Biology of Sex Differences

Introduction The aim of this study was to determine the annual age- and sex-specific prevalence of gender-affirming hormone and puberty blocker use among young people with a gender incongruence (GI) diagnosis in Norway. Methods We integrated data from multiple Norwegian national registers to perform a nationwide register-based study of individuals with known sex assigned at birth who were born in the period 1975-2017 and resident in Norway for all or part of the period 2008-2022. We first calculated the annual age- and sex-specific incidence of GI diagnoses in the population. Then, we calculated the annual age- and sex-specific prevalence of androgen, estrogen, and puberty blocker use among individuals with a GI diagnosis who were under age 25 (for androgens and estrogens) or 18 (for puberty blockers) in the year that they collected the prescription. Results The incidence of GI diagnoses has increased among youth in Norway, most notably since 2015 and with the largest increase among teens assigned female at birth. The prevalence of feminizing and masculinizing hormone therapy has increased in this period as well, but mainly among the oldest teens and young adults. The prevalence of puberty suppression is mostly low but has also increased since 2015, especially in recent years among teens assigned male at birth. Conclusion The prevalence of gender-affirming hormone and puberty blocker use has increased among transgender youth in Norway, concurrently with an increase in the incidence of GI diagnoses.

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.

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and a leading cause of female infertility, with complex genetic, metabolic, and hormonal etiologies. Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes, yet their roles in PCOS remain underexplored. Here, we identified and characterized PCOS differentially expressed gene-associated lncRNAs (PDEGAL) with an integrative approach combining expression data, genetic association, and evolutionary analysis. MethodsThirty-three PCOS-associated protein-coding genes were obtained from our prior study, and all their nearby and overlapping lncRNAs were annotated. These candidates were analyzed using UCSC Genome Browser-mapped annotations and datasets, including NCBI RefSeq, GENCODE, GTEx, GWAS SNPs, and conservation, as well as the FANTOM5 cap analysis of gene expression (CAGE) promoter data, to assess their expression, regulatory potential, genetic variant overlaps, and evolutionary conservation. ResultsTwenty-three PDEGALs (18 antisense to, and 5 sharing bidirectional promoters with, known PCOS-associated protein-coding genes) were identified. 17 PDEGALs contained GWAS SNPs with statistically significant disease associations, 9 of which were associated with PCOS-related traits. 5 PDEGALs demonstrated expression in the KGN granulosa cell model of PCOS. Key gene structure element (KGSE) analysis revealed that most PDEGALs are primate-specific. Integrating four criteria--GTEx expression, GWAS SNPs, FANTOM promoterome, and KGSE conservation--highlighted HELLPAR as the only lncRNA fulfilling all four, while five others--PGR-AS1, MTOR-AS1, ENSG00000265179, ENSG00000256218, and LOC105377276--fulfilled three of the four criteria. ConclusionsWe have systematically identified candidate PCOS regulatory lncRNAs with convergent genetic, expression, and evolutionary evidence. These results provide a framework for functional validation and highlight lncRNAs as potential biomarkers and therapeutic targets in PCOS that function by regulating their nearby and overlapping protein-coding genes.

Alternative polyadenylation (APA) is a common posttranscriptional mechanism to regulate gene expression. APA generates mRNAs with varying lengths of 3' UTRs or transcripts that encode distinct protein carboxy-terminal ends. APA is especially important in neurons, where different mRNA variants are often asymmetrically localized to dendrites and axons, and can be locally translated into proteins. Local protein synthesis is crucial for axon guidance, synaptic plasticity, and learning and memory, key processes associated with the development of alcohol use disorder (AUD). We investigated the role of APA in AUD using a mouse model of alcohol dependence characterized by increased voluntary drinking after chronic intermittent ethanol (CIE) exposure. We examined APA during protracted withdrawal from alcohol in three brain regions of male and female mice. Our analyses revealed hundreds of genes undergoing APA in males, but substantially fewer in females, suggesting sex-specific effects of CIE on APA. Notably, male and female mice displayed distinct APA signatures. APA genes were different from differentially expressed genes (DEGs), suggesting that these molecular processes are regulated independently. We also determined that the expression of APA genes was associated with neurons, while DEGs were associated with non-neuronal cells. Many of the APA genes were involved in synaptic integrity, neuroplasticity, and neuronal maintenance, which was consistent with their enrichment in neurons. Our study suggests that APA is a crucial sex- and cell type-specific mechanism in AUD with the potential to influence localized neuronal protein expression during protracted withdrawal and to modify alcohol consumption behavior. HIGHLIGHTSO_LIChronic ethanol exposure in mice results in profound changes of APA genes in brain. C_LIO_LICommonly regulated cleavage and polyadenylation sites and genes were identified in male but not in female mice. C_LIO_LIThere was a minimal overlap between APA and differentially expressed genes (DEGs). C_LIO_LIAPA genes were primarily associated with neurons, whereas DEGs were associated with non-neuronal cells. C_LI

