Genes, Brain and Behavior

Cocaine Use Disorder (CUD) poses a significant public health and socioeconomic challenge. Determining the genetic basis of predisposition for development of CUD is challenging in human populations but can be studied in Drosophila. We assessed cocaine consumption and cocaine preference of 74,875 flies from 598 sequenced, wild-derived, inbred lines from the expanded Drosophila melanogaster Genetic Reference Panel (DGRP3). We found significant genetic variation, sexual dimorphism, and genetic variation in sexual dimorphism for these traits. Whereas most lines showed cocaine avoidance, ~10% of the lines showed innate cocaine preference in at least one sex. Genome-wide association analyses for cocaine consumption, preference, and micro-environmental variance of these traits identified 2,155 polymorphisms in/near 866 genes that were enriched for Gene Ontology terms associated with neurogenesis, development, and behavior. Many of the associated genes had human orthologs with known associations with CUD and other substance use disorders as well as psychiatric and behavioral traits. We confirmed causal associations with cocaine preference for three polymorphisms with large effect sizes by assessing their effects in DGRP3 lines not included in the initial association analyses. Pairwise associations between these polymorphisms exhibited suppressing epistasis. These polymorphisms are in genes with human orthologs that fulfill essential functions in the nervous system, including the glucose transporter SLC2A8; KCNC2, a subunit of the voltage gated potassium channel; and CHRNA7, a nicotinic cholinergic receptor subunit. Thus, studies on Drosophila can provide insights into the genetic and neural mechanisms of CUD.

Motivational deficit is a prevalent symptom across a wide range of neurodegenerative and neuropsychiatric disorders. Despite its clinical importance, first-line treatments for these disorders fail to effectively treat this symptom domain. In animal models, motivation is typically assessed in the context of extrinsic reward, where reward is delivered for completing an effortful action. However, many motivated behaviours occur in the absence of a tangible reward and are instead driven by intrinsic motivation. Previous work has shown that an extrinsic motivation task (effort for reward (EfR)) and an intrinsic motivation task (effort based forage (EBF) task) show opposing responses to a range of pharmacological manipulations. However, it is not clear whether intrinsic and extrinsic motivation dissociate in the context of endogenous behavioural variation. We therefore investigated whether these tasks were sensitive to behavioural variation across three different strains of mice (C57Bl/6JJRi, 129S2/SvPasOrlRj and BALB/cJRi) and whether strain profiles diverged across tasks. Here, we found that BALB/c mice showed the lowest levels of foraging in the EBF task, indicative of a low intrinsic motivational state but showed the highest levels of high effort responding in the EfR task, indicative of a high extrinsic motivational state. These differences were not driven by an anxiety-related phenotype and were therefore indicative of a motivation phenotype divergence across tasks. This work highlights the importance of moving away from considering motivation on a single axis, as findings can diverge depending on the nature of the motivational process. This has important implications for both phenotypic interpretation and the development of treatments targeting motivational dysfunction.

Addiction is a complex and heritable trait which progresses through several developmental stages, each of which is presumably influenced by multiple partially overlapping genetic factors. Cocaine initially produces rewarding effects, followed by aversive effects including anxiety, craving, anhedonia, and withdrawal. These aversive effects have been suggested to contribute to the etiology of cocaine use disorders (CUD), as repeated exposure is thought to desensitize the rewarding effects and sensitize the aversive effects through a process involving both aberrant reward-based learning and aberrant avoidance-based learning. We examined the genetic basis of aversion learning using both food-based and cocaine-based behavioral assays in outbred Heterogenous Stock (HS) rats. A total of 1,074 HS rats (35.3% male) underwent runway operant cocaine-seeking, food-based progressive ratio and punishment testing, and locomotion testing. These phenotypes were significantly heritable (with h2 estimates as high as 0.307) and identified significant (p < 0.05) genetic loci related to avoidance-based learning including from the punishment task on Chromosomes 2, 3, 5, and 6, and the cocaine-operant runway latency task on Chromosome X. 172 positional candidate genes were identified from significant and suggestive loci, including Cdh10, Cdh12, Cdh18 which have previously been associated with smoking initiation from human GWAS, Adcy3, Cfap206, and Drc1 which are associated with primary neuronal cilia, as well as SNPs associated with novelty-related and social interaction phenotypes in independent samples of HS rats. Our results suggest that these aversion learning phenotypes are themselves complex heritable traits influenced by multiple genetic loci, which may pleiotropically affect other aspects of addiction biology.

