Genes, Brain and Behavior

Studies have shown that substance use liability is associated with novelty seeking, anxiety-like behavior, and pain sensitivity. We examined whether common genetic variation in outbred Sprague-Dawley rats explained variation in behavioral measures from three assays with established links to substance use: locomotor response to a novel environment, elevated plus maze, and tail flick. We estimated single-nucleotide polymorphism heritability and performed genome-wide association analyses using permutation-derived significance thresholds (N=534-654 rats across traits). Heritability estimates ranged from 0.14-0.38 across eleven traits. Three independent loci were identified: chromosome 1 for elevated plus maze open-arm behavior (=0.05), chromosome 14 for elevated plus maze immobility (=0.10), and chromosome 17 for tail flick latency (=0.05). Candidate genes included Slc18a2, Gfra1, and Pdzd8 (chromosome 1); Rel and Bcl11a (chromosome 14); and Eci2 and Eci3 (chromosome 17). We compared these loci with our genome wide association study of a F2 intercross of selectively bred high- and low-responder rats, originally derived from Sprague-Dawleys, that model individual differences in externalizing and internalizing behavior. The current loci are distinct from the ones identified in the bred lines. This difference likely reflects selection history in the high- and low-responder F2s, which focused on facets of exploratory locomotion, while loci for anxiety and pain sensitivity traits were identified in the outbreds. This highlights the benefit of using both outbred and selectively bred rats to probe causal variants contributing to individual differences in substance use liability. The current outbred findings implicate monoaminergic signaling, transcriptional control, and lipid metabolism as testable mechanisms for addiction-relevant behaviors.

BackgroundThe three-chamber test (3CT) is widely used to assess social behaviour in mice, based on the assumption that time spent near a conspecific reflects motivation for social contact. However, the design of the task constrains interpretation, as behaviour may reflect exploration, novelty seeking, or territorial investigation rather than affiliative social motivation. In addition, key biological factors such as sex differences and social hierarchy are often overlooked. AimsWe hypothesised that the 3CT overestimates sociability and used a direct-interaction phase to investigate motivation for affiliative social contact. We also integrated social status to determine if this modulated behavioural patterns and interacted with sex. MethodsAdult male and female C57BL/6 mice (n = 32) were tested in a standard 3CT, followed by removal of the cage barrier to permit direct contact. Behavioural parameters were quantified, and social status was determined using the tube test. ResultsMales exhibited higher social interest index scores than females. Once the barrier was removed, both sexes displayed a negative direct sociability index, indicating greater environmental exploration than social engagement. Correlation analysis revealed no association between indirect and direct measures. Sex differences emerged primarily among submissive mice, with submissive males showing greater social investigation than submissive females. ConclusionThese findings suggest that standard 3CT indices reflect exploratory rather than affiliative social motivation. The modified paradigm incorporating direct interaction provides a more realistic assessment of social behaviour and challenges assumptions about intrinsic sociability in mice.

Prairie voles (Microtus ochrogaster) are highly social rodents that have become a valuable animal model for studying social attachment, pair bonding, parental care, and the neurobiological mechanisms underlying social behavior. In recent years, due in part to the publication of the prairie vole genome and deeper mechanistic understanding of their social behavior, prairie voles have become a more popular research model, especially for translational research. However, generating reliable and reproducible findings requires effective colony management, including thoughtful breeding strategies, consistent husbandry practices, and clear documentation. In this paper, we describe the demographic history of and husbandry techniques employed in our prairie vole breeding colony at UC Davis from 2004 to 2020. Well-organized and transparent colony management allows for the preservation of informative behavioral traits in prairie voles and strengthens the impact of the prairie vole model across behavioral and biomedical science.

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.

Early life stress (ELS) in primates alters dopamine function, contributing to addiction, hyperactivity, cognitive deficits, aggression, and social subordinance. To assess whether dopamine receptor densities are affected by ELS, male juvenile rhesus monkeys (Macaca mulatta) were either mother-reared (MR, N=6) in a semi-natural environment or nursery-reared (NR, N=6) with peers in a laboratory. At 1 [1/2] years of age, subjects were sacrificed and the left prefrontal cortex (PFC), striatum (caudate and putamen), nucleus accumbens (NAcc), and claustrum (CLA) were explored through quantitative autoradiographic studies of dopamine receptor-1 (DRD1) and -2 (DRD2) conducted using [125I]-(+)-SCH 23982 and 125I-Epidepride, which have high affinity and selectivity for DRD1 and DRD2, respectively. No group differences emerged in striatal or NAcc receptor binding. However, MR monkeys exhibited significantly greater DRD1 binding in the left orbital PFC and significantly greater DRD2 binding in both the left medial PFC and right CLA compared to NR. These findings implicate the medial PFC (stress vulnerability, cognition), orbital PFC (reward valuation), and CLA (anxiety modulation) as critical sites disrupted by maternal deprivation. Therefore, we propose that nursery-rearing induces a hypodopaminergic prefrontal-claustral ecophenotype, underlying the cognitive, affective, and social impairments observed in NR monkeys.

