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

Abstract

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.

Keywords: Bed nucleus of stria terminalis; Oxytocin; Sex differences; Social anxiety; Social defeat; Stress.

Figure 1. Effects of systemic administration of OTA and social defeat stress on behavior

Timeline of experiment (A). Mean and SEM time spent in the interaction zone (B), in active investigation (C), and in corner zones (D) during the social interaction test. Mean and SEM of odor preference ratio during OP test (E). Left column shows diagrams representing behavior measured, middle column shows female data, right column shows male data, *=p < 0.05 effect of 5mg/Kg OTA vs. saline, †=p<0.01 effect of stress in saline treated animals, ***=p<0.01 effect of 1mg/Kg OTA vs. saline in stressed animals, ↓ p=0.08 effects of 5mg/Kg OTA vs. saline in stressed animals.

Figure 2. Effects of OTA on OTR activation of G-proteins and β-arrestins recruitment

Mean and SEM of BRET ligand effect of OT, PBS, OT+ OTA and three different concentrations of OTA for (A) Gq, (B) Gi1, (C) Gi2, (D) Gi3, (E) GoA, and (F) GoB activation and (G) β-arrestin 1 and (H) β-arrestin 2 recruitment *=p < 0.05 effect of drug treatment vs. PBS, ***=p < 0.001 effect of drug treatment vs. PBS.

Figure 3. OTR binding in naïve and stressed males and females

Timeline of experiment (A). Mean and SEM of OTR binding in (B) the dorsolateral nucleus accumbens core (NAcdl) core, (C) NAc shell, (D) dorsal lateral septum, (E) anteromedial bed nucleus of the stria terminalis (BNSTam), (F) paraventricular nucleus (PVN), and (G) central nucleus of the amygdala (CeA). † =p < 0.05 main effect of stress ***=p < 0.01 main effect of sex. Ac=anterior commissure, LV= lateral ventricle,3rdV= third ventricle, Ic= internal capsule.

Figure 4. Effects of intranasal administration of OT on EGR1 immunoreactivity in naïve males and females

Timeline of experiment (A). Mean and SEM of EGR1 positive cells per mm2 detected in BNSTam (B), and NAcdl (D), *=p < 0.05 effect of intranasal OT vs. saline. Diagrams of BNSTam (C) and NAc (E) quantification regions with representative photomicrographs and box placement. Ac=anterior commissure, LV=lateral ventricle

Figure 5. Effects of site-specific administration of OTA on behavior in stressed females

Timeline of experiment (A). Mean and SEM time spent in the interaction zone (B) and in corners (C) during social interaction. There were no differences in time spent in the interaction zone during acclimation (D) or in locomotor behavior during open field phase (E). Example of hits in Nissl stained slices in NAcdl (F) and BNSTam (G) with injections sites indicated by circles. Diagrams showing location of injections considered as hits for NAcdl (H) and BNSTam (I). Under each diagram, the number of animals receiving injection in each site is indicated. ***=p < 0.01 effect of OTA vs. saline in animals receiving injections in BNSTam.

Figure 6. Effects of OTA on risk assessment behavior

Drawing of focal mouse oriented towards target mouse (A). Mean and SEM time spent with head oriented towards the target mouse in control and stressed females receiving systemic injections of saline, 1 mg/kg OTA or 5 mg/Kg OTA (B). Stressed females receiving site specific injections of aCSF or OTA (C). Control and stressed males receiving systemic injections of saline, 1 or 5 mg/Kg OTA (D). There was no difference between control and stressed females during acclimation phase (E, when the cage was empty). Similarly, OTA infusion in to the BNSTam had no effect during the acclimation phase (F). *=p<0.05 main effect of OTA vs saline or aCSF, †=p=0.01 effect of stress vs. same drug treatment (aCSF or 1 mg/kg OTA).