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.

20% of Americans are at risk for environmental circadian rhythm disruptions (CRD) due to shift work, leading to substantial negative health outcomes. However, females are especially affected with greater vulnerability for substance use (SU) and adverse outcomes associated with pregnancy, including for offspring at birth and later in life. In mice, prenatal CRD (pCRD) recapitulates these risks, but it is unknown whether pCRD affects SU in mature offspring. To investigate this, C57BL/6J dams were disrupted by reversing the light/dark cycle during gestation. Following pCRD, reward- (cocaine conditioned place preference, intravenous self-administration) and mood-related behaviors (open field, elevated plus maze, light/dark box, forced swim) were measured in adult offspring. Adult female offspring of dams exposed to CRD developed an anhedonic-like phenotype with decreased food self-administration, cocaine intake and reinforcing properties of cocaine. Opposingly, pCRD male offspring showed a SU-like phenotype with increased cocaine preference, higher order food self-administration and cocaine reinforcement. Interestingly, these divergent behavioral outcomes were not specific to reward. While female pCRD mice showed increased anxiety-like behavior, pCRD males showed decreased anxiety/increased risk-taking behavior, as well as decreased immobility in the forced swim test. Rhythms in corticosterone were also sex-specifically affected by pCRD. These results suggest that pCRD may predispose individuals to distinct psychiatric disorders based on sex with mood disorders developing in females and SU disorders developing in males. By better understanding how disrupted rhythms during pregnancy affect behavior in adulthood, we can develop novel therapeutic approaches for SU and mood disorders in adults.

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The disease is characterized by movement disorders, and it also presents with personality changes, including apathy and aggression, along with cognitive decline. While most animal models for HD have been validated for motor deficits, less is known about alterations in other behavioral functions. Here, we performed a longitudinal study to analyze the behavior of a knock-in mouse model of HD with a chimeric mouse/human exon 1 containing 140 CAG repeats inserted in the murine huntingtin gene. We specifically inquired about the onset of cognitive impairments in knock-in mice and whether changes in various behavioral functions such as locomotion, anxiety, and cognition correlate at the individual level. Our data indicate that female and male knock-in mice exhibit reductions in body weight, novelty-induced locomotion, and remote spatial memory retrieval. However, social behavior, working, and short-term memory remain unaffected. Within knock-in mice, lower open-field activity correlated with poorer remote memory performance. Moreover, CAG repeat length negatively correlated with locomotor activity and spatial memory, indicating that greater repeat expansion predicts more severe behavioral impairment. These findings identify early affective changes, followed by selective long-term memory and locomotor deficits, in knock-in mice, supporting this model as a useful platform for studying prodromal HD and repeat-length-dependent disease variability. HighlightsO_LICAG140 knock-in mice show early anxiety, later reduced locomotion and memory deficits C_LIO_LILong-term and remote memory are impaired while short-term and working memory are spared C_LIO_LILower locomotion at the age of 8 months correlates with poorer memory at 14 months of age in individual CAG140 knock-in mice C_LIO_LIGreater CAG repeat length predicts worse locomotion and memory C_LI

Bryant-Li-Bhoj Syndrome (BLBS; OMIM: 619720, 619721) is a Mendelian neurogenetic condition, first described in 2020, with a mixed neurodevelopmental/neurodegenerative phenotype and variable systemic features. To date, 100 affected individuals with 74 unique causative variants have been published. Clinical data and prior functional work in multiple model systems have emphasized the utility of interrogating the pathogenesis of multiple causal variants to identify a convergent, therapeutically targetable mechanism. Additionally, the ability to evaluate the efficacy of future therapeutics relies on the availability of a robustly validated preclinical model. Here, we characterize the developmental and neurobehavioral phenotypes of a novel BLBS mouse model harboring one of the most recurrent causative variants (h3-3a p.T45I). H3.3T45I mice recapitulate the BLBS natural history: perinatal growth restriction, delayed developmental milestones, and progressive motor and gait impairments. Adult mice additionally display craniofacial differences, impaired nest building, hyperactivity in a social context, and male-specific elevated aggression. The non-invasive, clinically translatable endpoints established here provide a validated preclinical platform for evaluating therapeutics for a community whose current standard of care is symptom management. Summary StatementA new mouse model mirrors the developmental delays, motor decline, and behavioral changes seen in individuals with this rare, progressive genetic brain disorder, providing a foundation for testing future therapies.