Behavioral dysfunction is a common characteristic of many mental health disorders. While the causes of these disorders vary, aberrant behaviors may arise from alterations in transcriptional regulation already during early neural development. Because transcription factors (TFs) often belong to families of closely related members, disruption of a single TF may indirectly influence the functionality of other family members. Consequently, mutations in TFs within the same family may lead to overlapping, yet distinct, phenotypes. This feature of TF function has important implications for understanding behavioral phenotypes, but detailed analyses across a single TF family are still lacking. In this study, we present a comprehensive behavioral analysis of adult zebrafish harboring mutations in individual members of the TALE and Hox TF families, that are essential for nervous system development. Using a battery of validated behavioral assays, we uncover elevated stress responses among all TF mutant lines, as well as TF-specific dysregulation in coping strategies, social interaction, learning/memory, and endurance and locomotion. The shared behavioral abnormalities across mutants suggests TF family members converge on core developmental pathways for stress-related behavioral regulation, while mutation-specific phenotypes indicate unique roles for individual TFs in fine-tuning neural function. Our findings provide a systematic behavioral characterization of TALE and Hox mutants in a vertebrate model and provide a framework for understanding how genetic variation within TF families may differentially contribute to vulnerability for mental health disorders.

Prenatal alcohol exposure (PAE) disrupts embryonic development and gives rise to a variable fetal alcohol spectrum disorder (FASD) phenotype characterized by neurodevelopmental and dysmorphological defects. We investigated the effects of PAE on placental gene regulation by performing genome-wide DNA methylation (DNAm) microarray and gene expression (mRNA sequencing) analyses in 87 PAE, 77 unexposed control, and 11 smoking-exposed-only placentas. Significant alterations were identified in genes involved in synaptic function including both excitatory and inhibitory neurotransmission, and in genes previously associated with attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder, schizophrenia, and addiction. When placental molecular alterations were compared with neuropsychological and dysmorphological phenotypes of the same children assessed at six years of age, numerous correlations were observed between DNAm and gene expression, as well as head circumference, cognitive performances, ADHD traits, and dysmorphology. These included associations between DAOA methylation and verbal intellectual abilities and language development, GPHN methylation and working memory index, and GLI3 expression and both working memory index and midfacial hypoplasia. As these alterations were observed in the placenta, this tissue not only enables the identification of phenotype-specific FASD candidate genes but also represents a valuable tool for studying complex developmental disorders in human.

RASopathies are developmental conditions associated with cognitive and sensory processing impairments. They are caused by pathogenic variants in genes that result in overactivation of the RAS/MAPK signaling pathway. Genes linked to this pathway have been reported to be enriched among Drosophila models with habituation deficits, a behavioral phenotype reflecting sensory filtering. To identify hidden RASopathies - monogenic disorders that converge on RAS/MAPK overactivation without being classically linked to the pathway - we generated 89 and screened 41 viable habituation-deficient Drosophila RNAi models for RAS/MAPK overactivation, measured as an increased phosphorylated ERK to ERK ratio. This screen identified Sms, the ortholog of human spermine synthase (SMS), implicated in Snyder-Robinson syndrome. RAS/MAPK overactivation along with hyperreactivity and habituation impairments are confirmed in a full loss-of-function mutant. A RNAi screen targeting polyamine pathway genes identified Sat (human SAT1/2, SATL1) to reproduce these phenotypes. Knockdown of Sms or Sat in GABAergic neurons impaired habituation, implicating polyamine metabolism in inhibitory circuit function. These findings reveal previously unrecognized convergence between polyamine dysregulation and RASopathies, suggesting shared therapeutic opportunities through modulation of either pathway. SUMMARY STATEMENTUsing Drosophila, we uncovered polyamine metabolism genes, Sms and Sat, as modulators of RAS/MAPK and sensory processing, revealing a shared mechanism between polyaminopathies and RASopathies that may inform unified therapies.

