Genes, Brain and Behavior

Physical activity offers myriad benefits to health and well-being, in humans and other animals as well. In rodents, voluntary wheel running can attenuate the effects of both physical and social stressors on rodent social behavior. Whether wheel running affects rodent social behaviors per se remains less well understood. We conducted the current study to test whether home cage access to running wheels impacts the social behaviors of adult, group-housed C57BL/6J female mice during same-sex interactions with novel females. Group-housed females were either given continuous home cage running wheel access or a standard paper hut starting at weaning, and as adults, social behaviors were measured during interactions with novel females. In two cohorts, we found that 5 weeks of running wheel access during adolescence reduced the time that subject females spent investigating a novel female and also tended to reduce total ultrasonic vocalizations produced during interactions. These effects were not reversed by a 2-week period of running wheel removal but were recapitulated in a different cohort by 2 weeks of running wheel access in adulthood. Unexpectedly, we found that these effects on female social behavior were not due to wheel running per se, because females raised from weaning with immobile running wheels also showed low rates of social behaviors during same-sex interactions in adulthood. Overall, we find that the presence of a running wheel in the home cage has an enduring inhibitory influence on female social behavior during same-sex interactions, a finding that has implications for the design of studies that include same-sex interactions between female mice.

Neuroligin-3 (NLGN3) was first identified as a risk gene associated with autism spectrum disorder (ASD). The initial variant, p.R451C, associating NLGN3 with ASD has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. Furthermore, while most of the identified variants are present in males with maternally inherited variants from unaffected mothers, several de novo variants were observed in females, suggesting a possible functional difference between de novo and maternally inherited variants. To address the functional consequences of NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to one de novo variant (p.R175W) identified in one female proband, and two maternally inherited variants (p.R451C and p.R597W) identified in male probands. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the de novo p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology and sleep patterns, with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that de novo NLGN3 variants identified in females are likely gain-of-function, while maternally inherited variants have mixed loss-and gain-of-function effects. Moreover, the location of the variants may contribute to the distinct functional differences we observed. Some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author SummaryAutism spectrum disorder (ASD) is a common neurodevelopmental disorder. Mutations in the Neuroligin-3 (NLGN3) gene are associated with ASD but very few of these mutations have been characterized in animal models. Most of these mutations affect male individuals who maternally inherited their genetic mutation; however, more rarely female individuals may present with a genetic mutation that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different mutations, one mutation identified in a female and two mutations identified in males, affect the flys behavior and synapse development. We identified altered sleep patterns in some of our mutants which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and mutations in this gene can alter its function. However, different mutations can have differential effects. This demonstrates the need to assess multiple variants simultaneously because each variant may have distinct functional significances.

The exploration-exploitation trade-off refers to the conflict between using known strategies that reliably yield reward (exploitation) and sampling uncertain options that might yield better outcomes (exploration). Dysregulation of this balance is implicated in neuropsychiatric disease, and while sex differences in this balance have been described, the biological bases remain unclear. To quantify sex differences in this trade-off, we tested mice (n=74 male, 62 female) on four home-cage based foraging tasks with an operant pellet dispensing device, Feeding Experimentation Device 3 (FED3). Mice completed the tasks continuously over multiple days and the tasks were their only source of food. Across multiple tasks, males showed higher win-stay behaviour than females, indicating greater exploitation of previously rewarded actions, an effect that was modest in size but highly significant. Power analyses revealed that >30 mice per sex were needed to detect these modest but significant sex differences with 80% power. No consistent sex differences were observed in pellet intake, suggesting that differences in exploitation did not reflect differences in hunger drive or demand for pellets. Exploitation is a more efficient strategy when environmental parameters are fixed, while exploration can be more advantageous when parameters such as reward locations are changing and uncertain. We tested this idea by re-running our mice in a probabilistic foraging task, where actions led to uncertain probabilities of reward. While males continued to show higher levels of win-stay behaviour on this task, this no longer led to increases in accuracy. Behavioural modelling also supported this framework, demonstrating that stronger win-stay behaviour was most advantageous in deterministic models, and less advantageous in probabilistic models. Together, our findings demonstrate that male and female mice have small but significant differences in their exploration-exploitation balance, which leads to more accurate foraging in certain, but not uncertain, environments.

