Midbrain neurons regulate the production of courtship squeaks in female mice

Animals produce different vocalization types (e.g., contact calls, mating calls) to communicate in different behavioral contexts, but how the brain is organized to enable the production of different vocalization types remains poorly understood. Recent work found that specialized neurons in the midbrain periaqueductal gray (PAG) regulate the production of ultrasonic vocalizations (USVs) in female and male mice, but these PAG-USV neurons are not required for the production of broadband vocalizations (squeaks). To identify PAG neurons that may regulate the production of squeaks, we used an activity-dependent labeling strategy (TRAP2) to identify and manipulate PAG neurons that are active in association with the production of courtship squeaks in female mice. Optogenetic activation of courtship squeak-TRAPed PAG neurons elicits the production of squeaks, while ablation of TRAPed PAG neurons blocks the production of courtship squeaks without altering female non-vocal courtship behaviors. Surprisingly, ablation of courtship squeak-TRAPed PAG neurons did not affect the production of footshock-elicited squeaks. Moreover, non-selective PAG ablation, which blocked the production of courtship squeaks in females and USVs in both sexes, attenuated but did not block the production of footshock-elicited squeaks. Our results identify a population of PAG neurons that critically regulates the production of courtship squeaks in female mice and also suggest that a non-canonical, PAG-independent pathway regulates the production of pain-elicited vocalizations.