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.

Females have two X chromosomes, one of which is inactivated early in development with specific regions and genes escaping inactivation. Thus, X chromosome loss putatively results in decreased dosage of X chromosome escapee and pseudoautosomal genes, impacting downstream pathways. Evidence from Turner syndrome indicates that X chromosome monosomy results in consistent neuroanatomical and cognitive phenotypes. However, it remains unclear whether mosaic karyotypes, with mixed proportions of 45X and 46XX cells, attenuate these phenotypes. We examined whether X chromosome mosaicism is predicted by neuroanatomical and cognitive features. Higher proportion of 46XX cells was significantly predicted by structural properties in somatosensory, motor, visual, and language brain areas, and by performance in visuospatial, fine-motor, and language tasks. Thus, mosaicism partially rescues phenotypes linked to full 45X monosomy and may explain the role of the X chromosome not only across heterogeneous phenotypic expression in females, but also in sex differences observed in neuropsychiatric conditions.

Changes within neurotransmitter systems are associated with variation in anxiety-related behavior. The adenosine signaling pathway has been associated with anxiety and caffeine has been utilized as a modulator. However, studies have not considered the impact of an individuals stress coping style (e.g. proactive, reactive) and corresponding differences in neuromolecular signaling that can influence the behavioral responses. To assess the role of adenosine signaling, we acutely treated reactive and proactive zebrafish with 50 mg/L caffeine and evaluated anxiety-like behavior using a novel tank diving test (NTDT). We then quantified whole-brain gene expression of genes representing distinct parts of the adenosine signaling pathway: adenosine receptors A1B, A2Aa, A2Ab, and A2B (adora1b, adora2aa, adora2ab, and adora2b, respectively) and enzymes adenosine deaminase (ada) and ecto-5-nucleotidase (nt5e). We found significant main effects of coping style, sex, treatment, and coping style by sex by treatment interaction effect on stress behaviors. Specifically, compared to controls, caffeine reduced stress behavior in only reactive males. We also observed significant differential baseline gene expression within the adenosine signaling pathway between the reactive and proactive strains, where reactive zebrafish expressed higher levels of adenosine receptors A1B, A2Ab, A2B, and adenosine deaminase and lower levels of adenosine receptor A2Aa than proactive zebrafish. These findings indicate that variation in adenosine signaling between the stress coping styles and sexes may be contributing to differences in anxiety-related behavior.

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.

BackgroundEarly-life adversity can alter emotional and social development and increase vulnerability to later life stress. We investigated how early adverse experiences (EAE) and later adverse experiences (LAE) shape adult emotional contagion (EC) responses in female and male rats. MethodsEAE was induced using the limited bedding and nesting model during the first postnatal week. LAE was induced via footshocks during adolescence. In adulthood, male and female rats underwent an EC test in which observers witnessed a conspecific receiving footshocks. ResultsAdolescence-footshock exposed observers showed cingulate cortex-associated increased immobility, proximity, and attention toward distressed conspecifics during adulthood, compared to adult-exposed and sham animals, both in male and female animals. While EAE did alter maternal care, pup stress physiology, and pup weight, we found evidence that it did not alter immobility during EC. However, female demonstrators paired with EAE observers showed increased immobility, linked to a reduced rate and lower frequency of the observers 50-kHz vocalizations. Mediation analysis revealed that a shift toward lower-frequency 50-kHz vocalizations specifically mediated this effect, suggesting a sex-specific pathway by which early adversity shapes social behavior. ConclusionsEarly and adolescent adversity influenced distinct aspects of emotional contagion: EAE mediated an observer-to-demonstrator emotional transfer during EC, while LAE impacted a demonstrator-to-observer transfer, with no evidence of additive effects. Our results highlight developmentally specific and sex-dependent mechanisms by which early and later adversity alter social-affective responses in adulthood.