We assessed whether pigs provide consolation, referring to targeted affiliation that attenuates a partners stress, under experimental conditions that manipulated exposure to stressed partners. Using a within-subject design, 74 pigs were tested in three contexts: a helping task in which group members could observe and help a trapped focal pig to return to the group, a direct-reunion, in which group members were naive to the experience of a separated focal pig until reunion, and an undisturbed control. We measured affiliative and non-affiliative interactions, anxiety behaviours and changes in salivary cortisol. Only the helping context satisfied most consolation criteria: there were selective increases in unidirectional affiliative contacts from the observer to the focal pig, non-affiliative interactions remained at baseline, and focal pigs showed fewer anxiety behaviours. In contrast, direct-reunions triggered increases in affiliative and non-affiliative interactions and higher anxiety. Cortisol increased during both direct-reunions and helping, but its level was not linked to affiliation. Results add to growing evidence for consolation behaviour in pigs and suggest best practices for reintegrating pigs into groups. Graded reintroductions that allow observers to assess the emotional state of targets may promote social buffering, whereas abrupt regrouping may trigger more generalized arousal or personal distress.

BackgroundSteroid sulfatase (STS) cleaves sulfate groups from steroid hormones. In humans, STS deficiency is associated with X-linked ichthyosis (a dermatological disorder), neurodevelopmental/mood conditions, and cardiac arrhythmias. Until recently, no single-gene knockout mammalian model existed to investigate these associations; previous work in such a model has been limited to skin phenotypes. MethodsWe generated a novel C57BL/6J mouse model with a deletion in critical exon 2 of Sts. We then examined gene expression and enzyme activity in liver and brain samples of homozygous mice, and assessed the breeding performance and health of male and female deletion-carriers. Subsequently, we compared performance across a range of behavioural paradigms in wildtype and homozygous male and female mice: elevated plus maze, open field, rotarod, spontaneous alternation, and acoustic startle/prepulse inhibition. We also investigated serum steroid hormone levels by liquid chromatography-mass spectrometry and measured heart weights and two morphological indices (bodyweight/tibia length) post mortem. ResultsHomozygous mice almost completely lacked STS expression/activity. Genetically-altered mice exhibited grossly-normal breeding performance, health, and endocrinology. Homozygous mice were more active, and had higher normalised heart weights, than wildtype mice. We also found significant genotype x sex interactions on bodyweight, and on two behavioural measures (potentially reflecting lower anxiety in homozygous males and heightened anxiety in homozygous females). ConclusionsThe Sts-deletion mouse represents an experimentally-tractable model in which to identify and characterise phenotypes associated with STS deficiency. The mechanistic basis of the genotype-phenotype associations described here requires further investigation, and whether such associations translate to humans remains to be tested.

Internet gaming disorder (IGD) presents a significant public health challenge, yet its complex biopsychosocial mechanisms and dynamic risk trajectories remain poorly understood due to a scarcity of comprehensive longitudinal and multimodal cohorts. To address this critical gap, we established the Chinese College Student Gamers Cohort (CCSGC), a prospective, multimodal longitudinal study of 793 first-year undergraduates primarily playing Honor of Kings from 2022 Sept. The CCSGC integrates semi-annual psychosocial questionnaires, annual neuroimaging (EEG/fMRI), and biospecimen collection over multiple years. Baseline data revealed individuals with IGD (n=211) exhibited significantly higher gaming craving, psychological distress (depression, anxiety), impulsivity, and maladaptive motivational features compared to non-IGD gamers (regular players (RP) n=400; casual players (CP) n=182). Longitudinal analyses across four waves indicated bidirectional temporal associations between IGD severity and mental symptoms, and a stabilization of IGD incidence after an initial decrease. Furthermore, specific neurophysiological (e.g., N400 amplitude to game cues) and neuroimaging (e.g., superior parietal activation) markers were identified that correlated with IGD severity and predicted one-year outcomes in gaming disorder or social functioning. The CCSGC provides an invaluable resource for dissecting the heterogeneity, comorbidity, and intricate biopsychosocial mechanisms of IGD, holding significant potential to advance risk prediction, early identification, and targeted intervention strategies.