Negative cognitive biases in depression are more pronounced in females than in males. This sex difference emerges during adolescence, a sensitive developmental stage when chronic stress exposure increases the risk of depression in adulthood. The neurobiology linking adolescent stress to sex-specific cognitive bias and resting-state network reorganization in adults remain poorly understood. The study aimed to investigate the longitudinal effects of chronic restraint stress (CRS) during adolescence on cognitive bias and functional connectome in emerging adulthood. 28 Wistar rats (sex-balanced; aged five weeks on arrival) were trained on a judgment bias task with distinct tactile cues signalling differential rewards. Cognitive bias was quantified from responses to ambiguous probe trials. Following training, animals were randomly and equally assigned to CRS or control groups (sex-balanced). Offline resting-state functional MRI scans were conducted at adolescent baseline (pre-CRS) and again in adulthood (post-CRS), followed by probe trials to assess neural and behavioural changes. Following CRS, females showed a greater tendency to shift toward negative bias than males (ratio of odds ratio=3.67). Furthermore, CRS significantly reduced functional connectivity between the left cerebellar-auditory and hypothalamic-thalamic networks only in females. Repeated-measures correlation between cognitive bias and network connectivity were not statistically significant across sex-by-group strata, potentially due to offline imaging and small sample size. However, intra-individual association revealed sex-specific trends, with CRS females showing moderately positive correlations and CRS males exhibiting a weak negative association. The results could inform stratified connectome-based interventions targeting adolescent stress exposures to potentially reduce the risk of adult depression. Six keywords: Resting-State Functional MRI, Chronic Restraint Stress, Judgement Bias, Open Field Test, Sex Differences

BackgroundHormonal transition phases represent windows of increased neuroplasticity across the female lifespan. In this study, we aim to investigate the brain anatomical architecture of hormonal transition phases by directly comparing menarche, as a period of rising levels of steroid hormones, and menopause, as a time of declining levels. MethodsWe fit linear models on cross-sectional and linear mixed-effect models on longitudinal magnetic resonance imaging (MRI) datasets, to explore the effects of menarche onset (ABCD study data, Ncross-sectional=1274, Nlongitudinal=611) and transition into menopause (UK Biobank data, Ncross-sectional=1614, Nlongitudinal=212) on 66 cortical and 135 subcortical brain volumes, and to identify brain structures with opposing but regional overlapping effects in both periods. Models were adjusted for age and corrected for multiple comparison (P <.05; FDR-corrected). ResultsCross-sectionally, using a between-subject design, 83 brain volumes showed effects of menarche-onset and 17 volumes showed effects of menopause-transition. Of these, seven brain volumes were significantly affected by both transitional periods, showing opposing directional volume changes. Longitudinally, using a within-subject design, 56 brain volumes exhibited menarche effects, of which 46 replicated cross-sectionally. No menopause effect survived correction for multiple comparison, likely due to limited longitudinal sample size. ConclusionOur findings confirm regionally overlapping brain structural alteration between the two hormonal phases - menarche and menopause - showing the hypothesized opposite effect directions. Additionally, our results show the robustness of menarche effects, which converged across cross-sectional and longitudinal study designs. Taken together, our results contribute to a better understanding of hormone related neuroplasticity, emphasizing the importance of not only understanding individual phases, but understanding the overarching patterns across the female reproductive lifespan.

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.

