AbstractThe midbrain periaqueductal gray (PAG) region is a critical anatomical regulator of fear‐related species‐specific defensive reactions (SSDRs). Pituitary adenylate‐cyclase‐activating polypeptide (PACAP), and its main receptor PAC1, play an important role in fear‐related behavior and anxiety disorders. However, the function of the PACAP‐PAC1 system within the PAG with regards to SSDRs has received little attention. To address this gap, we used transgenic PAC1flox/flox mice to examine both conditional and unconditional defensive reactions. We performed conditional PAC1 gene deletion within the ventrolateral(vl)PAG of PAC1flox/flox mice using an adeno‐associated virus (AAV) coding for Cre recombinase. Following viral expression, we used a white noise fear conditioning preparation that produces both an unconditional activity burst to the onset of noise that is followed by conditional freezing. On Day 1, mice received five white noise foot‐shock pairings, whereas on Day 2, they were exposed to white noise five times without shock and we scored the activity burst and freezing to the white noise. Following behavioral testing, histology for immunofluorescent analysis was conducted in order to identify PACAP positive cells and stress‐induced c‐fos activity respectively. We found that PAC1 deletion in vlPAG increased the unconditional activity burst response but disrupted conditional freezing. PAC1 deletion was accompanied by higher c‐fos activity following the behavioral experiments. Furthermore, a significant portion of PACAP‐EGFP positive cells showed overlapping expression with VGAT, indicating their association with inhibitory neurons. The findings suggested that intact PACAP‐PAC1 mechanisms are essential for SSDRs in vlPAG. Therefore, midbrain PACAP contributes to the underlying molecular mechanisms regulating fear responses.