Stressors are commonly used in rats to induce models of anxiety or depression. The effectiveness of these stressors is often evaluated using specific behavioural tests. In a previous meta-analysis of chronic variable stress (CVS) procedures, we predicted that longer and more intensive stress procedures would result in larger effect sizes in behavioural tests. However, we found that the duration or intensity of CVS procedures did not correlate strongly with the magnitude of the effect sizes reported in behaviouraltests. In that study, we were concerned that the large and unexplained diversity in CVS procedure design, both in terms of duration and the types of stressors used, made it challenging to detect the factors that were influencing effect size. In an effort to address this, we explore here the use of a much simpler stress procedure - chronic restraint stress (CRS) - to study the relationship between the duration of CRS procedures and the effect sizes obtained in subsequent behavioural tests. We searched PubMed for articles using CRS procedures with rats, systematically documented the total duration of restraint, and carried out a meta-analysis of the effect sizes obtained in four behavioural tests: the forced swim test (FST), the sucrose preference test (SPT), the elevated plus maze (EPM) and the open field test (OFT). We found that chronic restraint stress increased immobility in the FST, decreased sucrose preference in the SPT, decreased time spent in the open arms of the EPM but had no effect on time spent in the centre of the OFT. However, the effect sizes in all behavioural tests, except the SPT, were not moderated by the duration of the CRS procedure, indicating that longer CRS procedures are associated with larger effect sizes in the SPT but not in the FST or EPM.

Alzheimers disease (AD) is a devastating neurodegenerative disorder characterized by memory loss and a decline in cognitive function. Hallmarks of AD include an age-dependent accumulation of toxic amyloid beta (A{beta}) 42 in the brain, energy dyshomeostasis caused by mitochondrial dysfunction, and iron overload. However, the role of iron overload and mitochondrial dysfunction in AD pathology is unknown and their precise relationship with A{beta} 42 toxicity in AD pathology is unclear. C. elegans provide a powerful model system to untangle and clarify these relationships. In this study, we quantify the temperature-dependence of iron toxicity (16, 20 and 25C) in neurons and muscle of C. elegans that overexpress A{beta} 42. We found that A{beta} 42, regardless of the cell-type expression, caused accelerated paralysis compared to age-matched WT worms with the greatest degree of paralysis observed at an elevated temperature (25C). Moreover, the combination of iron toxicity and A{beta} 42 results in an enhanced paralytic phenotype at 16C. Thus, iron exposure potentiates A{beta} toxicity observed at low temperatures. Iron toxicity stimulated both maximum (State 3) and leak (State 4) respiration in WT and A{beta} 42 worms. A{beta} 42 worms also exhibited increased leak respiration at baseline that was further exacerbated by iron toxicity. Iron burden and sensitivity increased A{beta} 42 peptide toxicity. A{beta} 42 worms exhibited reduced levels of Ca, Zn, Mn, and K. Overall, our results suggest that iron potentiates A{beta} toxicity at low temperature and enhances A{beta} peptide mediated mitochondrial bioenergetic dysfunction in C. elegans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/714217v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9eaf46org.highwire.dtl.DTLVardef@542eforg.highwire.dtl.DTLVardef@16d9678org.highwire.dtl.DTLVardef@1b1b16d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITemperature stress modulates the synergetic interactions of iron toxicity and A{beta} 42 pathology C_LIO_LIIron sensitivity drives increased cell-type specific A{beta} 42 pathology C_LIO_LIEnergy dyshomeostasis via impaired mitochondrial function and increased proton leak contributes to iron- and A{beta}-induced pathology C_LI

