The temporal organization of mouse ultrasonic vocalizations

House mice, like many tetrapods, produce multielement calls consisting of individual vocalizations repeated in rhythmic series. In this study, we examine the multielement ultrasonic vocalizations (USVs) of adult male C57Bl/6J mice and specifically assess their temporal properties and organization. We found that male mice produce two classes of USVs which display unique temporal features and arise from discrete respiratory patterns. We also observed that nearly all USVs were produced in repetitive series exhibiting a hierarchical organization and a stereotyped rhythmic structure. Furthermore, series rhythmicity alone was determined to be sufficient for the mathematical discrimination of USVs produced by adult males, adult females, and pups, underscoring the known importance of call timing in USV perception. Finally, the gross spectrotemporal features of male USVs were found to develop continuously from birth and stabilize by P50, suggesting that USV production in infants and adults relies on common biological mechanisms. In conclusion, we demonstrate that the temporal organization of multielement mouse USVs is both stable and informative, and we propose that call timing be explicitly assessed when examining mouse USV production. Furthermore, this is the first report of putative USV classes arising from distinct articulatory patterns in mice, and is the first to empirically define multielement USV series and provide a detailed description of their temporal structure and development. This study therefore represents an important point of reference for the analysis of mouse USVs, a commonly used metric of social behavior in mouse models of human disease, and furthers the understanding of vocalization production in an accessible mammalian species.