Background: Adolescence is a critical period of neurodevelopment with the emergence of chronic medical conditions and increasing exposure to long-term medications. P-tau217 is a sensitive blood-based biomarker of neuropathology in older adults, yet its developmental behavior and susceptibility to common clinical factors in youth are unclear. Here we tested whether p-tau217 varies with age, comorbidity, or medication use during adolescence; and whether collection method (venous vs Tasso+ capillary) yields comparable concentrations. Methods: In an adolescent cohort, plasma p-tau217 was measured by Simoa-X. Paired venous and Tasso+ capillary samples were also analyzed from adult volunteers for methodological comparison Results: In adolescents (n=41; mean age 16{+/-}2.6 years), p-tau217 did not correlate with age or BMI z-score and did not differ by psychiatric, cardiometabolic, or gastrointestinal comorbidity, nor by corresponding medication use. In contrast, p-tau217 concentrations were >10-fold higher in Tasso+ capillary plasma than venous plasma, a discordance replicated in paired adult samples. Conclusion: Plasma p-tau217 appears physiologically stable across common clinical variables in adolescence, but highly sensitive to biospecimen collection method. Venous and Tasso+ capillary plasma should not be directly compared or pooled until methodological differences are resolved. These data provide a developmental baseline and critical methodological caution for pediatric neuroscience and decentralized biomarker studies.

Cannabis (marijuana) is the most widely used recreational drug in the USA accounting for about 62 million users in 2024. Among cannabis users, 26% are of prime reproductive age (18-25 years). Delta-9 tetrahydrocannabinol (THC) is the principal psychoactive component of cannabis and has been detected in human seminal fluids. Although abundant evidence indicates adverse effects of THC exposure on spermatogenesis in different species, acute effects of THC on postejaculatory sperm including fertilization potential and subsequent carryover effects on embryo development are largely unknown. The present study was designed to provide missing information on structural and mechanistic effects of THC exposure to postejaculatory sperm function by evaluating sperm indices often overlooked or masked during clinical evaluation. A bovine embryo continuum model was employed to determine effects of THC on sperm structure, kinematics, bioenergetics, and binding mechanisms. Effects of THC on the sperm genomic and epigenomic landscape were determined, complemented by paternal carry over effects on embryo development as a human translational model to elucidate paternal effects on future development, and to mirror sperm exposure during transport within the female reproductive tract. Cryopreserved bovine sperm from three bulls were independently exposed to physiologically relevant concentrations of THC (0 and 32nM, n = 2 individual replicates/bull) for 24 h under non-capacitating conditions at 25{degrees}C followed by quantification of sperm kinematics at 37{degrees}C. Samples of THC-exposed sperm and vehicle-control (0.1% DMSO) were collected in replicate following immediate addition of THC (0 h) and again at 24 h. DNA damage, acrosome integrity, bioenergetics, changes to DNA methylation and embryo development were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. Computer-assisted sperm assessment revealed a reduction in progressive motility of THC-exposed sperm after 24 h while other parameters were not affected. Acrosome integrity as determined by flowcytometric analysis with FITC-PSA was severely compromised in THC-exposed sperm (P [&le;] 0.05), despite no detectable difference in capacitation status using merocyanine staining. Similarly, DNA integrity as determined by TUNEL assay was significantly impaired after 24 h of THC exposure (P [&le;] 0.05). Mechanistic effects of THC were explored through characterization of the transmembrane G-protein coupled cannabinoid 1 receptor (CB1). CB1 is expressed in the post-acrosomal region and its abundance decreased as compared to unexposed sperm. Alterations to the methylation landscape of sperm were then determined after 24 h of THC exposure through whole-genome Enzymatic Methyl Sequencing. PCA analysis indicated that sperm from different males formed distinct clusters, implying individual differences among bulls, while the effects of THC exposure produced tighter clusters. Paternal carryover effects on embryos derived by in vitro fertilization from THC exposed sperm had reduced 2-cell cleavage, 8-16 cell morula development, and reduced blastocyst development compared to unexposed sperm (46% vs. 33%). In conclusion, post-ejaculatory mammalian sperm exposure to THC compromises acrosome integrity, induces DNA damage, changes the sperm methylome, and reduces developmental potential. Collectively, these data implicate new considerations for recreational and clinical use of cannabis that impact cellular and molecular mechanisms important for sperm function with detrimental consequences for gamete interaction and embryo development.