A large subset of ASD associated genes, almost 50% of the highest confidence risk genes listed on the Simons Foundation Autism Research Institute database, are epigenetic modifiers. This suggests that the organization of sensory biology and its coupling to underlying genetic control are an important element underpinning this discord. Furthermore, sensory processing changes in individuals with autism spectrum disorder (ASD) has been a growing area of study in recent years. C. elegans have robust readouts for both developmental and sensory biology allowing these signatures of ASD to be systematically modelled. 52 epigenetic modifiers (65 strains) were selected for study in C. elegans based on gene function, presence of orthologues in C. elegans and the availability of viable putative null strains. This highlighted significant changes to reproduction, gross development and sensory processing across the range of epigenetic modifiers. Each strain was filtered against selective criteria for significant sensory and developmental phenotypes allowing for selective phenotypic profiles to emerge. These were three primary groups, those with sensory perturbations but unaffected gross development (6), developmentally affected genes with intact sensory function (10) and finally genes with impaired gross development and sensory function (11). Thus, this study provides a link between sensory and developmental outcomes in ASD associated mutant strains and suggests that more regular sensory testing should be performed in human cohorts to further refine sub-categorisation of ASD cohorts.

Atypical social functioning is a core feature of autism, yet findings remain fragmented across components and development. We aimed to systematically integrate this literature and characterize the organization, development, and moderators of social functioning in autism. We conducted a systematic review and meta-analysis of behavioral studies published between January 1990 and August 2025, identified through PubMed, Web of Science, and prior reviews, including studies with clinically diagnosed autistic individuals and neurotypical controls. A qualitative synthesis and two complementary quantitative meta-analyses were performed, with risk of bias evaluated through study-level characteristics. A total of 2,622 studies (94,114 autistic and 172,847 neurotypical individuals across 32 countries) were included, covering 22 social components that clustered into five domains. Overall group differences were substantial (Hedges g = -0.744, 95% CI [-0.797, -0.690]). Differences emerged earliest in motivation-based processes ([~]6 months), followed by motor, emotion, and inference domains, and showed age-related divergence alongside improvement in some skills. Cross-domain analyses revealed stronger interdependencies in autism and an organizational pattern most consistent with serial relationships among domains. These findings should be interpreted in light of methodological heterogeneity, underpowered samples, and uneven cultural representation. Together, the results provide an integrative framework for understanding the organization and development of social functioning in autism, with implications for precision subtyping, developmentally timed interventions, and neurodiversity-informed research and policy. This study was pre-registered (PROSPERO: CRD42024566141).

Actigraphy is a non-invasive and cost-effective method for monitoring behavioral rhythms under real-world conditions by collecting time-resolved measurements of locomotor activity, light exposure, and temperature. Although several open-source packages support specific aspects of actigraphy analysis, aspects such as preprocessing, metric calculation, and mathematical modeling are often distributed across separate software packages, limiting interoperability and increasing programming overhead. Here we introduce circStudio, a Python package that unifies actigraphy data processing and mathematical modeling of circadian rhythms within a single framework. Built from the pyActigraphy codebase and integrating circadian models from the Arcascope circadian package, circStudio provides flexible preprocessing tools, support for multiple actigraphy file formats through adaptor classes, standalone functions for computing commonly used actigraphy metrics, and implementations of several mathematical models of circadian rhythms. The package enables users to move efficiently from raw wearable data to physiologically interpretable circadian outputs. Ultimately, circStudio aims to facilitate reproducible workflows and to provide a flexible foundation for research applications across circadian biology, sleep science, and digital health.