Behavioral test batteries are increasingly used to characterize multiple functional domains in zebrafish, yet the potential impact of test sequence on behavioral outcomes remains poorly defined. Here, we systematically evaluated whether test order influences behavioral responses in a three-assay battery comprising the novel tank test (NTT), mirror-induced aggression (MIA), and social preference (SP) test. Adult zebrafish (Danio rerio) were exposed to all possible permutations of the three assays in a fully counterbalanced design, allowing assessment of order effects across locomotor, anxiety-like, aggression-related, and social behaviors. Test order produced modest and parameter-specific effects, primarily affecting locomotor activity in the NTT and social proximity in the SP assay. Time-course analysis revealed within-test behavioral dynamics, with limited evidence that test order modulates early adaptation or late engagement with the testing environment but does not alter overall temporal response profiles. Sex-dependent effects were assay-specific and most pronounced in the NTT, with no consistent sex differences observed in MIA or SP. To evaluate the global structure of behavioral variation, Principal Component Analysis (PCA) was performed across assays. Despite localized effects of test order, no clear multivariate separation between test sequences was observed, indicating that sequential testing does not produce distinct baseline phenotypes. Together, these findings support the robustness and reproducibility of multidomain behavioral batteries while highlighting the importance of standardized test-order reporting to improve cross-study comparability.

Operant paradigms are powerful tools to quantify motivation and reward. Traditionally, operant conditioning research has been limited to food and drug reinforcers. Recent advances in commercially available operant equipment, however, allow for the quantification of social motivation. These operant assays are an improvement over commonly used social preference tasks, as they enable direct measurement of the effort and motivation driving social behavior. Based on a design by Venniro et al. (2020), the MedPC social operant boxes modify the traditional operant box setup for social interactions. The experimental rat can lever-press to raise a door for an interaction with a target rat behind a porous barrier. These social operant boxes have been widely adapted to test social behavior in adult and adolescent rodents and investigate how a range of conditions (e.g. stress, drug taking, etc.) affect social motivation. However, there is a gap in assessing maternal motivation for pups during the postpartum period, despite ample evidence that postpartum social behavior is highly relevant for offspring health outcomes. Here, we detail 3D-printed modifications to the standard Med PC social operant boxes to adapt the social target chamber to safely house neonatal pups. We have also developed testing protocols to assess motivation during the limited postpartum period. These data demonstrate that, with simple modifications to social operant chambers and testing protocols, the field can implement advanced behavioral approaches to directly assess maternal motivation.

Long-term opioid therapy is limited by analgesic tolerance and opioid-induced hyperalgesia, but the roles of genetic background, sex, and drug exposure remain unclear. We used 20 inbred strains from the Hybrid Rat Diversity Panel to examine thermal sensitivity, oxycodone analgesia, tolerance, and hyperalgesia-like changes following voluntary intravenous oxycodone or saline self-administration. Rats underwent tail-immersion testing before self-administration (Pre-SA) and after self-administration (Post-SA). Oxycodone analgesia was assessed using the percent maximum possible effect time course and the corresponding area under the curve. Pre-SA thermal sensitivity differed across strains and between sexes, and Pre-SA oxycodone analgesia also differed across strains. Oxycodone self-administration produced a sex-dependent increase in thermal sensitivity that was most evident in males. During Post-SA testing, oxycodone self-administering rats showed reduced analgesic responsiveness compared with saline controls, and the magnitude of this difference varied across strains. Within-strain Pre-SA-to-Post-SA comparisons identified tolerance-like reductions in several strains. Across strains and sexes, oxycodone self-administering rats showed a greater Pre-SA-to-Post-SA reduction in analgesic responsiveness than saline controls, consistent with analgesic tolerance. Total oxycodone intake was not associated with tolerance at either the strain-mean or individual-animal level. Heritability estimates were higher for thermal sensitivity and analgesia (H2 {approx} 0.28-0.40) than for changes in thermal sensitivity and tolerance (H2 {approx} 0.18-0.27). These findings demonstrate strain variation in thermal sensitivity and oxycodone analgesia, sex-dependent hyperalgesia-like effects, and reduced analgesic responsiveness following voluntary oxycodone intake.

