Ultrasonic vocalizations (USVs) are believed to play a critical role in mouse communication. Although mice produce USVs in multiple contexts, signals emitted in reproductive contexts are typically attributed solely to the male mouse. Only recently has evidence emerged showing that female mice are also vocally active during mixed-sex interactions. Therefore, this study aimed to systematically quantify and compare vocalizations emitted by female and male mice as the animals freely interacted. Using an eight-channel microphone array to determine which mouse emitted specific vocalizations during unrestrained social interaction, we recorded 13 mixed-sex pairs of mice. We report here that females vocalized significantly less often than males during dyadic interactions, with females accounting for approximately one sixth of all emitted signals. Moreover, the acoustic features of female and male signals differed. We found that the bandwidths (i.e., the range of frequencies that a signal spanned) of female-emitted signals were smaller than signals produced by males. When examining how the frequency of each signal changed over time, the slopes of male-emitted signals decreased more rapidly than female signals. Further, we revealed notable differences between male and female vocal signals when the animals were performing the same behaviors. Our study provides evidence that a female mouse does in fact vocalize during interactions with a male and that the acoustic features of female and male vocalizations differ during specific behavioral contexts.

Animals engage in complex social encounters that influence social groups and resource allocation. During these encounters, acoustic signals, used at both short and long ranges, play pivotal roles in regulating the behavior of conspecifics. Mice, for instance, emit ultrasonic vocalizations, signals above the range of human hearing, during close-range social interactions. How these signals shape behavior, however, is unknown due to the difficulty in discerning which mouse in a group is vocalizing. To overcome this impediment, we used an eight-channel microphone array system to determine which mouse emitted individual vocal signals during 30 minutes of unrestrained social interaction between a female and a single male or female conspecific. Females modulated both the timing and context of vocal emission based upon their social partner. Compared to opposite-sex pairings, females in samesex pairs vocalized when closer to a social partner and later in the 30 minutes of social engagement. Remarkably, we found that female mice exhibited no immediate changes in acceleration (movement) to male-emitted vocal signals. Both males and females, in contrast, modulated their behavior following female-emitted vocal signals in a context-dependent manner. Thus, our results suggest female vocal signals function as a means of ultrashort-range communication that shapes mouse social behavior. Acoustic signaling is a vital means of both intra-and inter-species communication across the animal kingdom, allowing the transfer of information without limitations of light availability or physical proximity between individuals 1,2. Unlike other communication modalities, acoustic communication is effective over a wide range of distances, and vocalizations are often grouped into two categories: short-and long-range signals 3. Short-range sounds are emitted by most species that vocalize (e.g., marmosets 4 , rats 5 , and moths 6), often used for interpersonal communication and to promote social cohesion 7,8. Long-range signals, while not ubiquitous, are common across the animal kingdom (e.g., wolves 9 , whales 10 , and birds 11). These signals are generally used to warn others, communicate with distant members, or indicate territoriality 9,12,13. Research on long-distance calling often focuses on males 14,15 , even though many species show long-range calling from both sexes 16-19. In some species, females actually emit more long-distance calls than males 20,21. Female elephants, for instance, are more vocal than males and emit long-range calls to communicate with social partners 22. These signals are believed to facilitate social recognition over great distances 23. However, in many animal species the function and range of female-emitted signals is less clear. In mice, the propagation and behavioral impact of female-emitted signals is less established. Adult mice (Mus musculus), while predominately silent in isolation 24 , emit ultrasonic vocalizations, signals spanning 30-110 kHz in frequency 25 , during aggressive and affiliative behaviors 26-30. ...

Adult mice emit ultrasonic vocalizations (USVs), sounds above the range of human hearing, during social encounters. While mice alter their vocal emissions between isolated and social contexts, technological impediments have hampered our ability to assess how individual mice vocalize in group social settings. We overcame this challenge by implementing an 8-channel microphone array system, allowing us to determine which mouse emitted individual vocalizations across multiple social contexts. This technology, in conjunction with a new approach for extracting and categorizing a complex, full repertoire of vocalizations, facilitated our ability to directly compare how mice modulate their vocal emissions between isolated, dyadic and group social environments. When comparing vocal emission during isolated and social settings, we found that socializing male mice increase the proportion of vocalizations with turning points in frequency modulation and instantaneous jumps in frequency. Moreover, males change the types of vocalizations emitted between social and isolated contexts. In contrast, there was no difference in male vocal emission between dyadic and group social contexts. Female vocal emission, while predominantly absent in isolation, was also similar during dyadic and group interactions. In particular, there were no differences in the proportion of vocalizations with frequency jumps or turning points. Taken together, the findings lay the groundwork necessary for elucidating the stimuli underlying specific features of vocal emission in mice.

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