ImportancePolysubstance use is common, but substance use associations with neuroimaging measures have largely been investigated within individual drug types. Whether effects are substance-specific or -general, and how predispositional risk and exposure contribute, remains unclear. ObjectiveIdentify shared and unique associations between substance use and brain structure, and characterize the contributions of predispositional risk and environmental exposure, in a large sample of young adults in the US. DesignThis cross-sectional family-based study used data from the Human Connectome Project (2017 release, collected from 2012-2015). SettingData were collected at Washington University in St. Louis, MO, USA. ParticipantsTwins, non-twin siblings, and singletons with magnetic resonance imaging (MRI) and substance use self-report were included in the analysis. Data were analyzed in 2025. ExposureHistory of substance use was assessed using the Semi-Structured Assessment for the Genetics of Alcoholism. Variables included lifetime use, heavy or past-year hazardous use, and age of use onset for alcohol, marijuana, tobacco, and illicit drugs. Additionally, alcohol and marijuana dependence were assessed. Main Outcomes and MeasuresLinear mixed-effect models examined associations between substance use and brain structure, with an initial focus on past-year hazardous alcohol use, as 95% of the sample endorsed lifetime alcohol use. Analyses then tested associations with other substance use variables, and whether effects were shared or substance-specific. Between-family, within-family, and genetic variance component analyses tested risk and exposure effects. Results1,113 participants (N = 445 families; ages 22 - 37; M=28.8, SD=3.7) had no missing data for the primary analyses. Hazardous alcohol use was negatively associated with global brain thickness ({beta} = -0.12, p < 0.001), which explained all other regional and global associations. Of the drugs with a shared-effect on global brain thickness, only lifetime marijuana use explained unique variance over alcohol ({beta} = -0.08, p = 0.013). Within-family analyses found evidence for unique putative exposure effects of both alcohol ({beta} = -0.11, p < 0.001) and marijuana use ({beta} = -0.07, p = 0.002) on global thickness. Marijuana use further showed a predispositional effect, both in between-family comparisons ({beta} = -0.11, p = 0.007) and genetic variance component analyses ({rho}G = -0.2, p = 0.004), which were not explained by alcohol use. Conclusions and RelevanceBrain structural associations with substance use reflect substance-general and -specific effects, as well as a combination of predispositional and exposure effects. Findings suggest that the negative consequences of polysubstance use may reflect the additive effects of multiple unique exposures.

Background: Attention problems are common transdiagnostic symptoms of psychiatric illness. Although environmental exposures and experiences influence attention during adolescent development, the underlying neural pathways by which they do so is unclear. Methods: We measured attention problems, attention-related brain networks, and multidimensional environmental experiences (the exposome) using data from the ABCD Study (N = 11,878). We tested whether the exposome is associated with 9-10-year-olds attention-related brain network strength and current and future attention problems. We further examined cross-sectional indirect pathways linking the exposome, brain network strength, and attention problems. Results: The exposome predicted youths current and future self-, caregiver-, and teacher-reported attention problems as well as their current attention-related brain network strength. This brain network signature of sustained attention also predicted attention problems from all three reporters. Indirect effects models revealed that the exposome was associated with current reported attention problems both directly and indirectly though this brain signature. Conversely, predictive brain network strength was related to attention problems both directly and indirectly through the exposome. Conclusion: Interactions between environmental exposures, experiences, and brain network organization are associated with attention problems in early adolescence. These findings support a bidirectional framework linking the environment and functional brain networks in the development of attention problems.

Nearly one third of women of reproductive age in the United States are prescribed opioids annually; 14% of women fill an opioid prescription during pregnancy, and one in five report misuse. Opioid use during pregnancy has given rise to an increasing population of infants born with gestational opioid exposure. Although substantial clinical work has focused on treating these infants as they experience opioid withdrawal symptoms at the time of birth, notably few studies have examined the effects of gestational opioid exposure on brain development and long-term cognitive function. During typical brain development, endogenous opioids and their receptors are highly expressed by neural progenitor cells, neurons, and glia where they modulate cell proliferation, differentiation, and maturation. Thus, any disruption to the endogenous opioid system during the critical period of brain development may have lasting consequences on brain cell populations and the behaviors they influence. Indeed, opioid-exposed infants have smaller brains than age-matched peers and show significant neurodevelopmental impairment; they also have higher rates of learning disability at school age. To investigate how exposure to exogenous opioids during brain development affects neural maturation in the hippocampus, a brain region critical for learning and memory, our lab has developed a clinically relevant perigestational morphine exposure rat model. The current study reports that perigestational exposure to morphine delays postnatal hippocampal neuronal maturation, alters astrocyte and oligodendrocyte proliferation, and alters expression of brain-derived neurotrophic factor (BDNF), a protein crucial for healthy brain growth. Furthermore, we show that environmental enrichment rescues BDNF deficits, offering evidence for the effectiveness of non-invasive, non-pharmacological intervention for developmental consequences of perigestational opioid exposure.