Phelan-McDermid syndrome (PMS) is a genetic neurodevelopmental disorder caused by a microdeletion within chromosome 22q13.3 1-6, and accounts for [~]1-3% of cases of autism spectrum disorders (ASD). Individuals with PMS typically present with neonatal hypotonia, severe speech delay, intellectual disability, motor impairments, and autism-related features, although additional manifestations such as epilepsy, sleep disturbances, and developmental regression are common. As in ASD, there is considerable heterogeneity in cognitive and behavioral impairments in PMS, making it difficult to determine which features arise directly from SHANK3/Shank3 deficiency and how deficits manifest across development. In the present study, we transferred the targeted Shank3 mutation, previously characterized on the Sprague Dawley background, onto the Long-Evans strain and performed a longitudinal behavioral assessment of Shank3-deficient rats from infancy to adulthood. The rats were evaluated for delays in early sensory and motor development, and ultrasonic vocalizations as neonates, social behavior, short term memory and gait as juveniles, and cognitive-executive dysfunction using a touchscreen-based visual discrimination and reversal learning task as adults. Despite profound reductions and/or complete loss of Shank3 protein expression, heterozygous and homozygous male and female rats showed normal physical and neurological reflexes across development, yet female Shank3 knockout pups showed a selective reduction in distress-associated ultrasonic vocalizations. As juveniles, subtle abnormalities emerged in short-term memory and limb coordination, while social preference for novelty and recognition was normal. Prominent behavioral abnormalities were observed in adults characterized by rapid and error-prone responding to visual stimuli during discrimination learning and reversal. These data suggest that rats with complete or partial Shank3 deficiency produce a selective behavioral profile in which high-order cognitive dysfunction is more pronounced than deficits in basic sensorimotor or select social behaviors. More broadly, these findings highlight executive dysfunction as a key consequence of the loss of Shank3. For the first time, we report the loss of Shank3 expression on a Long-Evans background strain as a valuable translational tool for investigating cognitive dysfunction and therapeutic windows in Shank3-associated disorders.

Background and aimVery preterm birth (VPT; [&le;]32 weeks gestation) is associated with an increased risk of later psychiatric disorders, including psychosis. Although psychosis typically emerges in adulthood, subclinical early signs along the psychosis continuum, such as psychotic-like experiences (PLEs), can be observed much earlier. We therefore aimed to study PLEs in childhood in VPT individuals recruited from a clinical cohort compared with full-term (FT) controls. We subsequently investigated whether findings could be replicated in an independent population-based cohort. MethodsPrimary analyses were conducted in the Brain, Immunity and Psychopathology (BIPP) study, including 197 children born VPT recruited through Neonatal Intensive Care Units and 72 FT controls assessed at a mean age of 10.50{+/-}1.77 years. Between-group differences in PLEs were then examined in the Adolescent Brain Cognitive Development (ABCD) study, including 149 children born VPT and 9519 FT controls assessed at a mean age of 9.94 {+/-} 0.63 years. PLEs were assessed using the Prodromal Questionnaire-Brief Child Version (PQ-BC), yielding frequency and distress-related scores for both the total scale and three specific domains (unusual thought content, perceptual abnormalities, disorganised speech). Regression models tested associations between birth status (VPT and control) and PQ-BC scores adjusting for age, sex, and socio-economic status, with secondary models additionally adjusting for cognitive ability and broader psychopathology. Pooled analyses examined cohort effects (BIPP and ABCD) and cohort-by-group status (VPT and control) interactions. ResultsIn BIPP, VPT birth was associated with higher PQ-BC total ({beta}=1.61, p=0.004) and distress scores ({beta}=0.78, p=0.039), with the strongest and most consistent associations observed for perceptual abnormalities across total score (sum of endorsed items), distressing items, and distress severity scores (all p[&le;]0.01). These associations were attenuated but largely persisted after adjustment for cognitive ability and broader psychopathology, particularly for perceptual abnormalities. In ABCD, VPT birth was not significantly associated with global or domain-specific PQ-BC outcomes. DiscussionVPT birth is associated with increased vulnerability to PLEs in childhood, particularly in the domain of perceptual abnormalities. The lack of clear replication in the population-based ABCD cohort may reflect differences in the composition of its VPT subgroup, which may not fully represent VPT individuals typically seen in clinical cohorts.