Children with Down syndrome (DS) experience delays in cognitive, physical, and motor development. Overexpression of Dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), a gene on human chromosome 21 (Hsa21) and triplicated in individuals with Trisomy 21, contributes to neurodevelopmental delays associated with DS, and is a candidate for therapies to improve neurodevelopmental phenotypes. Male and female Ts65Dn DS model pups are trisomic for [~]100 Hsa21 orthologs including Dyrk1a, and both sexes show significant DYRK1A overexpression on postnatal day 6 (P6) in the hippocampus, cerebral cortex, and cerebellum. This study tested the hypothesis that normalization of Dyrk1a copy number in Ts65Dn pups prior to P6 would diminish physical and behavioral developmental outcomes in Ts65Dn mice, thus providing a standard of comparison for success of interventions targeting Dyrk1a. At P3-P21, Ts65Dn compared to euploid pups showed sex-specific deficits in physical, motor, and behavioral development. Male Ts,Dyrk1a+/+/Dox-Cre mice showed improved emergence to running on P19, and both sexes of Ts,Dyrk1a+/+/Dox-Cre mice exhibited reduced isolation-induced ultrasonic vocalizations during the second postnatal week. Dyrk1a normalization in Ts65Dn pups did not improve all abnormal phenotypes, perhaps because of developmental dysregulation between Dyrk1a RNA and DYRK1A protein levels, involvement of other trisomic genes, or improvements in only adult mice.

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.

IntroductionEnvironmental factors are increasingly implicated in the etiology of autism spectrum disorder (ASD). Polybrominated diphenyl ethers (PBDEs) are anthropogenic toxicants added as flame retardants to consumer products that have become ubiquitous environmental contaminants and disrupt thyroid hormone (TH) and neuroendocrine systems. We have previously shown that developmental PBDE exposure produces ASD-like traits with involvement of oxytocin (OXT)-thyroid hormone signaling. Limosilactobacillus reuteri (LR), a widely used probiotic bacterium, has been shown to improve social functioning and increase TH and OXT levels in murine models. Therefore, we tested the hypothesis that LR supplementation (LR) prevents PBDE-induced deficits in socioemotional behavior with concomitant modulation of TH signaling genes on hypothalamic OXT neurons. MethodsC57BL/6N mouse offspring were exposed to a commercial penta-mixture of PBDE congeners, DE-71, at an environmentally realistic concentration, 0.1 mg/kg/d (DE-71), or to corn oil vehicle (VEH/CON) via their mothers during gestation and lactation. Offspring received supplementation with LR ATCC PTA 6475 (107-108 CFU/mL, po) indirectly via the dam or continuation directly through adulthood. Unsupplemented controls were given saline. ResultsFecal microbiome analysis in offspring confirmed colonization of LR at postnatal day (P) 40 and depletion by P104. LR treatment increased plasma total thyroxine in DE-71 and plasma OXT in VEH/CON dams. In DE-71 offspring of both sexes, LR normalized deficient scores on social novelty preference and emotional recognition in adult females and males and deficient long-term social recognition memory (SRM) in adult DE-71 females; DE-71 males were normal. Reduced olfactory dishabituation between two social odors may partly explain the compromised socioemotional behavior produced by DE-71 in an LR-dependent manner. Multiplex RNA in situ hybridization performed on immunoreactive OXT-ergic neurons in the paraventricular hypothalamic nucleus (PVH) revealed significant upregulation of TH transporter monocarboxylate transporter 8 (Mct8) and downregulation of iodothyronine deiodinase 3 (Dio3) in DE-71 relative to VEH/CON females. This toxicant-induced reprogramming was prevented by probiotic treatment. DE-71 males expressed reduction in Mct8 and Dio3 transcripts on OXT-ergic neurons with minimal LR protection. In the female supraoptic nucleus (SON), Mct8 and Dio3 were downregulated by DE-71 and normalized in DE-71+LR; there were no group effects on transcript levels in male SON. Results of fecal 16S rRNA sequencing indicated reduced -diversity and altered {beta}-diversity in the gut bacterial community of female but not male DE-71 exposed offspring; most changes were correctable by LR. Alterations in taxa-level abundance caused by DE-71 and reversed by LR were observed in both sexes. These involved Bifidobacterium, Coprococcus, Desulfovibrio, Oscillospira, and Peptococcaceae in females and Desulfovibrionaceae, Rikenella, and Turicibacter in males. Exposed dams showed no detriment in - and {beta}-diversity while showing reduced abundance of several Firmicutes and Proteobacteria taxa that could be rescued by LR. The relative abundance of Lactobacillus was upregulated in DE-71 males and DE-71+LR males and dams. ConclusionsThese results indicate that developmental probiotic supplementation effectively mitigated organohalogen-induced ASD-like deficits in socioemotional behavior and partially corrected dysbiosis of gut bacterial communities in exposed offspring of both sexes. Concomitantly, PBDEs altered the expression of TH regulatory genes Mct8 and Dio3 in PVH OXT neurons in a sex-dependent manner, suggesting that TH regulation of OXT neuroendocrine cells may modulate the emergence of toxicant-induced ASD-relevant behavior. While LR reinstated normal behavioral outcomes in PBDE-exposed offspring of both sexes, coincident normalization of hypothalamic TH signaling transcripts occurred more broadly in females, indicating the existence of unique parallel processes influencing the preventive effects of LR on ASD-relevant behavioral deficits in both sexes.

