Heterogeneous effects of maternal separation on the acoustic structure of mouse pup vocalizations

Early life adversity is a major risk factor for the development of social and communicative impairment. Maternal separation (MS) has been studied in rodent models; however, its effects on the developmental trajectory of vocal communication remain unclear. In this study, we investigated the effects of MS on ultrasonic vocalization (USVs) in ICR mice. Pups underwent 24 h of MS on Postnatal day (P) 4, and USVs were recorded on P4, P6, P8, P11, and P14. Each syllable was analyzed for duration, mean frequency, and coefficient of variation in frequency (cvfreq), which reflected the range of frequency modulation. The USVs were categorized into low- and high-frequency clusters using k-means clustering. While both groups exhibited similar overall developmental patterns, specific differences emerged by P6, immediately following separation. In the high-frequency cluster, the MS group produced fewer USVs than the control group, with a marked reduction in the chevron category. In the low-frequency cluster, the MS group showed a reduced cvfreq with a prominent decrease in the step-down category. These changes occurred without significant group differences in other acoustic parameters or call categories at the later time points. Our findings highlight the call type-specific nature of vocal impairment following early life stress. Rather than producing a general suppression or excitation of vocal behavior, maternal separation appears to selectively alter the complexity and quantity of specific USV types. Such alterations highlight critical targets for future studies linking early-life stress-related neuroendocrine signaling to systematic changes in vocalization.