Frontiers in Behavioral Neuroscience

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.

Zebrafish are widely used to study learning and memory. Here, we describe zAcademy, a programmable conditioning arena released in full as open hardware and open software. zAcademy combines a 3D-printed dual-chamber arena, bottom-mounted LED matrices that generate visual patterns under software control, graphite rod electrodes gated by an Arduino-controlled relay, an overhead infrared camera, and a position-based analysis workflow. In one 180-min session, adult zebrafish were classically conditioned to associate a checkerboard light pattern beneath the arena (conditioned stimulus, CS) with a mild shock (unconditioned stimulus, US). Conditioned avoidance was assessed in six unreinforced CS-alone probes: one immediately after training to test learning, the remaining five at 30-min intervals to test memory. Spatial preference was quantified as a performance index (PI; occupancy of the dark zone). Wild-type fish shifted toward the dark zone immediately after training, whereas shank3 KO fish did not. The wild-type response was carried primarily by females rather than males. In addition, among initial responders, females sustained elevated PI at more later probes. zAcademy provides an inexpensive, fully documented, and readily modified platform for comparing experience-dependent behavioral change across genotypes and sexes.

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.

Anxiety disorders are among the most common psychiatric illnesses, with a significantly higher prevalence in females than in males. The open field (OF) and the elevated plus maze (EPM) are the two assays used most often to model anxiety-like behavior in mice. It is usually assumed that they index a common underlying trait, yet few studies have investigated this in a large group of male and female mice using an advanced analytical approach. The goal of these studies was to determine sex differences in locomotor activity and anxiety-like behavior in the OF and the EPM, and to establish which behavioral parameters correlate between the tests. Adult male and female C57BL/6 mice (n = 61; 29 male, 32 female) were given a single 15 min session in the OF, analyzed as a whole and in three consecutive 5 min blocks, and a single 5 min EPM trial. Males and females did not differ on any OF measure over the cumulative 15-minute session. Locomotion and stereotypy declined over time with females habituating faster than males while center exploration did increase. In the EPM, females made more closed-arm entries than males while open-arm exploration was identical. Every significant correlation between the two tests involved an activity measure. All four pairs formed between OF and EPM activity parameters were significant whereas none of the six pairs formed between OF center parameters and EPM open-arm parameters were significant. Hierarchical clustering revealed the same structure, placing the activity measures of both tests in one cluster, the EPM open-arm measures in a second, and the two OF center measures in a third. The cross-test activity correlation was strongest during the initial five minutes of the OF and weakened as the session progressed. These studies indicate that the OF and EPM yield convergent measures of activity, but divergent measures of anxiety-like behavior.

The rapidly increasing number of video tracking-based behavioral summary tools and methods raises the question as to the most suitable approaches for pharmacological fingerprinting in pre-clinical research. We have recently shown that social context has a strong effect on behavioral syntax in mice, further suggesting that treatment effects can be context dependent. Here, we aim to answer the question whether there is an optimal combination of context and behavioral summary method for effect detection and discrimination of different psychoactive substances in a controlled environment. To this end, we applied eight different treatment-dosage pairs (amphetamine 1.5,3,6 mg/kg; modafinil 5,10,50 mg/kg; seltorexant 3,10 mg/kg) and evaluated five different approaches to behavioral summary: parametric aggregation, unsupervised segmentation in Keypoint-MoSeq (KPMS) & Variational Animal Motion Encoding (VAME), and supervised segmentation in Simple Behavioral Analysis (SimBA) & A-SOiD, across two different contexts (Solitary & Social) in 314 recordings of freely moving mice in an open-field arena. Surprisingly, our results show no significant differences in performance across models and context. Across treatment effect detection to treatment-dose discrimination, all models showed performance significantly above chance that was insensitive to various data limitations and extensions. Overall, our study shows that under the tested conditions and treatments, the choice of a behavioral summary model does not meaningfully affect the description of treatment effects. Simple aggregate measures from tracking data and machine learning based behavioral summary approaches that are expensive, in terms of training data and computational resources, performed equally well. Our findings taken together with literature suggest that the fuller decomposition of complex behavior through unsupervised machine learning might be necessary for the description of large-scale datasets but does not necessarily align with the goals present in smaller-scale treatment discrimination tasks common in pre-clinical research.