Background and aimsPerseverative behaviours are commonly assessed using operant paradigms in which rodents work for drugs or food under physiological deprivation, limiting translational relevance to some behavioural addictions. Here we validated an operant paradigm in which the acquired behaviour is driven neither by physiological needs nor hedonic responses. MethodsMice were trained to lever-press for green light. Exp.1 used a within-subjects design to examine lever discrimination and whether responding could be "satiated" by light preexposure. Exp.2 examined instrumental contingency using a between-subjects design, with light delivery equated between contingent and non-contingent groups. Exp.3 replaced green light with dim red light producing less retinal photoreceptor excitation but comparable heat to assess non-photic cues. Exp.4 examined whether green light could affect food seeking different motivational states. ResultsIn Exp.1, green light supported lever discrimination. Among high responders, the satiation effect was modest (<15% reduction) and did not deter lever pressing. In Exp.2, instrumental contingency promoted response acquisition whereas random light delivery did not. In Exp.3, dim red light failed to sustain behaviour, producing [~]50% response decrement. In Exp.4, light potentiated food seeking under ad libitum feeding. Discussion and conclusionsResponse-contingent light serves as a reward to establish operant responding, which cannot be explained by alerting effects or thermal cues. Our study bridges the gap between animal models and findings from humans that coloured light may exacerbate smartphone use and that light therapy may reshape reward circuits in individuals with Internet gaming disorder symptoms [Li et al. (2026) Advanced Science 13:e14044].

ABSTRACT Background and aims: Mental and behavioral disorders due to use of tobacco (MBDT) present a critical challenge to global health, yet modifiable lifestyle factors for reducing its risk remain poorly understood. Given that dietary fibre can affect mental health through gut-brain communication, we sought to explore how fibre intake relates to MBDT risks in smokers. Methods: We specifically evaluated the link between dietary fibre intake and MBDT within a smoking population. Utilizing the UK Biobank (UKB) database, we performed cross-sectional (N=19,943) and prospective cohort (N=19,885) evaluations applying logistic and Cox proportional hazards models, respectively. To determine potential causality, two-sample Mendelian randomization (MR) was applied, relying on GWAS summary data derived from the IEU Open GWAS Project and FinnGen repositories. Results: Cross-sectional findings indicated that individuals in the top quartile (Q4) of fibre intake exhibited decreased MBDT risks relative to the bottom quartile (Q1) (OR: 0.32, 95% CI: 0.13-0.79). Over a median observation time of 12.84 years, the prospective evaluation demonstrated a notable inverse correlation (Q4 HR: 0.46, 95% CI: 0.40-0.54). Non-linear modeling via restricted cubic splines uncovered an L-shaped dose-response curve. Furthermore, MR results confirmed a genetically predicted protective causality (IVW OR: 0.68, 95% CI: 0.49-0.95), which remained consistent across sensitivity validations. Conclusions: Among smokers, higher dietary fibre intake is robustly associated with a reduced risk of mental and behavioral disorders due to the use of tobacco, offering a modifiable dietary target for public health interventions.

Introduction: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation with complex etiology involving both genetic and environmental factors. Epigenetic mechanisms may mediate environmental contributions, but separating genetic from environmental effects remains challenging. Methods: We present an epigenome-wide association study with 32 monozygotic and 22 dizygotic twin pairs discordant for NSCL/P on blood and saliva samples. Differential methylation analysis was conducted using linear models to identify CpG sites showing significant methylation differences between affected and unaffected twins followed by functional annotation and pathway enrichment analysis. Results: The top-ranked finding is a differentially methylated region comprising two CpG sites at the CYP26A1 locus, cg12110262 (P = 3.21x10-7) and cg15055355 (P = 1.39x10-3). CYP26A1 is essential for retinoic acid catabolism and craniofacial patterning. The chromatin regulator ANKRD11, which causes KBG syndrome featuring cleft palate was the second best hit. Differentially methylated CpG sites showed significant enrichment in craniofacial enhancers and overlap with multiple GWAS-validated cleft genes including VAX1, PVRL1, SMAD3, and PRDM16. Conclusions: Our findings implicate retinoic acid signaling and chromatin regulation in NSCL/P etiology and demonstrate the value of discordant twin designs for distinguishing environmental from genetic epigenetic contributions to complex malformations.