Adolescent binge drinking is a strong predictor of alcohol use disorder and related mental health outcomes in adulthood, which may be due to disruptions in myelination during this dynamic period of brain development. White matter expansion in frontal regions during adolescence is essential for mature decision-making and stress regulation, yet the cellular mechanisms by which alcohol disrupts this process remain poorly understood. We used multi-label immunofluorescence and confocal microscopy to visualize proteins in oligodendrocyte lineage cells and myelin ensheathment of axons in the anterior cingulate cortex (Cg1) and corpus callosum (CC) following four weeks of episodic voluntary binge drinking using the Drinking-in-the-Dark model in adolescent male and female C57BL/6NJ mice beginning on postnatal day 28. Contrary to our initial hypothesis that alcohol targets early-stage oligodendrocyte precursor cells (OPCs), binge drinking selectively depleted mature oligodendrocytes expressing aspartoacylase (ASPA) in the Cg1 and CC of male mice, but not females. This enzyme is essential for lipid biosynthesis and myelin production, and this cell-specific loss was accompanied by significant hypomyelination of axons only in males. These findings identify a later maturational stage of oligodendroglial development as a sex-dependent target of alcohol, advancing our mechanistic understanding of prefrontal myelin deficits in adolescent drinking. Furthermore, ASPA emerges as a potential therapeutic target for alcohol use disorder and demyelinating diseases, with differential vulnerability across sex carrying important implications for adult neurodevelopmental outcomes.

Astrocytes play essential roles in maintaining brain homeostasis and in contributing to synaptic functions, but, in response to injury, infection, or disease, astrocytes can downregulate their homeostatic and physiological functions while increasing neuroinflammatory responses. The central amygdala (CeA) is important for stress responsivity and the development of alcohol (ethanol) dependence. Using a multi-omics approach in Aldh1l1-EGFP/Rpl10a mice and the chronic intermittent ethanol two-bottle choice (CIE-2BC) model, we have characterized the translational response of CeA astrocytes, as well as the proteomic and phosphoproteomic changes in ethanol dependent, non-dependent, and naive mice. We identified astrocyte-specific alterations in neuroimmune functions and antioxidant/oxidative stress pathways in ethanol dependent mice as well as cytoskeletal plasticity related pathways in non-dependent mice. Proteomic analysis showed down-regulation of astrocyte physiological functions in dependent animals while phosphoproteomic analysis identified pathways associated with cytoskeleton remodeling in both dependent and non-dependent mice. Reconstructions of astrocyte morphologies demonstrated increased CeA astrocyte complexity in dependent and non-dependent groups compared to naive mice. The astrocyte-specific activation of neuroimmune and antioxidant pathways, down-regulation of homeostatic functions, alteration in protein phosphorylation-mediated cytoskeleton remodeling, and increased astrocyte morphological complexity demonstrate that ethanol dependence induces astrocyte reactivity in the CeA consistent with both adaptive and maladaptive changes. These findings highlight the role of CeA astrocytes in the progression from alcohol intake to dependence and represent a first step toward identifying astrocyte-specific therapeutic strategies to treat Alcohol Use Disorder (AUD) aimed at potentiating reactive astrocyte adaptive changes and inhibiting maladaptive responses.

Objectives: Accurate classification of menopausal stage is fundamental to midlife health research. Although the Stages of Reproductive Ageing Workshop (STRAW+10) criteria provide gold-standard criteria, their application in research settings is inconsistent. Classification challenges are compounded in individuals without observable menstrual cycles due to surgical or contraceptive-induced amenorrhoea. The Reproductive Ageing in Women (RAW) Questionnaire and accompanying classification Framework was developed and validated to improve consistency and inclusivity when classifying menopausal stage. Study design: A multi-phase study was conducted between May 2022 and July 2025. Phase one involved questionnaire development based on STRAW+10 and the Menopause-Specific Quality of Life Questionnaire. Phase two assessed content validity via expert review (n=3). Phase three evaluated face validity using think-aloud interviews and focus groups (n=14). Phase four validated RAW within a cross-sectional cohort study (n=156), and assessed construct validity (n=30), test-retest reliability (6-21 days; n=128; Kendall's Tau-b and Cohen's kappa), and biological validity using follicle stimulating hormone (FSH). Results: Feedback supported clarity, relevance and usability, with refinements improving inclusivity for surgical and contraceptive-induced amenorrhoea. Construct validity demonstrated consistent application of classification criteria. Questionnaire classification showed 93% concordance with self-identified menopausal status in the construct sample and 87.8% agreement within the cohort sample. Test-retest reliability was excellent ({tau}, p=0.940, p<0.001). Follicle stimulating hormone levels differed across RAW-classified stages (p<0.001), with 96.1% concordance between RAW pre and postmenopausal classifications and FSH thresholds. Conclusions: Prioritising menstrual characteristics while incorporating age and symptom criteria improves methodological consistency and inclusivity in menopausal stage classification. Longitudinal validation is warranted to assess temporal sensitivity across the menopausal transition.