It is estimated that approximately fifty percent of the population will encounter at least one traumatic event during their lifetime; however, only six percent develop Posttraumatic Stress Disorder (PTSD), a trauma-related disorder characterized by persistent alterations in affect and cognition, including an exaggerated fear response. It is well established that females (9.7%) are more than twice as likely as males (3.6%) to be diagnosed with PTSD. Nevertheless, a significant challenge remains in understanding how the severity of traumas experienced in childhood versus adulthood influences PTSD development across sexes. To address this challenge effectively, the adoption of preclinical animal models that accurately reflect these key characteristics is crucial. The Stress Enhanced Fear Learning (SEFL) paradigm in rodents encapsulates some of these complexities associated with PTSD, such as persistent fear sensitization following exposure to acute trauma. This study investigates how stress severity and developmental timing affect the long-term maintenance of SEFL in male and female rats. Juvenile and adult rats were initially subjected to no stress (control, context only), low stress (1 footshock), moderate stress (3 footshocks), or high stress (15 footshocks), followed by an extinction session either one day (recent) or sixty days (remote) later. Subsequently, to evaluate SEFL in a new context, all subjects received a single footshock and were tested the following day. Shortly after stress induction, all groups displayed SEFL. Although the effects persisted for sixty days in adult males and juvenile females, even at the lowest stress level, they were significantly reduced in juvenile males and adult females. These findings offer novel insights into sex-related differences in the persistence of SEFL and introduce new considerations regarding how the timing of developmental stressors influences future threat encoding and fear responses.

Despite its strong link to neuropsychiatric conditions, frustration remains critically understudied in humans and animals alike. Therefore, there is an urgent need to develop tools to understand and therapeutically target frustration-related functions. Interestingly, humans and rats respond similarly during frustrative nonreward by increasing barpress durations. We previously validated barpress duration in rat operant tasks as a reliable measure of frustration-related behavior; however, it is wellknown that in addition to duration of responding, emotional states such as frustration alter other aspects of responding such as force of pressing. One-dimensional, static measures such as maximum force could miss rich information contained within operant data. Thus, the objective of this study is to apply machine learning (ML) to force/time profiles to discriminate frustration-related barpresses from non-frustration-related barpresses. Results showed an AUROC for FR1 (i.e., non-frustrated) vs. extinction (frustrated condition) for individual barpresses of 0.65 that improved to 0.84 with a chunk size of 10. The model generalized well to progressive ratio responding, a different kind of frustration procedure. We conclude that force/time profiling does provide utility beyond one dimensional measures of duration or force separately, meaning that we can indeed infer the internal state of frustration from behavior using ML techniques. Importantly, this project will also serve as proof-of-concept for applying ML to predict other internal states from barpress data.

In recent years, rodent touchscreen-based paradigms have gained popularity for their flexible task design, automated data collection and improved standardization. These devices minimize experimenter intervention and contribute to more replicable and reliable research. However, the high costs and proprietary hardware / software associated with commercially available systems present a financial barrier for smaller labs or researchers with limited resources. To address this issue, we developed "NC4Touch", an open-source scalable, modular rodent testing apparatus. It features three independent touchscreens and a feeding port for easy use in a wide array of tests comparable to those available with standard operant chambers, with the added benefit that visual stimuli can be highly customized. The system includes a user-friendly graphical interface that offers real-time control, task customization, video recording, and data management. We demonstrate its effectiveness in a visual discrimination task using both rats and mice. Several devices can be operated in parallel from the same computer and user interface, allowing high- throughput data collection. We provide detailed assembly instructions for the hardware and well- documented and easily-configurable software. We hope that the affordable open-source nature of NC4Touch will expand the scope of behavioral testing, allowing researchers to overcome traditional financial barriers and create a more collaborative community. Significance StatementTouchscreen-based testing is a powerful tool for assessing cognition in rodent models, however commercial systems remain cost-prohibitive for many researchers. NC4Touch is a rodent touchscreen apparatus that provides an open-source, customizable, and affordable alternative that enables high-throughput, cognitive testing in both mice and rats. By lowering financial barriers and promoting hackability, this system enables greater accessibility and collaboration in research.

