Divergent Effects of Peripheral vs. Central Oxytocin Administration on Observational Fear Behavior in Male and Female Mice

<b>Background:</b> Observational fear, a form of empathic response to others' distress, exhibits marked sex differences. Oxytocin (OT) is a key modulator of social and emotional behaviors, but its role in observational fear-and how this varies by sex and administration route-remains controversial. <b>Methods:</b> We studied behavioral responses in male and female mice during observational fear. We first blocked systemic oxytocin (OT) signaling with a peripheral antagonist. We then tested different routes of OT administration (intranasal, intraperitoneal). Further, we microinjected OT directly into the anterior insular cortex (AIC). Finally, we used a chemogenetics strategy to selectively activate or inhibit OT neurons. <b>Results:</b> Male mice exhibited sustained freezing behavior and elevated corticosterone levels in response to observational fear. In contrast, females more quickly resumed baseline activity levels and showed an increased number of interactions. Systemic blockade of oxytocin (OT) signaling selectively reduced fear expression in males. Strikingly, intranasal OT administration elicited heightened fear-related responses in both sexes, whereas intraperitoneal OT administration induced anxiolytic-like effects. Direct OT microinjection into the anterior insular cortex (AIC) produced sex-divergent reductions in fear responses: decreasing freezing duration in males and reducing avoidance behaviors in females. Chemogenetic activation of OTergic neurons replicated these anxiolytic effects, while inhibition had no effect. <b>Conclusions:</b> OT bidirectionally regulates observational fear in a sex-, route-, and site-specific manner, challenging the simplistic view of OT as universally prosocial. The AIC is a critical node in empathetic fear circuits. These findings underscore the necessity for precision in targeting the OT system for treating stress-related psychiatric disorders.