Endocannabinoid (eCB) signaling is a key regulator of reward-related dopaminergic signaling, particularly in response to drugs of abuse, such as cocaine. To date, our understanding of this mechanism has primarily been limited to male subjects. Prior work establishes that female cocaine users have more adverse outcomes, and female rats show greater sensitivity to cannabinoid type 1 receptor (CB1R) regulation of cocaine self-administration. Therefore, we hypothesize that female rats exhibit enhanced eCB regulation of cocaine-evoked dopamine (DA). We used in vivo fiber photometry recording of the dopamine biosensor, dLight 1.3b, in the nucleus accumbens medial shell (NAcms) in response to cocaine in male and female rats. Rats were pretreated with cannabinoid-targeting drugs to investigate the effects of CB1R inactivation or augmentation of the eCB 2-AG on cocaine-evoked DA. Our results revealed that CB1R inactivation attenuates cocaine-evoked DA in male and female rats, but females showed enhanced sensitivity for CB1R regulation of cocaine-evoked DA. Cocaine-evoked DA was enhanced by augmenting 2-AG levels, and females again showed increased sensitivity to this manipulation. Finally, females show greater cocaine-evoked DA when in a non-estrous cycle compared to estrous, reinforcing that estrous cycle is a determinant of cocaine-evoked DA. These data indicate that females show enhanced eCB regulation of cocaine-evoked DA signaling, underscoring the importance of sex as a biological variable in our understanding of endocannabinoid regulation of drug reward. HighlightsO_LICB1R inactivation attenuates cocaine-evoked DA in NAcms, preferentially in females C_LIO_LI2-AG augmentation via MAGL inhibition enhances cocaine-evoked DA, with female bias C_LIO_LIEstrous phase modulates the dopamine response to a high dose of cocaine in females C_LIO_LIMale and female rats show similar baseline DA and locomotor responses to cocaine C_LI

Male odor induces various behavioral and physiological responses across the reproductive cycle in female mice. Although male odor preference in females is reduced during pregnancy, how it changes across later stages of the reproductive cycle, including nursing and weaning, remains unclear. Here, we found that male odor preference is lost during pregnancy and nursing. To identify the olfactory systems involved in these changes, we examined neural activity using c-Fos immunohistochemistry. Male odor exposure during nursing increased neural activity in the accessory olfactory bulb and the posteroventral medial amygdala (MeApv), a key node of the accessory olfactory system, as well as in subdivisions of the central amygdala, but not in the ventromedial hypothalamus or the bed nucleus of the stria terminalis. Finally, lesions of the MeApv prevented the loss of male preference during nursing, indicating that the MeApv is required for suppression of male preference during this stage.

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.

BackgroundSubstance use initiation in adolescence is influenced by both genetic and environmental factors; however, large-scale genetic studies often treat initiation as a binary outcome and underuse longitudinal timing information. MethodsWe conducted time-to-event (survival) genome-wide association analyses (GWAS) of initiation for four outcomes--alcohol, nicotine, cannabis, and any substance use--using longitudinal follow-up data from the Adolescent Brain Cognitive Development (ABCD) Study. We performed ancestry-stratified GWAS within European (EUR), African (AFR), and Hispanic (HISP) groups, applying consistent quality control and covariate adjustment. Summary statistics were harmonized across ancestries and meta-analyzed using inverse-variance weighted fixed-effects and DerSimonian-Laird random-effects models. We evaluated genomic inflation and heterogeneity (Cochrans Q and I2), identified independent lead variants at genome-wide and suggestive significance thresholds, and assessed cross-trait overlap of associated loci. ResultsIn the multi-ancestry meta-analysis, we observed suggestive association signals across traits (minimum p-values: alcohol [~] 1 x 10-7, any [~] 1 x 10-7, cannabis [~] 5 x 10-8, nicotine [~] 1 x 10-8). Nicotine initiation showed one genome-wide significant variant in both fixed- and random-effects meta-analyses (p < 5 x 10-8). Across traits, suggestive loci demonstrated limited overlap, with the strongest concordance between alcohol and any substance use, consistent with shared liability. Heterogeneity statistics indicated that some loci exhibited cross-ancestry variation in effect estimates. ConclusionsSurvival GWAS leveraging initiation timing can identify genetic signals that may be missed by binary designs and enables principled multi-ancestry synthesis. Our results highlight both shared and trait-specific genetic contributions to early substance initiation and provide a foundation for downstream functional annotation and integrative modeling with environmental risk factors. These findings demonstrate the value of incorporating developmental timing into genetic discovery and provide a framework for integrating longitudinal risk modeling with genomic analyses.