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.

Executive functions are a set of cognitive processes that regulate behavior and adapt responses to changing demands. Impairments in these processes are common in various neuropsychiatric conditions, including obsessive-compulsive disorder. Mice overexpressing the neuronal glutamate transporter EAAT3 in the forebrain (EAAT3glo/CaMKII) exhibit increased compulsive behaviors and synaptic alterations relevant to OCD. However, whether EAAT3 overexpression affects executive functioning across multiple cognitive domains has not been studied. Using operant conditioning and visuospatial learning tasks, we evaluated the performance of EAAT3glo/CaMKII mice across three behavioral domains commonly associated with executive function: cognitive flexibility, working memory, and inhibitory control. EAAT3glo/CaMKII mice showed impaired cognitive flexibility in both operant extinction and reversal learning. EAAT3glo/CaMKII mice also failed to acquire the Trial-unique, delayed nonmatching-to-location task, suggesting impaired working memory, and showed increased impulsivity in the five-choice serial reaction time task, as evidenced by elevated premature responses. Perseverative responses, response accuracy, and attentional performance were unaffected in EAAT3glo/CaMKII mice. Collectively, these findings suggest that EAAT3 overexpression in forebrain neurons is associated with a cognitive profile characterized by difficulties in flexibility and task acquisition, alongside increased impulsivity, while attentional performance and motivational measures remain intact in a model of compulsive behavior.

Defensive survival behaviors are shaped by both danger attributes and internal physiological states. Well-characterized defensive responses depend not only on whether danger is potential or perceived, but also on its spatial and or temporal proximity. The relationship between danger imminence and defensive behaviors is referred to as the threat imminence continuum, which provides a conceptual framework bridging ecology and behavioral neuroscience. Although this theoretical framework is gaining momentum, another much less explored danger attribute is the extent to which the environment may be controllable. Most paradigms assessing defensive behaviors rely on conditioned stimuli as danger warning-signals in stable environments that hamper the study of environment controllability and its impact on defensive behaviors. To address this question, we repurposed the active place avoidance (APA) paradigm combined with supervised machine learning to characterize multivariate exploratory and defensive behaviors in male and female mice. One important benefit of APA is that it allows the study of active avoidance when the platform is rotating as well as deliberative avoidance when the platform is immobile. As such, APA is very well suited to test the impact of environment controllability on the expression of defensive behaviors. Using this unified preparation, we systematically compared quantitative danger attributes (punishment intensity, behavioral agency) across danger states (absence, presence and omission of punishment) and learning experience (early and late learning stages), while considering sex as a biological variable. Our results demonstrate that defensive behaviors depend on punishment intensity in a sex-dependent manner in APA. Defensive responses can be classified as proactive (risk assessment) or reactive (freezing, escape) in APA. Their response probability depends on behavioral agency, is mutually exclusive and depends on danger spatial proximity. Acute treatment with norepinephrine transporter blocker selectively increases reactive defensive behaviors while sparing proactive defensive behavior. Collectively, these results provide experimental evidence that environmental stability calibrate proactive and reactive defensive behaviors whose neural correlates may be divergent.

