Whole-Brain Mapping of Neuronal Activity Associated with Vocal Socialization Behaviors in Adult Mice

Vocal communication is essential for social behavior, yet the distributed brain networks underlying vocal production remain elusive. Male mice produce ultrasonic vocalizations (USVs) during courtship, providing a rodent model for investigating neural circuits underlying innate vocal communication. Here, we used a double-labelling strategy that combined genetic activity tagging (TRAP2) and c-Fos immunohistochemistry to generate an unbiased, whole-brain map of neuronal ensembles activated during courtship-induced USV production in adult male mice. By tracking neuronal activity across 25 brain regions during two independent courtship sessions, we identified populations consistently recruited during social vocalization. Quantitative analyses revealed robust activation in the caudal periaqueductal gray, confirming its established role as a hub for vocal motor control. Importantly, correlation analyses between neuronal activity and USV count distinguished regions specifically linked to vocal output from those associated with general social interaction. The medial prefrontal cortex, particularly the prelimbic and cingulate cortices, exhibited strong positive correlations with USV production, suggesting an integrative role in translating the social and motivational context into vocal commands. Notably, the rostral striatum showed a strong correlation with USV count, with vocalization-related activity preferentially localized within striosome compartments of the medial striatum. Given that striosomes receive limbic cortical input and are implicated in motivational processing, these findings suggest that corticostriatal limbic-motor circuits modulate innate vocalization contingent on emotional and social contexts. Collectively, our study proposes a comprehensive neuroanatomical framework linking cortical motivation centers, striatal limbic-motor pathways, and brainstem vocal motor circuits, providing insights into the distributed neural architecture underlying mammalian social communication. Significance Statement Mouse ultrasonic vocalizations (USVs) provide a mammalian model for investigating the neural circuits underlying social communication. Using whole-brain activity mapping with double labeling of TRAP2 and c-Fos, we identified a distributed network extending from the medial prefrontal cortex and striatum to the periaqueductal gray that is recruited during courtship-induced vocalization. The discovery that USV-related neurons in the striatum are preferentially localized to striosomal compartments reveals a potential limbic-motor interface for integrating social motivation with vocal motor output. Together, these findings identify a multi-level neural network that may be evolutionarily conserved for integrating motivation and motor control in mammalian communication.