The measurement of inbreeding has gained significance across diverse fields, including population and conservation genetics, agricultural genetics, breeding programs for animals and plants, and wildlife management. This is due to the fact that inbreeding leads to increased homozygosity and results in lower genetic diversity, rendering populations more vulnerable to environmental changes, diseases, and other stressors. High or mid-coverage whole genome sequencing (WGS) has been widely used for inbreeding estimation, but it is resource-intensive. We aimed to investigate the use of ultra low-coverage whole genome sequencing (ulcWGS) as a cost-effective alternative for inbreeding analysis. Domestic dogs were used for our study as their extensive breeding histories lead to populations with a wide range of inbreeding levels. We constructed a multi-breed reference panel from high-coverage WGS samples. Inbreeding in independent ulcWGS samples was then estimated using runs of homozygosity (RoH) and inbreeding coefficients (F). We modeled the relationship between these measures and sequencing depth using nonlinear regression, to generate inbreeding estimates relative to sequencing depth. Resulting relative RoH and F measurements were significantly correlated, with purebred dogs exhibiting more runs of homozygosity and higher inbreeding coefficients compared to mixed-breed dogs. Our findings demonstrate that ulcWGS can provide reliable and economical estimations of inbreeding, expanding accessibility to genetic monitoring.

Opioid and methamphetamine use disorders (OUD and MUD) are characterized by enduring neural adaptations within brain reward circuitry, yet the cell-type-specific post-transcriptional mechanisms underlying these changes remain poorly understood. While microglia are essential for maintaining central nervous system homeostasis and modulating neuroinflammatory responses to drugs of abuse, their alternative splicing (AS) programs have not been defined in the context of addiction. This study characterized the microglial AS landscape in the mouse dorsal striatum during morphine and methamphetamine intravenous self-administration (IVSA), as well as following a 21-day period of abstinence. Analysis of RNA-sequencing data using rMATS and DEXSeq revealed that both drugs significantly dysregulate core splicing machinery, with skipped exons (SE) emerging as the most prevalent splicing event. Notably, morphine exposure induced a robust persistent splicing signature, comprising 736 exonic regions in 221 genes that remained altered through abstinence, whereas methamphetamine-induced changes were primarily reversible. Functional annotation predicted that approximately 27.5% of these events induce frameshifts, potentially impacting critical microglial pathways such as autophagy (Wdr81), chromatin remodeling (Chd4, Kmt2c), and RNA processing (Hnrnpl, Mbnl2, Tia1). These findings identify previously unrecognized post-transcriptional neuroimmune mechanisms and suggest that persistent splicing dysregulation in microglia may contribute to the long-term pathophysiology of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/716002v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c30f01org.highwire.dtl.DTLVardef@10e16d7org.highwire.dtl.DTLVardef@1fd80dforg.highwire.dtl.DTLVardef@17c80f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Wellbeing is commonly defined as the combination of feeling good and functioning well and typically conceptualized as two related but distinct components. Hedonic wellbeing emphasizes pleasure, happiness, and life satisfaction, while eudaimonic wellbeing focuses on meaning, personal growth, flourishing, and the realization of ones potential. The Mental Health Continuum-Short Form was developed as a comprehensive measure of wellbeing and includes three subscales assessing emotional, social, and psychological wellbeing. Although the Mental Health Continuum total score is often interpreted as an indicator of overall wellbeing, the underlying genetic structure of its three subscales and its genetic overlap with other commonly used wellbeing measures remains unclear. Using data from 5,212 individuals from the Netherlands Twin Register (72% female, mean age 36.4), we fitted multivariate twin models to examine the genetic architecture of the Mental Health Continuum and its associations with other wellbeing measures (quality of life, life satisfaction, subjective happiness, and flourishing). Results indicate that, at the genetic level, the Mental Health Continuum is best explained by its three distinct subscales rather than by a latent factor. When considering the Mental Health Continuum together with the other wellbeing measures, we found moderate to high genetic correlations (r = 0.52 - 0.83), indicating substantial overlap in the genetics underlying the wellbeing constructs. However, we did not find evidence for a single common genetic factor underlying all constructs. These findings highlight the multidimensional structure of wellbeing, but the moderate to high genetic correlations across measures suggest that it is important to align the level of measurement (phenotypic vs genetic) with the research question.