Characterization of ultrasonic-vocalization-modulated-neurons in rat motor cortex based on their activity modulation and axonal projection to periaqueductal gray.

Vocalization, a means of social communication, is prevalent among many species, including humans. Both rats and mice use ultrasonic vocalizations (USVs) in various social contexts and affective states. The motor cortex is hypothesized to be involved in precisely controlling USVs through connections with critical regions of the brain for vocalization, such as the periaqueductal grey matter (PAG). However, it is unclear how neurons in the motor cortex are modulated during USVs. Moreover, the relationship between USV modulation of neurons and anatomical connections from the motor cortex to PAG is also not clearly understood. In this study, we first characterized the activity patterns of neurons in the primary and secondary motor cortices during emission of USVs in rats using large-scale electrophysiological recordings. We also examined the axonal projection of the motor cortex to PAG using retrograde labelling and identified two clusters of PAG-projecting neurons in the anterior and posterior parts of the motor cortex. The neural activity patterns around the emission of USVs differed between the anterior and posterior regions, which were divided based on the distribution of PAG-projecting neurons in the motor cortex. Furthermore, using optogenetic tagging, we recorded the USV modulation of PAG-projecting neurons in the posterior part of the motor cortex and found that they showed predominantly sustained excitatory responses during USVs. These results contribute to our understanding of the involvement of the motor cortex in the generation of USV at the neuronal and circuit levels.Significance Statement USVs in rodents have been widely used as experimental models to study neural mechanisms and deficits in social, emotional, and motor functions in mammals. However, the involvement of the motor cortex has not yet been fully characterized. In this study, we investigated the neural activity around USVs across the rat motor cortex and its relationship with projections to the PAG, which plays a central role in producing USV. The results demonstrated strong involvement of the motor cortex in USVs at both neuronal and circuit levels. This study provides a basis for future studies examining the cortical control of USVs using genetic and physiological manipulations.