Production of mouse ultrasonic vocalizations and distress calls is associated with different patterns of Fos expression in the nucleus retroambiguus

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm. Significance Statement Animals produce multiple vocalization types that differ in their acoustic features, but how the brainstem is organized to enable the production of different vocalization types remains unknown. In the current study, we performed immediate early gene mapping in the nucleus retroambiguus, a vocalization-related hindbrain region, to compare patterns of neural activity in mice that produced social calls vs. distress calls. Our findings suggest that even in premotor regions, largely non-overlapping sets of neurons may be recruited during the production of different vocalization types.