Project Summary/Abstract Learning when to fear and when not to fear significantly impacts how we interact with our environment. Failure to appropriately regulate fear in situations that should be considered safe can be observed in stress disorders, such as Post-Traumatic Stress Disorder (PTSD). Even in individuals without a stress disorder, arousal is positively correlated with how threatening a safety cue is perceived. Restoring compromised behavioral control over fear under safety/threat conflict appears to rely on the strength of the safety cue as an inhibitor. This effectiveness as an inhibitor is critical to understand as presenting safety cues during exposure therapy could potentially enhance treatment outcomes in reducing maladaptive fear. However, the neural circuits promoting adaptive responding to safety cues that are well discriminated from fear cues are poorly understood. Therefore, identifying the behavioral and neural circuit mechanisms that regulate safety signaling is a critical and unmet medical need. Our extensive experience with safety conditioning paradigms makes us the best qualified to complete the proposed experiments. More specifically, we have the technical ability to articulate the essential neural circuitry for discriminating amongst safety, fear and reward cues, as well as conditioned inhibition of fear and reward. Our prior work has shown separate networks exist within the infralimbic cortex (IL) and basolateral amygdala (BLA) that either respond selectively to safety under threat conflict (safety-specific), or respond to both the safety conflict and reward cue (safety/reward). We propose that safety-specific signaling is encoded within the BLA and IL inputs to the central amygdala (CeA) (Aim 1), while the overlapping safety/reward signal is within the BLA, IL, and prelimbic (PL) inputs to the nucleus accumbens (NAC) (Aim 2). We also propose that the anterior cingulate cortex (ACC) and the retrosplenial cortex (RSC) will participate in the transfer of the fear reducing effects of a learned safety cue to novel contexts since they have been implicated in generalizing memories across contexts and integrating cues with contexts (Aim 3). In the proposed work we will be following individual cells longitudinally across reward cue learning, safety cue discrimination learning, tests for conditioned inhibition, cued fear extinction, and retardation of fear acquisition to determine how individual cells of this circuit generalize across different types of cued safety, cued reward and new contexts. Together, this work will impact the field of safety learning by identifying and teasing apart circuit mechanisms of how a safety cue suppresses fear, how it gains rewarding properties, and how it may regulate fear in unfamiliar contexts, all of which are highly relevant to many psychiatric disorders. In sum, we have a unique and novel translational approach to better understand the neural mechanisms of conditional discrimination that ma...