An increasing number of epidemiological and experimental studies have demonstrated a bidirectional relationship between mood disorders and the circadian system, with disrupted circadian rhythms contributing to depressive states, and their restoration playing a key role in antidepressants effects. In this context, we sought to examine whether key molecular targets of antidepressants exhibit diurnal regulatory patterns. Naive adult male and female C57BL/6 mice were euthanized at 3-hour intervals beginning at Zeitgeber Time 0 (ZT0), and hippocampal (HC) and medial prefrontal cortex (mPFC) tissues were collected for RT-qPCR and western blot analyses. We observed statistically significant diurnal rhythmicity in all analyzed transcripts (cFos, Arc, Nr4a1, Dusp1, Dusp5, and Dusp6) in both HC and mPFC samples, with peak expression occurring during the dark (active) phase (ZT15-18). Phosphorylation levels of TrkBY816 (tropomyosin-related kinase) and GSK3{beta}S9 (glycogen synthase kinase 3{beta}) also showed periodic rhythmicity, peaking during the light (inactive) phase. Levels of p-ERK2T185/Y187 (extracellular-signal regulated kinase) did not display rhythmicity, but peaked during the light phase in the HC, especially in males. Collectively, these findings demonstrate that antidepressant targets are subject to diurnal regulation, highlighting the importance of integrating circadian biology and time-of-day as relevant variables in the development of translationally relevant antidepressant research. HighlightsO_LIKey molecular targets of antidepressants exhibit diurnal regulation in adult mice C_LIO_LIDiurnal patterns were conserved across targets, sexes, and brain regions (HC&PFC) C_LIO_LIcFos, Arc, Nr4a1, Dusp1,5,6 mRNAs display peak expression during the dark phase C_LIO_LITrkBY816 and GSK3{beta}S9 phosphorylation peak during the light (inactive) phase C_LIO_LIAntidepressant mechanisms may be linked with circadian and sleep-wake dynamics C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/716906v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1e65e60org.highwire.dtl.DTLVardef@13e302corg.highwire.dtl.DTLVardef@1ccc25forg.highwire.dtl.DTLVardef@1ed10d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundExposure to prenatal maternal inflammation (PNMI) has been linked to neurodevelopmental alterations in human offspring. Preclinical studies suggest that PNMI disrupts reward circuitry, particularly within mesolimbic circuits. However, the effects of PNMI on mesolimbic circuits (i.e, ventral tegmental area (VTA) projections to the hippocampus (VTA-H) and limbic striatum (VTA-LS)) in humans are not yet known. MethodsData for PNMI biomarkers [interleukin (IL)-6, IL-8, IL-1 receptor antagonist (IL-1ra), soluble TNF receptor-II (sTNF-RII)] from first trimester (T1) and second trimester (T2) maternal sera, and offspring MRI brain scans in late midlife (aged 57-63 years), were available for 89 mother-offspring dyads. Probabilistic tractography delineated bilateral VTA-H and VTA-LS tracts. Macrostructural tract measures were examined using hierarchical linear regressions. Microstructural integrity was assessed using neurite orientation dispersion and density imaging, and permutation-based cluster analyses. ResultsHigher T2 IL-1ra was associated with increased macrostructure (left VTA-H tract), whereas higher T2 sTNF-RII was associated with reduced macrostructure (right VTA-H and VTA-LS tracts) and higher T2 IL-8 (bilateral VTA-LS tracts). Microstructurally, higher T2 IL-6 was associated with increased neurite density (distal cluster, right VTA-H tract), while higher T1 IL-8 was associated with reduced neurite density (near the hippocampus in the left VTA-H tract, near the VTA in bilateral VTA-LS tracts). ConclusionsPNMI was associated with altered mesolimbic reward circuitry in offspring. This suggests that prenatal inflammation may contribute to affective and motivational disorders in offspring via alterations in mesolimbic circuitry.

