Safety learning produces rapid fear suppression and distinct amygdala-prefrontal engagement

Animals detect and evaluate signs of danger and safety in their environment to ensure survival, yet the neural mechanisms that distinguish safety learning from other forms of conditioned inhibition, remain poorly understood. Here, we directly compared fear and safety learning in male rats. Fear conditioned rats showed high freezing to the tone and the conditioning context, whereas safety conditioned rats showed significant tone-specific reduction in freezing. This safety cue could also generalize to a novel, previously unassociated threat context leading to suppressed freezing when presented demonstrating that inhibitory actions of safety cues are not tied to its original training environment but can modify fear expression across settings. Fear and safety learning also produced unique patterns of neuronal activation and glutamatergic receptor expression in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and central amygdala (CeA), as measured by c-Fos immunohistochemistry and Western blotting. Fear conditioning induced greater Fos expression in the BLA and CeA, as well as elevated amygdalar NMDA receptor (GluN1) levels, whereas safety learning increased amygdalar PSD-95 and AMPA receptor (GluA1) expression. Both safety and fear learning increased mPFC Fos expression without affecting glutamatergic receptors levels. Finally, safety conditioning was associated with lower tone-evoked freezing than fear conditioned rats across early extinction sessions and was accompanied by distinct patterns of prefrontal and amygdala activation across extinction. Together, these findings suggest that safety learning engages neural and behavioral mechanisms distinct from fear learning and extinction, while modifying amygdala-prefrontal circuits towards more rapid fear suppression.