Copper is an essential micronutrient required by enzymes that catalyze oxygen-dependent reactions, but toxic in excess. Mutations in the ATP7A and ATP7B copper transporters cause neuropsychiatric symptoms and neurodegeneration by mechanisms that remain to be elucidated. We previously reported that the ATP7A biochemical interactome is enriched in Parkinsons disease (PD) and neurodegeneration associated proteins, yet the functional outcomes of these interactions are unknown. Using Drosophila, we tested genetic interactions between ATP7 mutants that alter copper levels and a subset of these PD and neurodegeneration causative genes and found sex differences with some candidate genes enhancing ATP7 deleterious phenotypes in both sexes, while others were sex specific. Most notably, we found that Lrrk2 (Lrrk), the most commonly mutated gene in familial forms of PD, protects against ATP7 dysfunction in epidermal epithelial cells with a stronger effect in males than females. However, in dopaminergic neurons Lrrk plays a role in intracellular copper induced toxicity in females but not males, supporting context dependent interactions between ATP7A and PD-associated genes to protect against disruptions in copper homeostasis. Summary StatementWe performed a genetic interaction screen to explore the relationship between copper homeostasis and Parkinsons disease and other neurodegeneration associated genes.

Genetic variation in the oxytocin receptor gene (Oxtr) has been linked to differences in brain OXTR expression and long-term social bonds, but whether it shapes the moment-to-moment dynamics of early social interactions is unclear. Here we examined how the intronic Oxtr single nucleotide polymorphism (SNP) NT213739 in female prairie voles (Microtus ochrogaster) shapes their dynamic social interactions with an opposite-sex conspecific. Leveraging a computational pipeline to analyze the movements of freely interacting voles, we found that C/C females, which expressed higher OXTR levels in the nucleus accumbens than T/T females, spent more time socially observing novel males from a distance, especially early in interactions. This genotype-phenotype relationship persisted in multiple contexts, including the social preference test. Thus, natural Oxtr variation biases social observation in females toward unfamiliar males before bonds form, consistent with models where accumbens OXTR enhances the salience of social cues. These findings show that SNPs can shape subtle behavioral dimensions in early social encounters, with important implications for the role of oxytocin in the study of social attachment.

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.

7q11.23 Duplication Syndrome (7Dup) is a type of syndromic autism spectrum disorder caused by duplication of a typically 1.5-1.8 Mb segment in section q11.23 of chromosome 7, including 25-27 genes. Previous work has highlighted the GTF2I gene as playing a major role in the phenotype of 7Dup patients. Accordingly, mice with Gtf2i duplication (Gtf2i+/dup) are commonly used as an animal model of 7Dup. We previously reported deficits in several behavioral and physiological modalities, which were associated with Gtf2i dosage in mice conducting a battery of social discrimination tests. Here, we report the effect of treating Gtf2i+/dup mice with Baicalin, a naturally occurring flavonoid added to the mices drinking water (0.15 mg/ml), on these deficits. We found that Baicalin treatment ameliorated the higher surface temperature observed in Gtf2i+/dup males and the lower tail temperature observed in Gtf2i+/dup females during the social behavior tests. It also prevented the increased defecation rate exhibited by Gtf2i+/dup mice during the social preference test. We further analyzed the effect of Baicalin treatment on cortical neurons differentiated from 7Dup patient-derived IPSCs. Using whole cell patch clamp and calcium imaging, we found an increased rate of excitatory postsynaptic currents in Baicalin-treated cells, without a change in their firing rate, indicating a stronger synaptic activity in the Baicalin-treated cells. Altogether, our results reveal that Baicalin administration alleviates some of the behavioral and physiological effects of Gtf2i duplication in mice, and affects neuronal activity in cultured 7Dup human neurons. Thus, Baicalin administration has the potential to serve as a treatment for 7Dup patients.