Background: Sleep problems are common in autism, emerge early in life and reduce quality of life, yet the mechanistic link between autism and poor sleep remains unclear. Human and rodent data indicate that difficulty falling asleep is a core feature of autistic insomnia, pointing to impaired responses to sleepiness as the underlying cause. We previously showed that adult mice carrying a mutation in the high-confidence autism gene Shank3 (Shank3{Delta}C) recapitulate this insomnia phenotype and struggle to respond to sleepiness after acute sleep deprivation. Here, we used Shank3{Delta}C mice to examine the molecular basis of sleepiness and how this autism-associated mutation alters it to inform understanding of sleep problems in autistic individuals. Methods: This study used RNA-sequencing and bioinformatics to identify molecular targets underlying the effect of the Shank3{Delta}C mutation on the molecular basis of sleepiness across development in male mice. We first compared cortical genome-wide gene expression following acute sleep deprivation and recovery sleep in adult wild-type (WT) and mutant mice. We then used polysomnography and RNA-sequencing to assess the response to increased sleepiness in WT and mutant mice at postnatal days 24 and 30. Results: The neurotypical response to acute sleep deprivation shifted from upregulating neuronal growth and development pathways at P24/P30 to upregulating DNA damage repair and neuronal activity-dependent transcription in adulthood. The Shank3{Delta}C mutation largely blocked recruitment of these pathways at P24 and in adulthood while paradoxically increasing the magnitude of the mutant response at P30. In addition, mutants consistently upregulated oxidative stress pathways linked to neurodegeneration and protein synthesis regardless of age, whereas WT animals downregulated these functions. Limitations: This study examined gene expression only in male mice, used a single autism rodent model, and averaged signals across mixed cortical cell types. Future work should include females, additional autism models, and single-cell approaches in additional brain regions to further characterize the cellular effects of sleep deprivation and autism-associated mutations. Conclusions: The Shank3{Delta}C mutation impairs the molecular accumulation of and response to sleepiness, both by elevating oxidative stress responses and by blocking the age-typical upregulation of pathways that differ between juveniles and adults.

In mammals, sex differences in the brain arise from genetic and hormonal factors, including organizational effects of perinatal testosterone. Epigenetic mechanisms including DNA methylation and demethylation have emerged as critical mediators of brain masculinization; specifically, their regulatory enzymes are upregulated in neonatal mice during the critical period of sexual differentiation, with their inhibition abolishing sex-specific cellular phenotypes. Here, we assessed sex differences in gene expression of the DNA demethylation machinery (Tet1, Tet2, Tet3, Gadd45a, Gadd45b and Tdg) during and after the critical period, and examined how these differences relate to the oxytocinergic system. mRNA expression was measured in the prefrontal cortex (PFC), preoptic area (POA) and paraventricular nucleus of the hypothalamus (PVN) at postnatal day (P) 7 and P18. At P7, males showed higher expression of all six genes than females in PFC, with no differences in POA or PVN; by P18, no regional differences remained. Oxytocin (OXT) immunoreactivity was surveyed across periventricular nucleus (Pe), anteroventral periventricular nucleus (AVPe), POA, PVN and supraoptic nucleus (SON). OXT was undetectable in the POA, AVPe and Pe at P7, and no sex differences were found in PVN or SON at either age, or in AVPe at P18. At P18, females showed higher OXT-immunoreactivity in the Pe and POA, than males. For Oxtr, qPCR revealed higher mRNA expression in the PFC of males at P7, with no other regional differences and none remaining at P18. Together, these findings suggest that sex differences in oxytocinergic regions arise from sex-specific epigenetic regulation during the critical period, and that perinatal testosterone may program DNA methylation dynamics underlying sex-specific gene expression in the developing brain. Our results support a model in which testosterone-dependent epigenetic mechanisms contribute to the sexual differentiation of neuroendocrine circuits, linking hormonal signals to long-term brain organization.