Developmental coordination disorder (DCD) is a common neurodevelopmental condition characterized by impaired motor coordination and learning, yet its neurobiological and genetic bases remain poorly understood. Here, we leverage the BXD recombinant inbred mouse panel to model the polygenic architecture of DCD and link behaviour, brain structure, and genotype. High-resolution ex vivo MRI across 14 strains revealed that DCD-like mice have modestly reduced total brain volume, with a distinct neuroanatomical profile characterized by enlarged cortical regions alongside reduced cerebellar, thalamic, and other subcortical volumes. These structural differences closely mirror findings reported in human DCD. Across strains, variation in brain structure strongly correlated with motor behaviours, with coordinated patterns linking increased cortical and decreased subcortical volumes to poorer motor coordination, while more focal associations were observed for motor learning. Multivariate analysis identified a dominant brain-behaviour axis capturing this cortical-subcortical trade-off. Quantitative trait locus (QTL) mapping revealed multiple loci influencing regional brain volumes, including a prominent locus on chromosome 12 regulating cerebellar structures, but did not identify single loci driving the main multivariate brain-behaviour relationships, consistent with a distributed genetic architecture. Together, these findings demonstrate that DCD-like motor impairments arise from coordinated alterations across distributed brain systems under polygenic control. This work establishes a translational framework linking genetic variation to brain organization and motor function, and suggests that DCD reflects the extreme of a continuous spectrum of neurobiological variation rather than a discrete condition.

Alcohol use disorder demonstrates ~50% heritability, much of which remains unexplained by genetic sequence alone. Chronic alcohol exposure before conception changes offspring phenotypes through epigenetic mechanisms that are still being elucidated. Preconception ethanol exposure studies have focused on paternal exposure, neglecting maternal and biparental exposure. To address this, we exposed adult male and female mice to five cycles of chronic intermittent ethanol vapor interleaved with two bottle choice ethanol drinking and mated them to produce male and female F1 offspring with paternal, maternal, or biparental preconception ethanol exposure or controls. Whole blood and medial prefrontal cortex from adult, ethanol-naive offspring underwent RNA-sequencing. We also analyzed previously unpublished RNA-sequencing data from male and female preimplantation embryos derived from preconception ethanol-exposed sires. Here, we report transcriptomic patterns of preconception ethanol exposure that depend on the exposed parent, offspring sex, and tissue which suggest metabolic and immune dysfunction in offspring.