We previously reported, using in-vivo fiber photometry, that operant responding reinforced by access to a peer (social self-administration) is associated with phasic dopamine increases in nucleus accumbens (NAc) core following lever insertion (reward-availability cue) and gradual increases preceding lever-pressing. Here, we sought to replicate these findings and determine whether dopamine signals (1) generalize to responding for high-carbohydrate palatable food, (2) show opposite patterns during negative reinforcement (shock avoidance/escape), and (3) depend on whether reinforcers are experienced alone or together. We trained rats (n=11; 6 females) to lever-press for access to a same-sex peer (15 s/trial) and palatable food (45-mg pellet/trial), followed by shock avoidance/escape (0.18-0.26 mA). After training, we expressed the dopamine sensor GRAB-DA2m and implanted optic fibers into NAc core. We measured dopamine activity during sessions with either one- or three-reinforcers. During social self-administration, dopamine activity showed phasic increases following lever insertion and gradual increases preceding lever-pressing; responses were moderately greater during sessions with all three reinforcers. Palatable food self-administration showed a similar pattern, but responses were approximately twofold greater during single-reinforcer sessions. During shock avoidance/escape, dopamine activity showed phasic decreases at warning onset, lever insertion, and shock onset; responses were also greater during single-reinforcer sessions. Results suggest that NAc core dopamine signaling distinguishes positive from negative reinforcement and is modulated by reinforcer availability. Compared with single-reinforcer sessions, dopamine responses during food self-administration and shock avoidance/escape were reduced during sessions with all three reinforcers, whereas responses during social self-administration modestly increased. Significance statementNucleus accumbens (NAc) dopamine is critical for processing the valence of positive reinforcers, whereas its role in negative reinforcement is less well understood. Here, we extended our prior work on operant social self-administration to determine whether these findings generalize to food reinforcement, show the opposite pattern during operant negative reinforcement (shock avoidance/escape), and depend on whether reinforcers are experienced alone or together. Results suggest that NAc core dopamine activity differentiates positive and negative reinforcement and is modulated by the presence of other available reinforcers. Specifically, dopamine responses during food self-administration and shock avoidance/escape were reduced when all three reinforcers were available together compared with when each reinforcer was experienced alone, whereas responses during social self-administration showed the opposite pattern.

Stressful experiences are a major risk factor for the development of neuropsychiatric disorders in vulnerable individuals. Identifying features that can distinguish stress-susceptible from resilient individuals remains a central goal in stress neurobiology. Due to the routine exclusion of female subjects from stress research or the use of male-defined criteria in behavioral characterization of subjects post-stress, female stress responses and vulnerability profiles remain poorly understood. This work aimed to gain a better understanding of sex-specific stress responses to social defeat stress. Here, we used a novel paired male-female social defeat paradigm and tracked subjects longitudinally in a ''two-hit'' design to evaluate responses to both acute social defeat stress (ASDS) and chronic social defeat stress (CSDS), and to determine how prior stress exposure influences subsequent vulnerability. We found that both ASDS and CSDS induces changes in social avoidance/approach behavior in males but not females, as measured by a classic social interaction ratio. Prior ASDS exposure enhanced susceptibility to subsequent CSDS most clearly in males, with females displaying specific changes in corner zone occupancy. Furthermore, we observed divergent anxiety profiles between males and females in a post-stress behavioral battery. Finally, females exhibited specific stress-related locomotor responses following ASDS. Together, these findings suggest that females may adopt distinct behavioral strategies following social defeat stress.

ROR{gamma}t, encoded by Rorc, is a key transcriptional regulator of T helper 17 (Th17) cell differentiation, and IL-17A signaling has been implicated in neural development and behavior. We examined behavioral phenotypes in male ROR{gamma}t-transgenic mice with T-cell-biased ROR{gamma}t overexpression and their wild-type littermates. Mice were assessed using the resident-intruder paradigm and the light-dark box test, followed by body-weight measurement. ROR{gamma}t-transgenic mice had significantly lower body weight than wild-type mice. No genotype differences were detected in attack latency, attack frequency, light-compartment occupancy, light-dark transitions, or transition latency. In contrast, analysis of locomotor activity in 1-min bins revealed a significant genotype x time interaction. Transgenic mice showed nominally reduced locomotor activity during the first minute of the light-dark box test, although this single-bin comparison did not remain significant after correction for multiple comparisons. These findings indicate that T-cell-biased ROR{gamma}t overexpression is associated with altered temporal dynamics of locomotor activity in a novel environment rather than a generalized anxiety-like or aggression-related phenotype.