Chronic stress is a major risk factor for psychiatric disorders such as depression and anxiety, yet the biological basis of individual differences in stress susceptibility and resilience remains poorly understood. Here, we examined physiological, behavioral, and medial prefrontal cortex (mPFC) transcriptomic responses to chronic restraint stress (CRS) in male BALB/c and C57BL/6J mice. After 21 days of CRS, BALB/c mice exhibited greater stress-related changes than C57BL/6J mice, including greater body weight loss, elevated serum corticosterone, reduced serum antioxidant capacity, and more pronounced depression-like behaviors. RNA sequencing showed largely strain-specific transcriptional changes in the mPFC. Strain x stress interaction analysis, followed by canonical pathway analysis using Ingenuity Pathway Analysis (IPA), identified strain-dependent molecular signatures. The most prominent differences involved extracellular matrix (ECM) organization and remodeling and neuroinflammatory signaling pathways, with greater predicted activation in BALB/c mice. IPA upstream regulator analysis further predicted multiple candidate regulators associated with these pathways, including TGF-{beta}/SMAD, C4a/C4b, and MAPK14. Among genes associated with these pathways, several ECM-related genes were preferentially upregulated in BALB/c mice, whereas activity-dependent immediate early genes were preferentially downregulated in C57BL/6J mice. These findings suggest that the strain-dependent mPFC transcriptional programs identified here may contribute to differential stress susceptibility and resilience.

BackgroundCircadian disruption is strongly associated with alcohol use disorder (AUD), but insight into the underlying brain-region and sex-specific mechanisms is limited. The function of the circadian clock gene Bmal1 within the striatum has been linked to alcohol drinking, yet its role within functionally distinct striatal subregions has not been systematically examined. MethodsWe deleted Bmal1 in medium spiny neurons of the dorsomedial striatum (DMS) or dorsolateral striatum (DLS). Male and female mice were tested for anxiety-like behavior, depressive-like behavior, and motor coordination. Voluntary alcohol intake was measured with an intermittent two-bottle choice paradigm, followed by sucrose preference and quinine-adulterated alcohol tests. To assess hormonal contributions, a subset of female mice underwent ovariectomy before behavioral testing. ResultsDeletion of Bmal1 in the DLS did not alter alcohol intake, alcohol preference, or quinine-adulterated alcohol intake in either sex. In contrast, DMS Bmal1 deletion significantly reduced alcohol consumption and alcohol preference in female mice, with no effect in males. These effects were not accompanied by changes in depressive-like behavior or motor coordination and were not explained by generalized reward changes, as sucrose preference was unaffected. Ovariectomy eliminated the effect of DMS Bmal1 deletion on alcohol intake, indicating dependence on ovarian hormones. ConclusionsThe DMS is a critical site at which Bmal1 regulates alcohol consumption in a sex-specific manner. These findings support an interaction between local circadian mechanisms and ovarian hormones in controlling alcohol drinking and highlight a potential target for sex-specific therapeutics in AUD.