Early life adversity can produce persistent changes during development that increase vulnerability to neuropsychiatric disorders. Although disruptions in reward processing are widely recognized as a hallmark of these disorders, reward is not a single construct. Distinct forms of reward including social interaction and primary rewards such as food rely on overlapping but dissociable neural circuitry and may be differentially affected by adverse experiences. Here, we used a rodent model of neonatal predator odor exposure (POE) to determine how early life threat influences motivation for social and sucrose reward in adulthood using operant procedures. Neonatal POE reduced adult motivation for a social reinforcer during an operant social self-administration task, whereas motivation for a sucrose reinforcer was unchanged. However, we did find a significant difference in sucrose self-administration with POE females pressing more for sucrose than control females. These findings demonstrate that neonatal threat does not produce a generalized deficit in motivation but rather selectively alters motivation across distinct reward domains. Together, this work identifies social reward as a particularly vulnerable behavioral domain following early life threat and provides new insight into how adverse developmental experiences shape adult reward-related behavior.

Social isolation is commonly used to model social stress and is a known risk factor for depression, with impacts on hippocampal function and postnatal neurogenesis. However, most studies focus on social isolation in juvenile mice isolation during adolescence, leaving the effects of prolonged adult isolation less understood. Post-transcriptional regulation of gene expression by microRNAs (miRNAs) plays a role in hippocampal function, and altered miRNA, as well as gene expression, has been reported in the hippocampus of mice exposed to social isolation. A single-nucleotide polymorphism in miR-30e in humans is associated with increased expression of the mature miRNA, impaired cognition, electroencephalogram waveform latency, depression, and schizophrenia. We investigated whether adult isolation in mice alters gene regulation via microRNAs, particularly miR-30e-5p, and affects hippocampal function. In adult BALB/c male mice, 10 weeks of isolation increased miR-30e-5p expression in the ventral hippocampus, reduced its target gene Neurod1, and impaired hippocampal-dependent cognition (object pattern separation), without clear anxiety- or depression-like behaviours. Isolated mice also showed a blunted response to acute stress. These findings suggest that adult social isolation affects hippocampal function through post-transcriptional gene regulation, highlighting a role for miR-30e-5p in neurogenesis and cognition in response to psychological stress.

Risk for opioid use disorder (OUD) is substantially heritable, yet its genetic architecture remains only partly understood. This study examined oxycodone intake in two nearly isogenic rat strains, Wistar Kyoto More Immobile (WMI) and Less Immobile (WLI), and their reciprocal female F1 offspring. The parental strains differ in depression-like behavior and substance use vulnerability, with WMI rats consuming more oxycodone than WLI controls. Voluntary consumption was measured with an operant licking self-administration protocol that delivered 60 l drug per reward. Across four experimental stages, oxycodone concentrations increased from 0.025 to 0.1 mg/ml, and session durations increased from 1 to 4 hours. Female offspring showed a parent-of-origin effect. F1 females sired by WMI fathers (WLIxWMI) displayed accelerated escalation during the transition from 1-hour to 4-hour sessions in Stage 2 and consumed more oxycodone than reciprocal WMIxWLI females across expanded-access stages. This vulnerability was associated with increased licking during the drug-unavailable timeout period. In WMI and reciprocal WMIxWLI female, consumption was regulated by the drugs subjective value, as measured by lick microstructure, during Stages 1 and 2. This relationship was absent in WLIxWMI females during Stage 2. Together, these findings suggest that paternal WMI lineage is associated with a rapid transition to high oxycodone intake and cue-directed drug seeking, and identify a parent-of-origin effect that may contribute to female vulnerability to addiction.

Alcohol use disorder (AUD) is a widespread psychiatric condition, yet the molecular mechanisms underlying its development remain poorly understood. While prior studies have largely focused on protein-coding genes, long non-coding RNAs (lncRNAs) remain underexplored in AUD. Malat1, a highly abundant and evolutionarily conserved lncRNA, is elevated in post-mortem brain tissue of human AUD subjects and rodents chronically exposed to ethanol; however, its causal contribution to AUD-relevant behaviors remains unknown. Using CRISPR/Cas9 genome editing, we generated two complementary global Malat1 knockout models to assess its role in alcohol intake and related phenotypes. Constitutive knockout selectively attenuated acute functional tolerance rate and every-other-day two-bottle-choice alcohol intake in females. These results were supported by an inducible adult conditional global knockout model, which reduced ethanol consumption in females without altering taste preference. Together, our findings provide the first causal evidence that Malat1 regulates alcohol consumption in a sex-specific manner, supporting further investigation into its underlying mechanisms in AUD.