The nucleus accumbens (NAc) and its excitatory input from the medial prefrontal cortex (mPFC) form a critical circuit underlying drug-induced plasticity associated with addiction-related behaviors. However, baseline differences in excitatory signaling across NAc subcircuits and sex-specific neuroadaptations following opioid self-administration remain poorly understood. Here, we examined synaptic signaling in mPFC-NAc pathways in drug-naive mice and after abstinence from remifentanil self-administration. Under drug-naive conditions, AMPA receptor- mediated glutamatergic signaling was generally elevated in D2 medium spiny neurons (MSNs) of both the NAc core and shell across sexes, while females exhibited greater excitatory signaling in D1 MSNs of the NAc core compared with males. Pathway-specific analyses revealed that prelimbic cortex (PL) inputs to NAc core D2 MSNs displayed enhanced calcium-permeable AMPA receptor (CP-AMPAR) signaling and increased presynaptic release relative to D1 MSNs. Following abstinence from remifentanil self-administration, miniature excitatory postsynaptic current analyses showed increased excitatory drive at D1 MSNs and decreased drive at D2 MSNs, largely restricted to the NAc core. At PL-Core D1 MSN synapses, remifentanil reduced AMPA/NMDA ratios, consistent with increased CP-AMPAR incorporation in males and females, while increasing presynaptic signaling exclusively in males. In contrast, PL-Core D2 MSN synapses showed a reduction in presynaptic signaling across sex, while ostensibly weakening postsynaptic signaling selectively in males through reductions in CP-AMPAR signaling. At infralimbic cortex (IL)-shell inputs, a reduction in AMPAR rectification indices at D1 MSN synapses was produced by remifentanil, while release probability was decreased at D2 MSN synapses in males only. Together, these findings reveal sex- and pathway-specific synaptic adaptations within mPFC-NAc circuits that may be obscured by global measures of excitatory transmission and identify baseline circuit differences that may shape opioid-induced plasticity.

Background Diverse epigenetic clocks are known to capture health risks associated with increased adiposity, but their estimates have never been combined to represent a holistic estimate of biological age acceleration (BAA). There is also a gap in research using epigenetic clocks to study adiposity in lower-middle income Asian countries. Methods and Findings Data from 1,745 participants (21.7{+/-}0.3 years old, 45% female) of the Cebu (Philippines) Longitudinal Health and Nutrition Survey were analyzed. BAA was calculated using PCHorvath 2, PCHannum, PCPhenoAge, PCGrimAge, PCDNAmTL, and DunedinPACE. After ascertaining suitability for factor analysis (Kaiser-Meyer-Olkin 0.81), factor analysis was used to create PCFactorAge. Analogously, FactorAge was created using Horvath, Hannum, PhenoAge, GrimAge, DNAmTL, and DunedinPACE. BMI, waist circumference (WC), and waist-to-height ratio (WHtR) were used to represent adiposity. Linear regression was used to test the association of each adiposity measure with each BAA measure. BMI, WC, and WHtR were positively associated with both BAA combinations: 5 kg/m2 higher BMI corresponded to 0.097 (p=0.015) standard deviation (SD) increase in FactorAge and 0.099 (p=0.004) SD increase in PCFactorAge; 10 cm increase in WC--with 0.091 (p=0.005) SD increase in FactorAge and 0.094 (p<0.001) SD increase in PCFactorAge; 0.1 increase in WHtR--with 0.164 (p=0.001) SD increase in FactorAge and 0.163 (p<0.001) SD increase in PCFactorAge. Additionally, WHtR was associated with meaningful increases in PhenoAge, PCPhenoAge, PCHorvath 2, PCHannum, PCGrimAge, and DunedinPACE. WC was positively associated with PCHorvath 2, PCHannum, PCPhenoAge, and DunedinPACE. BMI was positively associated with PCHannum, PCPhenoAge, and DunedinPACE. Conclusions Our study presents a novel approach to creating a BAA estimate using multiple epigenetic clocks and shows that adiposity measures predict this factor in a young Filipino cohort.