MEF2C haploinsufficiency syndrome is a severe neurodevelopmental disorder for which no disease-directed treatment is available. We investigated whether neuron-directed adeno- associated virus (AAV) delivery of a functional MEF2C coding sequence during the juvenile period could modify disease-relevant phenotypes in mice heterozygous for a Mef2c exon 4 deletion. Transcript-level analysis identified a brain-enriched MEF2C isoform containing the 1 and {beta} regions (nMEF2C) and a skeletal-muscle-enriched isoform containing 2 but lacking {beta} (mMEF2C). Separate human-synapsin-driven AAV vectors encoding either isoform were administered at postnatal day 28. Control-treated Mef2c heterozygous mice retained baseline sociability but lacked social-novelty preference. Mice treated with either nMEF2C or mMEF2C displayed social-novelty preference and improved selected responses to a new social partner, whereas open-field effects were limited. nMEF2C replacement also corrected dark-phase wakefulness and non-rapid eye movement sleep abnormalities and modified selected state- dependent electroencephalographic ratios, without broadly changing absolute band amplitudes or social-contact electroencephalographic activity. Atlas-based whole-brain mapping revealed region-selective reductions in parvalbumin-immunoreactive profiles; direct statistical evidence of cellular rescue was confined to the secondary motor area after nMEF2C treatment. These findings show that selected MEF2C-dependent phenotypes remain modifiable during the juvenile period and support further optimization of MEF2C gene replacement with respect to isoform, dose, expression control, and cellular targeting.

The prevalence of neurodevelopmental disorders (NDDs) has increased dramatically, with growing evidence linking prenatal maternal stress exposure to NDDs. Across diverse maternal stressors, immune dysregulation emerges as a common feature, suggesting that fetal microglia may detect changes in the intrauterine environment and influence neurodevelopment. Accordingly, we utilized a mouse model of prenatal maternal cold stress to investigate the impact of maternal stress during pregnancy on fetal microglia morphology, cellular interactions, and phagocytic behaviors. Pregnant mice were exposed to cold stress from embryonic day 11.5 (E11.5) to E15.5 and fetal hypothalamic tissue was assessed from both male and female embryos. By adapting the morphology analysis toolset MicrogliaMorphology to assess fetal microglia, we demonstrate regional differences in microglial morphology in the fetal hypothalamus at baseline, with hypothalamic nuclei such as the paraventricular nucleus (PVN) containing fewer rod-like microglia compared to the broader hypothalamus. Interestingly, prenatal maternal cold stress induced a male-specific shift in microglial morphology from ameboid to ramified within the E15.5 PVN. Male embryos also displayed increased microglial-arginine vasopressin (AVP) neuronal interactions and microglial phagocytosis within the E15.5 PVN, but these changes were unique to microglia with a ramified morphology and were not observed when microglia with an ameboid or rod-like morphology were assessed. Using pHrodo bioparticles and flow cytometry, we further illustrate that prenatal maternal cold stress drives increased phagocytic activity in the E15.5 hypothalamus of male embryos, but not females. Together, these data demonstrate that prenatal maternal cold stress alters microglia morphology and drives morphology-dependent microglial interactions and phagocytic behaviors in male embryos which are unique to the hypothalamic PVN--a nuclei critical for social behaviors. Our findings also suggest that specific hypothalamic nuclei such as the PVN may be more sensitive to prenatal maternal stress, which has the potential to provide a cellular basis underlying the sex differences in microglia-dependent social deficits that were previously reported for this model.

Black carbon (BC), a combustion-derived component of fine particulate matter, has been linked to depressive symptoms, but controlled experimental evidence remains limited. We established a controlled BC inhalation model combined with chronic restraint stress (CRS) to determine whether inhaled BC alone induces depressive-like behavior and whether concurrent stress enhances behavioral and molecular vulnerability. Male C57BL/6J mice were assigned to Control, CRS, BC, or BC+CRS groups and exposed for 21 consecutive days, followed by behavioral testing and molecular analyses of plasma-depleted whole blood and stress-related brain regions. BC exposure alone induced depressive-like behavior, and the combined BC+CRS condition showed the most pronounced phenotype. These findings indicate that inhaled BC is sufficient to influence stress-relevant behavior and may heighten vulnerability under chronic stress. At the molecular level, BC shifted peripheral responses toward a stress- and inflammation-associated state with reduced plasticity-related signaling, whereas CRS preferentially engaged glucocorticoid-responsive regulation. Combined BC+CRS exposure further altered plasticity- and transcription-related regulatory programs in blood and stress-related brain regions, with prominent changes in the nucleus accumbens. These condition-dependent molecular patterns suggest that BC engages blood-brain stress-related pathways in a context- and region-specific manner. Together, these findings identify inhaled BC as a neurobehaviorally relevant environmental hazard.

