Approach. A secondary analysis of thermography data (n = 951 adults) compares inner-canthus maximum (TCEmax) and full-face maximum (Tmax) temperatures across five administrative categories via one-way analysis of variance (ANOVA), mixed-effects modeling, Bland-Altman analysis against oral reference, and Monte Carlo sensitivity analysis. Main results. ANOVA returns significant group differences (p < 0.001; eta-squared = 0.04 for TCEmax, 0.03 for Tmax), but the total spread (0.2 to 0.3 degrees Celsius) is comparable to device accuracy and within-subject variability. Individual variation accounts for 95% of TCEmax variance, and adding simulated Gaussian noise to participant-level temperatures (sigma = 0.10 to 0.30 degrees Celsius) preserves the complete group-mean rank order in as few as 37.5% of iterations. Significance. Testing whether a group difference survives perturbation at the scale of instrument accuracy offers a check against treating significance alone as evidence of physiology. Here, observed group differences are small relative to measurement variability, and their physiological interpretation remains uncertain, with implications for clinical tools that treat administrative categories as biologically meaningful.

Objectives: Opioids disrupt sleep, but how repeated exposure reorganizes thalamocortical networks is unknown. We asked whether morphine alters sleep architecture, regional oscillations, and phase synchronization, and how exposure history modifies these effects. Methods: From mice, we analyzed cortical electroencephalogram (EEG), central medial thalamic (CMT) local field potentials (LFP), and electromyography (EMG) for 24h after the first and fourth injections of morphine and in abstinence, and quantified vigilance-state occupancy, spectral power, state-power correlations, and the weighted phase-lag index (wPLI), which minimizes volume conduction. Results: Morphine produced hyperlocomotion, wake promotion persisting with repeated exposure, non-rapid eye movement (NREM) sleep suppression, complete rapid eye movement (REM) sleep loss, and rebound sleep. Cortex and thalamus dissociated: in NREM sleep after the first injection, cortical delta and low-gamma power increased, while CMT delta, theta, alpha, and beta power decreased. NREM occupancy coupled more strongly to CMT delta and theta power after both injections, whereas cortical wake correlations in theta, alpha, and beta fell and reversed during abstinence. Overall synchronization was altered only in theta and alpha: corticocortical alpha synchronization decreased acutely, and repeated exposure reduced thalamocortical theta synchronization in wake and NREM sleep. During abstinence, sleep architecture and synchronization largely normalized while spectral and state-power abnormalities persisted alongside mechanical hypersensitivity. Conclusions: We found that morphine does not merely reduce sleep; it reorganizes it: cortex and CMT move in opposite directions, thalamocortical phase synchronization is redistributed, and the resulting spectral and state-power abnormalities outlast the recovery of sleep architecture itself rather than producing tolerance.

Many studies using mouse models of human brain disorders rely on extended recordings of brain electrical activity through electroencephalogram, or EEG. The features that researchers measure in these recordings, such as seizures and epileptiform activity, are deeply influenced by whether the animal is awake, in non-rapid eye movement (NREM) sleep, or in rapid eye movement (REM) sleep. Because total sleep duration varies naturally across animals, rigorous experimental comparisons require classifying the sleep state of every few seconds of a recording. Manual annotation is highly labor-intensive, often requiring hours per subject. While automated scoring tools accelerate this process, they are typically trained exclusively on healthy cohorts and frequently fail when applied to disease models that exhibit atypical or fragmented sleep architecture. Somnus is an open-source Python package designed to automate rodent sleep staging using EEG, muscle activity (EMG), and optional video-tracking data. It includes a robust base model trained on diverse recordings from six independent laboratories, enabling out-of-the-box classification across various equipment setups without requiring prior manual labeling. A central feature of Somnus is its capacity for rapid adaptation to pathological sleep structure. The software provides a graphical interface that directs researchers to the specific epochs where the base model is least certain. Users can manually correct these targeted epochs in a built-in viewer by comparing the model's classification to the raw EEG signal and video, and then use their corrections to fine-tune the model. This workflow empowers researchers to generate accurate custom scorers for complex disease models or novel treatments with just a few minutes of manual correction, bypassing the need to hand-score entire experimental cohorts or develop their own models from scratch.