Early-life adversity durably alters neural development through complex mother-offspring interactions whose underlying mechanisms remain poorly understood. We investigated how neonatal maternal separation (NMS) affects the large repertoire of maternal behaviors and subsequently influences spinal nociceptive circuit development and pain responses in rat offspring. Rat dams underwent NMS from postnatal day 2 (P2) to P12, 3h/day, and maternal behaviors were assessed before and after the separation period. These behaviors were compared to those of control (non-separated) dams. Offspring spinal cord and dorsal root ganglia were analyzed at P14 and P24 for several neurotrophic, glutamatergic, and GABAergic gene expression patterns. Offspring nociceptive sensitivity was also assessed at P24. NMS induced increased maternal behaviors (including longer arched-back nursing, higher nest occupancy, and better pup retrieval efficiency), alongside reduced self-care behaviors. These behavioral adaptations were correlated with spinal gene reprogramming in offspring, characterized by a biphasic developmental pattern. At P14, we observed elevated neurotrophic signaling alongside increased GABAergic and glutamatergic markers. By P24, neurotrophic factors decreased while compensatory changes emerged, yet persistent excitatory-inhibitory imbalances remained evident. Parallel to these results, NMS rats also showed mechanical and thermal hot hypersensitivity at P24. These findings reveal that despite apparent maternal behavioral compensation following NMS, offspring exhibit neurotrophic-driven developmental dysregulation resulting in persistent spinal circuit alterations. The disconnect between maternal behavioral normalization and sustained molecular changes suggests that early separation stress triggers enduring neurobiological cascades independent of ongoing maternal care quantity, with long-term consequences for sensory processing and pain sensitivity.

Opioid Use Disorder (OUD) remains a prominent threat to global health. Genetic background influences the susceptibility of developing OUD, although specific genetic factors remain elusive. Rodent models that differ in susceptibility to escalation and dysregulation of opioid use are valuable tools to facilitate discovery of genetic pathways. Phenotypes associated with the development of OUD were compared in seven classic inbred rat strains (M520/N, WKY/NCrl, F344/NCrl, F344/Stm, LEW/Crl, LEW/SSNHsd, LE/Stm) from the Hybrid Rat Diversity Panel (HRDP). A two-phase self-administration paradigm was utilized to assess characteristics of the acquisition of oxycodone self-administration during daily 2-h sessions, and the escalation of oxycodone use during daily 12-h sessions. Genetic background influenced the acquisition of oxycodone self-administration as indicated by differences in the initiation of responding for oxycodone during each session and different amounts of oxycodone intake. We observed that escalation of oxycodone intake between-sessions was strain dependent, and the within-session distribution of oxycodone intake was strongly influenced by strain. The M520/N strain engaged in a unique pattern of intake, characterized by rapid initiation of oxycodone responding during the acquisition phase and a significant burst-like responding during escalation. Strain-dependent sex differences were also observed in several acquisition and escalation metrics. Of interest, burst responding was more prevalent in females of the M520/N strain compared to males. Together, these data indicate that genetic background influences not only overall oxycodone intake, but specific within- and between-session metrics that capture patterns of consumption across the substance use trajectory.

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.

Pathogenic variants in MAPK8IP3 (JIP3) cause Neurodevelopmental Disorder with or without variable Brain Abnormalities (NEDBA), characterized by cognitive impairment, global developmental delay, motor dysfunction, abnormal muscle tone, behavioral dysregulation including autism and attention deficit hyperactivity disorder (ADHD), seizures, and structural brain abnormalities. While more than 30 largely de novo MAPK8IP3 variants have been reported, the functional impact of variants across JIP3 protein structural domains is poorly defined. To address this knowledge gap, we compare clinical features of individuals with a truncating (p.E27X) or one of two missense (p.R578C and p.R1146C) variants from distinct JIP3 functional protein domains to corresponding knock-in mouse models. Our findings showed that all individuals, regardless of variant type, exhibited delays in language and gross motor function, but individual variants were associated with distinct motor, cognitive, and psychiatric symptoms. Corresponding homozygous p.E27X and p.R1147C variant mice resulted in embryonic lethality, consistent with essential roles for JIP3 in early neurodevelopment. Behavioral characterization of viable heterozygous mice revealed variant-specific locomotor, motor coordination, and hindlimb clasping defects that closely recapitulate clinical observations. Heterozygous p.R579C mice exhibited reduced locomotion, impaired motor performance, and hypertonia-like clasping, mirroring human motor deficits. In contrast, p.R1147C mice displayed hyperactivity, hindlimb clasping, and decreased brain weight, paralleling human clinical features. Together, our findings demonstrate that while distinct MAPK8IP3 variants lead to some shared phenotypes, they are also associated with distinct phenotypes that could reveal domain-specific aspects of JIP3 function. This work establishes the first domain-resolved in vivo rodent models of NEDBA and provides a validated translational platform for mechanistic investigation and preclinical therapeutic testing.