The social anxiety disorder (SAD) is one of the most common mental health disorders, often developing in adolescence. It can severely impair educational achievement, career progression, and social functioning. Certain individuals within the Chinese Kunming dog (CKD) population display spontaneous, SAD-like behaviors towards strangers, offering a valuable model for genetic investigation. Using whole-genome sequencing data, we conducted a genome-wide association study on this SAD-like behavior in 100 CKDs, identifying several genes previously linked to human psychiatric disorders. Notably, SLC6A3 reached genome-wide significance, whereas CADPS2 exceeded only the suggestive threshold and is therefore considered a network-supported candidate locus. By integrating weighted gene co-expression network analysis with public RNA-seq data from the VTA of 38 mice (after quality control), we further showed that Slc6a3 and Cadps2 are part of a co-expression network module associated with the dopamine system. These findings suggest that this specific module may play a functional role in the stranger-directed anxiety-related behaviours in CKDs. Furthermore, we identified Nrip3 as a key candidate transcriptional coregulator in this specific module. Subsequent virus-mediated overexpression and behavioural experiments showed that Nrip3 regulates gene expression within this network and induces a spectrum of behavioural alterations in mice, including social avoidance, anhedonia, and increased locomotor activity. Collectively, this study highlights the involvement of dopamine system genes in canine social anxiety and suggests Nrip3 as an upstream regulator of Slc6a3 associated with anxiety. The identification of conserved anxiety-related signatures between humans and dogs further supports the dog as a valuable translational model for psychiatric research.

Fear conditioning studies have historically relied on freezing as the primary measure of conditioned fear despite evidence that defensive responding is behaviorally diverse and sexually dimorphic. The endogenous opioid system, particularly mu-opioid receptor (MOR) signaling, is known to regulate fear learning and conditioned analgesia, yet its role in alternative fear-related behaviors and sex differences remains unclear. Here, we investigated the effects of systemic naloxone administration prior to auditory fear conditioning on freezing, darting, shock responsivity, and ultrasonic vocalizations (USVs) in male and female rats. Adult Sprague Dawley rats received naloxone (5 mg/kg, i.p.) or saline prior to conditioning and underwent fear recall testing 24 hours later. Naloxone produced sex- and behavior-specific effects across conditioning and recall. During conditioning, naloxone increased freezing in males during baseline and early tone presentations, while females exhibited reduced shock-response velocity and increased post-shock freezing. Naloxone did not significantly alter darting or USV production during conditioning. During recall, freezing behavior did not differ across groups. Naloxone-treated females, however, exhibited a distinct alarm-calling pattern, with fewer callers overall but increased call output among those that vocalized. These findings suggest that MOR antagonism differentially alters distinct components of fear expression in a sex-dependent manner and support the idea that freezing and alarm calling may reflect separable aspects of fear processing.

Understanding the neurobiological systems that regulate alcohol cue-induced craving is of utmost importance for the development of novel intervention strategies for alcohol use disorders (AUDs). However, although a human experimenter is required to conduct alcohol self-administration studies in the lab, the cues associated with the experimenter are seldom if ever factored into the experimental design. Thus, although we have learned much to date about alcohol cue-induced behavior and neurobiology, and in particular about discrete cues presented many times throughout a single daily alcohol self-administration session, we know relatively little about how responses to alcohol availability cues might predict subsequent alcohol consumption. For the current experiment, mice were exposed daily to auditory cues that preceded 2 hours of alcohol or water access using drinking-in-the-dark (DID) methods. An additional control group experienced cues but were not otherwise manipulated. Importantly, cues were initiated remotely from outside the animal facility, avoiding the experimenter being the first cue predicting ethanol availability. Head direction, location in the home cage, and movement velocity were the primary variables on interest. Surprisingly, during the cue period, there were no significant differences between groups in any of these measures, despite meaningful alterations over days. However, we observed many significant correlations between behaviors and drinking variables. First, we observed significant positive associations between ambulatory velocity during cues and subsequent total alcohol (R2=0.14; p<0.0001) and total water (R2=0.12; p=0.0002) consumption, but only in females. We also observed a significant positive relationship (R2=0.25; p<0.0001) between the amount of time oriented toward the sipper port during the auditory cues and the average rate of subsequent alcohol consumption (i.e. front-loading), but only in females. In males, head direction was found to be positively associated with subsequent total water consumption (R2=-0.21; p<0.0001), but not alcohol (R2=-0.01; p=0.2267). We also observed a significant negative relationship (R2=-0.15; p<0.0001) between proximity to the sipper during the cue period and subsequent total 2-hour alcohol intake in males. Although these associations were modest in strength, they suggest potential sex-specific behavioral predictors of alcohol consumption that are regulated by different neural dynamics.