Both prenatal alcohol exposure (PAE) and adolescent alcohol exposure (AAE) persistently impair executive function in humans and animal models. Executive function encompasses multiple interrelated domains including working memory, inhibitory control, and behavioral flexibility. We hypothesized that a developmental "double hit" of PAE and AAE would produce more severe behavioral deficits associated with these executive domains compared to alcohol-naive and single-exposed animals. We tested this hypothesis in rats by assessing disinhibition (low-light elevated plus maze; LL-EPM), behavioral flexibility (attentional set shift test; ASST), and working memory (spontaneous alternations in a T-maze); we also tested behavioral flexibility (ASST) in mice. Pregnant Sprague Dawley rats received water or 5 g/kg alcohol from gestational day (GD)13.5-GD20.5, and offspring received water or 5 g/kg alcohol on a 2-day-on, 2-day-off paradigm from postnatal day (PD)25 to PD54. Pregnant C57BL/6J mice received water or 4.5 g/kg alcohol from GD13.5-GD17.5, and offspring received water or 4.5 g/kg alcohol on a 2-day-on, 2-day-off paradigm from PD25 to PD42. Offspring underwent behavioral testing in young adulthood. Double hit rats showed more exploration in the LL-EPM than controls and fewer alternations in the T-maze than AAE-only rats, suggesting deficits in disinhibition and spatial working memory, respectively. Double hit rats and mice exhibited more errors and/or more trials to criterion in the ASST, indicative of decreased behavioral flexibility. Overall, double hit animals showed altered performance on tests related to executive function, suggesting that the combined exposure alters executive function in a manner distinct from single-exposure models.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by a broad spectrum of behavioral impairments. While multiple genetic and environmental factors are attributed to its cause, biological underpinnings are still poorly understood. We investigated an ASD-associated gene, WAC, for its neurobehavioral aspects using C. elegans and mice. Studies of C. elegans with wac gene deletions (wac-1.1 and wac-1.2) showed enhanced acetylcholine-associated behavior, as indicated by the aldicarb assay. No alteration in acetylcholine levels or acetylcholinesterase activity was observed. Upon further investigation, we found that the elevated cholinergic transmission resulted from increased activity of nicotinic acetylcholine receptors (nAChRs). Additionally, we observed reduced motility and dopamine-associated behaviors, along with a reduced ability to switch from crawling to swimming, a serotonin-dependent behavior. Upregulation in mRNA expression of the lev-1 gene was observed. Conversely, a feedback-counterbalancing response in the form of downregulated genes, acr-2, unc-17, unc-63, and unc-50, was also observed. Surprisingly, lev-1 RNAi did not reverse the enhanced cholinergic transmission in PHX2587 worms, indicating the involvement of other players. To validate our findings, we also assessed CHRNA7 levels in Wac+/- mice. While some genetic compensation was observed in heterozygous mice, we found a direct, inverse correlation between Wac mRNA expression and CHRNA7 levels in the mouse brain cortex, corroborating our findings from C. elegans. Overall, these studies indicate that wac gene deletion in C. elegans exhibits a neurotransmitter alteration that is relatable to ASD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/709202v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1771dc4org.highwire.dtl.DTLVardef@1434b4eorg.highwire.dtl.DTLVardef@10525ecorg.highwire.dtl.DTLVardef@fcd8a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundSubstance use disorders (SUD) are associated with a high global burden of disease, with 5.4% of all disability-adjusted life years lost due to alcohol and illicit drugs. Highly prevalent multimorbidity includes polysubstance use, mental health conditions, and other non-communicable and infectious diseases. Where traditional treatments are insufficient alone, music therapy (MT) is highly engaging and improves motivation and reduces craving; however, its long-term effects are unknown. The present study aims to examine long-term effects of active music groups (AMG) and music listening groups (MLG) versus treatment as usual (TAU) on addiction severity, recovery, and other outcomes in people with SUD Immediate and short-term effects, as well as mechanisms of these interventions, will also be examined. MethodsIn individuals with SUD across a wide range of age, gender, socioeconomic, and cultural backgrounds, a parallel 3-arm assessor-blinded pragmatic multinational randomised controlled trial (RCT) with embedded exploratory trials and mechanistic studies will determine long-term effects of AMG and MLG versus TAU on addiction severity (primary endpoint: 1 year), recovery, and other outcomes. Embedded trials will examine immediate effects of AMG or MLG combined with individual components of TAU combined to determine the best combinations of interventions. Experimental studies will examine mechanisms using cognitive testing and brain imaging. With 600 participants in 7 countries randomised, the trial will have 80% power on the primary outcome. Patient representatives, health technology assessment (HTA) bodies, and interventionists have been involved from conception and will ensure feasibility and applicability of the intervention across Europe. DiscussionThis document describes the FALCO RCT, the main part of the FALCO project, which aims to reduce disease burden through innovative, effective, and affordable treatment, and will strengthen research and innovation expertise. Recommendations from FALCO will inform intervention delivery across Europe and beyond, leading to increased safety, effectiveness, and cost-effectiveness, and improved quality of life for individuals with SUD. Stakeholders will be involved in communicating findings across all European countries and regions and ensuring that findings are effectively implemented. Trial registrationClinicalTrials.gov, NCT07028983, registered 11th of June 2025. https://clinicaltrials.gov/study/NCT07028983