Individuals affected by anxiety disorders experience significant distress in social situations that can lead to self-isolation and avoidance behaviors (Abramowitz and Deacon, 2010). Women are twice as likely to develop anxiety disorders compared to men (Kessler et al., 2012). While there are existing pharmaceutical and behavioral therapeutic interventions, ~40 % do not respond to these treatments (Williams and Trainor, 2018). Oxytocin is a neuropeptide that modulates social and emotional behaviors (Gimpl and Fahrenholz, 2001) and has been considered as a potential alternative therapeutic to existing antidepressants (MacDonald and Feifel, 2013, MacDonald and Feifel, 2014; Modi and Young, 2012; Gutkowska and Jankowski, 2012; Giovanna et al., 2020; van Zuiden et al., 2017; Schultebraucks et al., 2022; Rashidi et al., 2025). However, the field has struggled to explain findings where intranasal oxytocin sometimes decreases anxiety symptoms but in other cases increases anxiety and other psychiatric symptoms (Tabak et al., 2022; Schultebraucks et al., 2022). A potential explanation for these results is the social salience hypothesis of oxytocin (Bartz et al., 2011; Shamay-Tsoory and Abu-Akel, 2016), which proposes that oxytocin can modulate behaviors by enhancing attention to both positive and negative social contexts. Several lines of evidence indicate that oxytocin promotes salience by acting in discrete neural circuits to drive these dynamic responses (Steinman et al., 2016, Steinman et al., 2019; Nasanbuyan et al., 2018; Osakada et al., 2024; Duque-Wilckens et al., 2018).

In appetitive social contexts, activation of oxytocin receptors (OTR) in the mesolimbic dopamine system play a key role in promoting social approach behaviors. The nucleus accumbens (NAc) modulates motivational and learning processes (Salgado and Kaplitt, 2015), and activation of OTR within the NAc facilitates social approach (Williams et al., 2020), social learning (Nardou et al., 2019), pair bonding (Liu and Wang, 2003; Rigney et al., 2025), and social novelty seeking (Smith et al., 2017). Activation of OTR in the ventral tegmental area (VTA) has also been found to modulate social motivation (Borland et al., 2017) and learning (Hung et al., 2017). It was hypothesized that oxytocin acting in the mesolimbic dopamine system facilitates social salience in aversive social contexts (Shamay-Tsoory and Abu-Akel, 2016). However, recent evidence suggests that OTR in the extended amygdala may play an important role during stressful social contexts. Social defeat stress activates a population of oxytocin neurons in the medioventral bed nucleus of the stria terminalis (BNSTmv) in Peromyscus californicus (California mice) (Steinman et al., 2016) and C57Bl/6 J mice (Nasanbuyan et al., 2018). The BNST modulates anxiety-related and social behaviors (Lebow and Chen, 2016) and several subregions have strong molecular (Gegenhuber et al., 2022) and anatomical (Campi et al., 2013; Allen and Gorski, 1990) sex differences. Antisense knockdown of oxytocin in the BNST prevented defeat stress-induced social avoidance and social vigilance in female California mice (Duque-Wilckens et al., 2020). These BNST oxytocin neurons project to the anteromedial BNST (BNSTam) where activation of OTR promotes avoidance and vigilance (Duque-Wilckens et al., 2018; Luo et al., 2022). Oxytocin can also promote aversive responses in non-social contexts, as oxytocin in the dorsolateral BNST (BNSTdl) enhanced fear-potentiated startle (FPS) response in rats (Moaddab and Dabrowska, 2017). Together these data suggested that distinct neural circuits mediate the effects of oxytocin in appetitive and aversive social contexts (Steinman et al., 2019).

Most of the studies reviewed above relied on pharmacological manipulations to target OTR function. Pharmacological approaches target both locally expressed post-synaptic receptors and pre-synaptic receptors. Importantly, at least some behavioral effects of OTR can be mediated by pre-synaptic receptors. Dölen and colleagues showed that the behavioral effects of OTR antagonists infused into the NAc were mediated by OTR expressed on presynaptic terminals originating from the dorsal raphe nucleus (Dölen et al., 2013; Nardou et al., 2023). These studies were performed in C57Bl/6 J mice and utilized transgenic mouse lines to specifically target different populations of OTR. The CRISPR/Cas9 gene-editing systems provides a mechanism to perform similar manipulations in species for which transgenic lines are not available. A viral-mediated CRISPR/Cas9-based tool was developed to target OTR expression across a variety of rodent species used as models to study social behavior (Boender et al., 2023). When a guide RNA targeting the Oxtr gene was expressed with Cas9, OTR binding was reduced across six different rodent species, including California mice. We used this gene editing system to determine whether locally expressed OTR in the BNST or NAc modulate social approach and social vigilance in female California mice. This viral construct was recently used in a male spiny mouse model in the NAc, which reduced huddling behaviors with strangers as well as some feeding behaviors identifying a role for OTRs in non-reproductive contexts (Fricker et al., 2025).

California mice are monogamous rodents in which paired males and females defend joint territories. The high levels of aggression in female California mice make this species ideal for studying the effects of social defeat stress (SDS) in both males and females without the use of CD1 aggressive male mice generally used in C57BI/6 J models (Steinman and Trainor, 2017; Lake and Trainor, 2024). In this study we assessed the impact of OTR knockdown on behavior before and after stress using two behavioral assays in female California mice. Focal mice were tested in a large arena with an unfamiliar female stress-naïve target mouse confined to a small wire cage, which allows for precise measurements of social approach and social vigilance. Social vigilance is a risk assessment behavior in which an individual avoids but attends to a social context (Walker et al., 2023; Wright et al., 2020). Social approach is assessed by time spent within one body length of the cage. Focal mice were also tested in a smaller arena with freely moving stress-naïve and aggressive SDS target mice. This test is ideal for the observation of affiliative and defensive behaviors. Previous studies showed that social approach and social vigilance are modulated by OTR acting within complementary networks that include the BNST and NAc (Duque-Wilckens et al., 2018, Duque-Wilckens et al., 2020; Luo et al., 2022; Williams et al., 2020). We used a viral strategy to preform CRISPR-Cas9 editing of the oxytocin receptor target gene. We refer to reductions of OTR binding in CRISPR treated groups as “knockdown” within a specific brain region and examined whether changes in locally expressed OTR corresponded to changes in social behavior before and after social defeat stress. We used two behavioral assays to assess the roles of OTR on social approach as well as ethological measures of social interaction.