Extrahypothalamic oxytocin neurons drive stress-induced social vigilance and avoidance.

Oxytocin increases the salience of both positive and negative social contexts and it is thought that these diverse actions on behavior are mediated in part through circuit-specific action. This hypothesis is based primarily on manipulations of oxytocin receptor function, leaving open the question of whether different populations of oxytocin neurons mediate different effects on behavior. Here we inhibited oxytocin synthesis in a stress-sensitive population of oxytocin neurons specifically within the medioventral bed nucleus of the stria terminalis (BNSTmv). Oxytocin knockdown prevented social stress-induced increases in social vigilance and decreases in social approach. Viral tracing of BNSTmv oxytocin neurons revealed fibers in regions controlling defensive behaviors, including lateral hypothalamus, anterior hypothalamus, and anteromedial BNST (BNSTam). Oxytocin infusion into BNSTam in stress naïve mice increased social vigilance and reduced social approach. These results show that a population of extrahypothalamic oxytocin neurons plays a key role in controlling stress-induced social anxiety behaviors.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis.

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

Enriched laboratory housing increases sensitivity to social stress in female California mice (Peromyscus californicus).

Domesticated mice and rats have shown to be powerful model systems for biomedical research, but there are cases in which the biology of species is a poor match for the hypotheses under study. The California mouse (Peromyscus californicus) has unique traits that make it an ideal model for studying biological mechanisms underlying human-relevant behaviors such as intra-female aggression, biparental care, and monogamy. Indeed, peer-reviewed scientific publications using California mouse as a model for behavioral research have more than doubled in the past decade. Critically, behavioral outcomes in captive animals can be profoundly affected by housing conditions, but there is very limited knowledge regarding species-specific housing needs in California mice. Currently, California mouse investigators have to rely on guidelines aimed for more common laboratory species that show vastly different physiology, behavior, and/or ecological niche. This not only could be suboptimal for animals' welfare, but also result in lack of standardization that could potentially compromise experimental reproducibility and replicability across laboratories. With the aim of assessing how different housing systems can affect California mouse behavior both in the home cage as well as the open field and social interaction tests before and after social defeat stress, here we tested three different caging systems: 1. Standard mouse cage, 2. Large cage, and 3. Large cage + environmental enrichment (EE), which focused on increasing vertical complexity based on observations that California mice are semiarboreal in the wild. We found that the effects of housing were largely sex specific: compared to standard cages, in females large + EE reduced home cage stereotypic-like backflipping and rearing behaviors, while large cage increased social interactions. In males, the large+EE cage reduced rearing and digging but did not significantly affect backflipping behavior. Interestingly, while there were no significant differences in the open field and social interaction pre-stress behaviors, large and large+EE housing increased the sensitivity of these tests to detect stress induced phenotypes in females. Together, these results suggest that increasing social and environmental complexity affects home cage behaviors in male and female California mice without interfering with, but rather increasing the magnitude of, the effects of defeat stress on the open field and social interaction tests.

Acute inhibition of kappa opioid receptors before stress blocks depression-like behaviors in California mice.

Kappa opioid receptors (KOR) are considered to be a promising therapeutic target for stress-induced psychiatric disorders such as anxiety and depression. Preclinical data show that KOR antagonists have greater efficacy if administered before stressful experiences as opposed to afterwards. However, almost all of these studies use long-acting antagonists, leaving it unclear whether inhibition of KOR after stress is required for efficacy. Here we show that administration of the short-acting KOR antagonist AZ-MTAB before episodes of social defeat stress block the induction of anhedonia (both males and females) and social avoidance responses (females) that persist two weeks after stress. In both males and females pre-stress AZ-MTAB treatment also blunted anticipatory autogrooming behavior immediately prior to the third episode of defeat. In contrast when AZ-MTAB was administered two weeks after defeat (immediately before behavior testing) in female California mice, it was ineffective at reversing anhedonia and social avoidance. These results suggest that short-acting KOR antagonists may have greater therapeutic potential if administered before exposure to psychosocial stressors.

Sex Differences in the Effects of a Kappa Opioid Receptor Antagonist in the Forced Swim Test.

There is growing evidence that kappa opioid receptor (KOR) antagonists could be a useful class of therapeutics for treating depression and anxiety. However, the overwhelming majority of preclinical investigations examining the behavioral effects of KOR antagonists have been in male rodents. Here, we examined the effects of the long-acting KOR antagonist nor-binaltophimine (norBNI) on immobility in the forced swim test in males and females of two different rodent species (C57Bl/6J and California mice). Consistent with previous reports, norBNI (10 mg/kg) decreased immobility in the forced swim test for male C57Bl/6J and California mice. Surprisingly, dose-response studies in female C57Bl/6J and California mice showed that norBNI did not reduce immobility. Pharmacokinetic analyses showed that metabolism and brain concentrations of norBNI were similar in male and female C57Bl/6J. In the nucleus accumbens of male but not female C57Bl/6J, norBNI increased phosphorylation of c-Jun N-terminal kinase (pJNK), a putative mechanism for norBNI action. However, no differences in pJNK were observed in male or female California mice. Together, these results suggest that immobility in the forced swim test is less dependent on endogenous KOR signaling in female rodents and highlight the importance of examining the effects of possible therapeutic agents in both males and females.

Oxytocin Receptors in the Anteromedial Bed Nucleus of the Stria Terminalis Promote Stress-Induced Social Avoidance in Female California Mice.

BACKGROUND: The neuropeptide oxytocin (OT) is a key regulator of social and emotional behaviors. The effects of OT are context dependent, and it has been proposed that OT increases the salience of both positive and negative social cues. Here we tested whether the bed nucleus of the stria terminalis (BNST) mediates anxiogenic effects of OT. METHODS: First, we studied the effects of systemic administration of an OT receptor (OTR) antagonist L-368,899 on social behavior in male and female California mice exposed to social defeat. We examined the effect of L-368,899 on G protein activation and used early growth response factor 1 immunohistochemistry to identify potential sites of OTR action. Finally, we examined the effects of L-368,899 infused in the BNST on behavior. RESULTS: A single dose of systemic L-368,899 increased social approach in stressed female mice and decreased social approach in male mice naïve to defeat. L-368,899 prevented OT activation of G proteins and did not activate G proteins in the absence of OT. Intranasal OT, which reduces social approach in female mice but not male mice, increased early growth response factor 1 immunoreactivity in the nucleus accumbens core and anteromedial BNST in female mice but not in male mice. Stressed female mice that received an infusion of L-368,899 into the anteromedial BNST but not the nucleus accumbens core increased social approach and decreased social vigilance responses. CONCLUSIONS: Our results suggest that OTR activation in anteromedial BNST induces a vigilance response in which individuals avoid, yet attend to, unfamiliar social contexts. Our results suggest that OTR antagonists may have unappreciated therapeutic potential for stress-induced psychiatric disorders.