Rapid inter-individual vocal interactions in freely behaving rats revealed by acoustic source localization

Ultrasonic vocalizations (USVs) in rodents have been widely used as indicators of affective states; however, their role in rapid temporal exchange between individuals remains largely unexplored, primarily due to technical limitations in identifying vocalizing individuals during free social interactions. Here, we combined acoustic source localization (USVCAM) with markerless multi-animal tracking to examine the temporal structure of vocal interactions in freely behaving rat dyads. In three dyads with sufficient vocal output, inter-individual vocalization intervals exhibited a clear peak at approximately 50-250 ms, indicating fast temporal exchanges of vocalizations between individuals. To test whether this pattern could arise from independent vocal production, we performed Monte Carlo randomized simulations based on empirically observed intra-individual intervals. The simulated distributions failed to reproduce the empirical peak and instead showed a concentration at shorter intervals, resulting in a significant distributional difference. These results reject the null hypothesis of independent vocal generation and suggest that the observed temporal structure reflects the influence of one individual's vocalizations on those of the other individual. Together, these findings extend the potential applications of rodent USVs, in addition to their established use as affective indicators, by providing a proof-of-principle demonstration of rapid inter-individual vocal exchanges and a tractable experimental framework for investigating the temporal dynamics of social vocal communication.