Chronic social isolation induces persistent behavioral and physiological changes across species. In humans, prolonged social isolation is associated with insomnia and increased risks of cognitive decline, cardiovascular disease, compromised immunity, and premature mortality. Yet whether these consequences are modified by the environment in which social isolation is experienced remains unclear. In particular, can access to social sensory information from conspecifics alleviate the effects of isolation and can various ambient environmental settings modify them? A related question arises during behavioral measurement because behavioral assays themselves impose a new environmental context. Whether these assays simply capture the enduring effects of prior social experience or further modify those effects remains unknown. Using social isolation-induced sleep loss in Drosophila as a model, we systematically examined how the social, spatial, and photoperiodic environments during isolation and behavioral monitoring influence the isolation phenotype. Providing isolated flies with visual, olfactory, or tactile social cues from conspecifics living behind a divider, either individually or in combination, was insufficient to alleviate sleep loss. Spatial confinement exacerbated isolation-induced sleep loss, while winter-like photoperiod in spatially confined Drosophila Activity Monitor assays revealed the clearest progressive decline in sleep during sleep monitoring. Together, these findings demonstrate that social isolation-induced sleep loss is both enduring and dynamic: it persists despite exposure to social cues yet remains sensitive to the environmental context in which isolation is experienced and behavior is measured. More broadly, our study establishes a tractable framework for investigating the environmental modulation of chronic social isolation.

Context processing is an essential function in the cognitive repertoire of numerous species, including humans and mice, allowing them to learn from their environment and adapt their future behavior. Impairments or alterations in context processing are hypothesized to represent one of the core etiological mechanisms underlying trauma- and stressor-related disorders such as post-traumatic stress disorder (PTSD). Individual behavioral traits are key determinants of post-stress responses; however, how they influence distinct phases of contextual encoding and processing remains incompletely understood. This knowledge gap is further exacerbated by the limited availability of contextual fear protocols based on social stress, even though the latter is among the most frequent and impactful forms of stress exposure. Here, based on social defeat paradigms, we establish a longitudinal social conditioned place aversion (sCPA) protocol that leads to avoidance of the defeat-associated chamber in adult male mice. Prior characterization of mice according to anxiety-related phenotypes revealed that mice displaying high novelty-induced thigmotaxic (i.e. high anxiety-like) behavior showed lasting long-term memory of the defeat-associated chamber as denoted by persistent avoidance. On the other hand, lower levels of exploration/locomotor behaviors prior to stress exposure predicted greater avoidance of the defeat-associated chamber in the short-term. Finally, we reveal that inhibiting the activity of VTAVgat neurons during conditioning sessions abolished sCPA acquisition, while disrupting the predictive ability of locomotor/exploratory profiles derived prior to the sCPA. Our data establishes a robust temporally structured social stress-based conditioning protocol and reveals how prior individual anxiety- and exploration/locomotion-related traits influence distinct stages of context processing.

Acute disturbances of the light-dark cycle may lead to cognitive impairments associated with disturbances in hippocampal functions in humans and rodent models that are potentially governed by subcortical modulation. In this study, we applied a jet-lag-like model in mice by introducing a six-hour delay of the switch towards the light, inactive phase of mice following a contextual fear conditioning training. Phase delay (PD) resulted in a reduced fear memory expression in male but not female, associated with a sex-specific activation of orexinergic neurons in the lateral hypothalamus (LH) as well as of cells in the supramammillary nucleus (SuM) and in the hilus of the dorsal hippocampal dentate gyrus (DG), as assessed by immunolabelling for the activity marker c-Fos. Mimicking the overactivation of SuM and DG by chemogenetic stimulation before contextual fear memory retrieval replicated the PD-induced phenotype, suggesting a direct contribution of the SuM and the DG on modulating fear expression after PD. Further circuit analysis by c-Fos revealed a reciprocal interaction between the SuM and the DG. In addition, orexinergic neurons in the LH were activated by chemogenetic stimulation of the SuM. Together, our results reveal that an acute, jet-lag-like phase shift applied during late consolidation stages induced deficits in fear memory expression associated with an overactivation of the SuM-DG pathway and the orexinergic system. These findings may provide insights into the subcortical modulation of memory-relevant circuits, with relevance for acute light-dark rhythm disruptions prevalent in modern societies as well as for disorders associated with memory disturbances. Significance statementAcute disturbances of the light-dark cycle, as experienced during jet lag or shift work, are increasingly common and can impair memory and cognitive function. Here, we identify a brain circuit underlying jet-lag-induced fear memory deficits that occurs selectively in male, but not female mice. A six-hour delay of the dark phase impaired recall of a previously learned fear memory in males, associated with overactivation of two interconnected brain regions, the supramammillary nucleus and the dentate gyrus of the hippocampus, as well as neurons producing the wake-promoting signal orexin. Artificially mimicking this overactivation was sufficient to reproduce the memory impairment, revealing a hypothalamo-hippocampal circuit that translates circadian disruption into memory deficits, with implications for cognitive disorders and sex-specific vulnerability.

