Project Summary Clinical disorders arising from maladaptive emotion regulation present a large burden on society worldwide. Many of these disorders show comorbidity, for example, addiction with anxiety disorders. Cues predicting something aversive elicit avoidance and fear behaviors whereas cues predicting reward elicit approach and reward-seeking behaviors. Cues signifying safety have the power to modulate fear and reward-seeking behaviors by informing the organism whether or not the environment is safe. Thus, safety, fear and reward behaviors, and the circuitries governing these behaviors, are intertwined. The majority of studies on reward and fear processing have been conducted in parallel, investigating the circuitries separately in primarily male subjects. If we hope to understand and treat comorbid disorders resulting from maladaptive emotion regulation increased efforts in investigating how these circuitries integrate their functions to influence behavior is needed in both male and female subjects. The proposed work uses a novel behavioral paradigm explicitly designed to assess safety, fear and reward cue learning together in order to investigate the multiple circuitries that regulate these learning processes in a rat model. Our results show that, compared to males, female rats do not show suppression of conditioned fear in the presence of the safety cue, indicating a failure to regulate fear in ‘safe’ conditions, and they are more reward responsive during the reward cue. Since women are more than twice as likely as men to develop emotion dysregulation disorders, this paradigm offers a great opportunity to tease apart the sex differences in neural circuitry that are generating the behavioral sex differences. Together, the proposed aims are designed to identify the neural circuitry and mechanisms of safety, fear and reward cue discrimination and how they may differ between males and females. Aims 1 and 2 focus on the infralimbic prefrontal cortex, prelimbic prefrontal cortex and basolateral amygdala in male and female rats to identify differences in neural activity and the critical projections among these brain regions during safety-fear-reward discrimination. Aim 3 focuses on the projection from the ventral tegmental area to the basolateral amygdala in male and female rats. We expect to advance our understanding of the neural circuitry mediating discrimination among emotionally relevant environmental cues in both male and female subjects, thereby increasing our knowledge at a systems/circuits level of possible sex differences in emotional disorders. Determining how neural circuit sex differences are generating sex differences in safety-fear-reward discrimination will improve future development of sex-specific behavioral and drug treatments in individuals diagnosed with emotion dysregulation disorders.