Although female rats are typically less aggressive than male rats, lactating females will vigorously defend their nests and pups against an intruder. Much attention has been directed at the consequences of this aggression on the intruder and less on the consequences for the mother and her subsequent interactions with her pups. Here, we exposed resident Sprague-Dawley dams to the resident-intruder paradigm twice daily for five consecutive days, beginning when the dam's (RES) pups were 7 days old, to assess social stress effects on maternal behavior and neurobiology. Controls were dams that had time-matched (TMC) separation from their pups but were not exposed to intruders, and naive moms which were never separated nor exposed to an intruder (CTL). We assessed the dam's subsequent behavior and interactions with her pups on Day 1 and Day 5, and Fos expression after Day 5 in select regions of the prefrontal cortex, amygdala, hypothalamus and periaqueductal gray of the midbrain. In separate cohorts, after pups were weaned, the dams underwent restraint stress and plasma corticosterone assayed. PCA analysis of the dam's behaviors identified three components: normal self-focused behaviors; nurturing behaviors and rough non-nurturing behaviors. Relative to CTL, RES dams exhibited more disrupted behaviors towards their pups, including, rough transport, stepping on pups, and flinging/tossing pups around the cage. In contrast, TMC Dams showed some, but fewer changes relative to CTL, suggesting that separation from pups alone does not account for all disrupted behavior in RES dams. The bulk of these behavioral effects occurred in the first 5-10 min after reunion with the pups and were seen on both the first and fifth day of testing. Of the brain regions examined, the prefrontal cortex was activated by both the defeat/intruder stress (RES) and separation stress (TMC), whereas the dorsal PAG was activated specifically by the defeat/intruder stress. The medial and basolateral amygdala exhibited differential neuronal activity between the RES defeat/intruder-exposed dams and the other two groups. The RES moms exhibited an insufficient adrenocortical response to acute restraint stress. The results suggest that amygdala-dPAG activity is important for dissociating disrupted maternal care in RES (due to defense of the nest against an intruder) from simple pup separation, both of which activate the mPFC. The experience of repeatedly defending the nest may induce subsequent disruptions in HPA responses. The amygdala-dPAG pathway may regulate aspects of stress and emotional regulation exhibited by mothers who defend their offspring against intruders.

Ambiguity represents a form of uncertainty in which outcome probabilities cannot be explicitly learned, making decisions dependent on emotional states and cognitive biases. Early-life stress (ELS) increases the risk of adverse mental and physical health outcomes and alters affective processing and learning. ELS may thus affect how ambiguous information is processed, which may depend on interactions with adulthood stress (AS) and mechanistically on bioenergetic mechanisms mediated by top-down cognitive control systems within the prefrontal cortex (PFC). The present study investigated the effects of AS in rats exposed to early maternal separation (MS), a rodent model of ELS, on a task assessing cognitive bias, together with putatively accompanying alterations in PFC mitochondrial function. Cognitive bias was assessed using an ambiguous cue task (ACT) in MS and non-separated control rats tested at baseline and following repeated unpredictable mild stress during adulthood. MS did not affect baseline cognitive bias but increased response latencies. Following AS, control animals showed a significant negative shift in cognitive bias, whereas MS animals were resistant to this shift. MS was also associated with greater PFC mitochondrial respiratory capacity and uncoupling of oxidative phosphorylation following AS. These findings suggest that ELS is associated with a recalibrated phenotype that buffers against the affective consequences of later stress. Enhanced PFC mitochondrial bioenergetics may underlie this resilience, highlighting the importance of developmental context in shaping affective-cognitive responses to stress.