Quantifying social behavior requires accurate assignment of posture and actions to individual animals, which is often hindered by close contact, occlusion, and identity switches. Meanwhile, current behavioral recognition pipelines still lack stable predictive accuracy. Here we developed SABER, a locally deployable framework that couples multianimal pose estimation with identity preserving tracking, interpretable behavioral factor mining, and multiscale temporal behavior prediction from a single overhead video stream. Across spontaneous two mice social interaction, mating, aggression, and four mice recordings, SABER improved pose estimation accuracy and tracking continuity relative to comparator pipelines. Its behavioral factor-mining procedure identified interpretable kinematic, postural, and social descriptors, and temporal integration improved classification of behavioral categories. SABER offers an intuitive, open-source interface to facilitate use. Applied to social defeat stress mice, SABER detected reduced approach behavior and a multivariate behavioral profile that distinguished depression susceptible from control animals. SABER outputs could also be synchronized with miniscope calcium recordings, enabling joint analysis of behavioral states and neuronal population activity. SABER therefore provides an accessible, identity resolved route from single view social interaction video to behavioral phenotyping and brain behavior analysis.

Head-fixed behavior in rodents is a foundational technique in systems neuroscience which enables use of sophisticated imaging techniques in combination with animal behavior. However, accessibility of head-fixed behavior techniques is limited. Animal training consumes a large amount of experimenter labor and commercial setups, when available, are largely inflexible and financially burdensome. Here, we present a low-cost, modular, and open-source hardware and software implementation for head-fixed rodent decision-making tasks. Our design lowers experimenter labor and enables large teams of researchers to participate in animal training with minimal experimenter error using a simple touchscreen GUI and automated training progression. We demonstrate the efficacy of the platform by training a cohort of animals in a two-choice probabilistic rapid-reversal task in which mice continuously update action choices based on recent reward history. The presented design lowers the barrier to entry for laboratories seeking to conduct head-fixed rodent behavior and provides modular solutions for developing custom rigs based on experimental demands.

Opioid withdrawal drives continued opioid use in opioid use disorder (OUD), which affected an estimated 4 million Americans in 2024. However, only one non-opioid medication is FDA-approved for withdrawal treatment, and there is great need for more diverse treatment options. Mice are powerful models to study opioid withdrawal because they allow for controlled drug and environmental exposure, express OUD-relevant phenotypes, and provide access to withdrawal-relevant tissue at exact time points. However, mouse models are hindered by their over reliance on a single genetic background (C57BL/6J). Using a single background risks limiting the translatability of preclinical studies as they are projected into genetically diverse human populations. Inferring withdrawal intensity in mice typically relies on single behaviors or indexes of behaviors, and applying these approaches across mouse genetic diversity faces a critical challenge: differing withdrawal responses could either encode differences in withdrawal severity, or differences in withdrawal-evoked behaviors independent of severity. To overcome this limitation, a more comprehensive assessment of opioid withdrawal behaviors across mouse genetic diversity is needed. Here we leverage computer vision and machine learning to automate the simultaneous quantification of many mouse behaviors during opioid withdrawal in eight diverse strains of mice. We reveal that all eight strains exhibit withdrawal-evoked behaviors, and these behaviors are sufficient to identify the withdrawal state of the mouse. However, the effect of withdrawal on individual behaviors or indexes of behaviors is not uniform across genetic diversity, and extending methods tuned in one strain risks misclassifying withdrawal in others. Finally our approach was able to distinguish graded withdrawal intensity, as defined by opioid exposurewithin3of4strainstested. We therefore conclude that opioid withdrawal behavior is profoundly shaped by genetic background, but withdrawal severity can nonetheless be established within a background. Combining automated computer-vision phenotyping with genotype-aware modeling provides a scalable path toward genetically informed withdrawal assessment in diverse mouse populations, with the ultimate goal of identifying the genetic and biological networks that modulate opioid withdrawal and may represent new targets for treating opioid use disorder.