Purpose: In clinical trials, treatment efficacy is commonly assessed by comparing control and treatment groups. However, in large samples, even small and clinically trivial differences may achieve statistical significance. Accordingly, the Minimal Clinically Important Difference (MCID) is used as a threshold to determine whether statistically-significant effects are also clinically meaningful to patients. The objective of this study was to estimate the MCID for the Childhood Autism Rating Scale Second-Edition (CARS2) using the Patient Impression of Change (PIC) as an external anchor. Methods: Single-item PICs are not well suited to characterizing improvement in a multifaceted disorder such as ASD. Accordingly, the 77-item Autism Treatment Evaluation Checklist (ATEC) was used as a multi-item PIC. CARS2 and ATEC were administered concurrently to 62 children with ASD, aged 1.8-7.9 years, with assessments conducted six months apart. Results: The correlation between changes in CARS2 and ATEC total scores was 0.41-0.44 (p<0.0001), supporting the use of ATEC as an anchor measure. Two anchor-based methods yielded MCIDs of 2.39-2.82 CARS2 points. Two distribution-based methods produced MCIDs of 1.33-3.32. Conclusions: Taken together, these approaches suggest that a between-group difference of 2.6 CARS2 points (the midpoint of the anchor-based estimates) may serve as the MCID in children with ASD.

Objectives To evaluate the positive and adverse effects of parent-mediated interventions (PMIs) versus care as usual for children with autism. Setting Systematic review and meta-analysis and Trial Sequential Analyses (TSA), following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Methods We searched for randomised clinical trials of PMIs for children with autism in the databases CENTRAL, EMBASE, LILACS, PsycINFO, MEDLINE, and SCI-EXPANDED (up to 13 August, 2025), complemented with manual searches. 12,359 articles were screened. Data were synthesised using meta-analyses and Trial Sequential Analyses (TSA), and risks of bias and certainty of the evidence were evaluated. Primary and secondary outcome measures The primary outcome was autism characteristics. Secondary outcomes were adverse effects, child adaptive functioning, child language, child and parent quality of life, and parental stress. Ten exploratory outcomes were included. Results 32 trials (N=1,625) comparing PMIs to usual care, waiting list, or no intervention were included. All trials had a high risk of bias. The multiplicity-adjusted threshold for statistical significance was p = 0.013 due to the number of outcomes. Meta-analyses and TSAs showed it could be rejected that PMIs reduced autism characteristics (MD = -0.88; 95% confidence interval -2.92 to 1.15; p = 0.05, 4 trials, N=353, low certainty), child adaptive functioning (7 trials, N=408), child language (4 trials, N=308), or parental stress (7 trials, N=385). Due to insufficient data, the remaining secondary meta-analyses could not be conducted. Meta-analyses of exploratory outcomes showed beneficial effects concerning child behaviour problems and parent sensitivity/synchronicity. Conclusions This meta-analysis found no benefits of PMIs on child autism characteristics, child adaptive functioning, child language, or parental stress. Benefits were found in reduction of child behaviour problems and improved parent sensitivity/synchronicity. The evidence remains uncertain, and more trials including outcomes of adverse effects and quality of life are needed.