Acoustic features of rat ultrasonic vocalizations underlying emotional responses

Adult rat ultrasonic vocalizations (USVs) are commonly classified into 22-kHz and 50-kHz calls, associated with negative and positive emotional contexts, respectively. However, the acoustic features underlying listeners’ responses remain unclear. We examined how mean frequency, call duration, and frequency modulation contribute to physiological and behavioral responses to rat USVs. Natural USVs and synthetic probe stimuli were presented to rats, and responses were assessed using heart-rate variability (HRV) and speaker-arm stay time in a radial-arm maze. Probe stimuli were generated by swapping mean frequency and/or call duration between positive 50-kHz and negative 22-kHz USVs, while frequency modulation was quantified from the resulting stimuli. Playback of negative 22-kHz USVs significantly increased mean R–R interval, indicating cardiac slowing, whereas positive 50-kHz USVs did not significantly alter HRV indices after correction for multiple comparisons. Bayesian modeling revealed that mean frequency and call duration independently predicted changes in mean R–R interval, whereas frequency modulation did not explain the tested HRV index. In the behavioral experiment, mean frequency and call duration also independently predicted speaker-arm stay time. These findings indicate that listeners’ responses to rat USVs are shaped primarily by mean frequency and call duration.