Mouse pups produce. ultrasonic vocalizations (USVs) in response to isolation from the nest (i.e., isolation USVs). Rates and acoustic features of isolation USVs change dramatically over the first two weeks of life, and there is also substantial variability in the rates and acoustic features of isolation USVs at a given postnatal age. The factors that contribute to within age variability in isolation USVs remain largely unknown. Here, we explore the extent to which non-vocal behaviors of mouse pups relate to the within age variability in rates and acoustic features of their USVs. We recorded non-vocal behaviors of isolated C57BL/6J mouse pups at four postnatal ages (postnatal days 5, 10, 15, and 20), measured rates of isolation USV production, and applied a combination of pre-defined acoustic feature measurements and an unsupervised machine learning-based vocal analysis method to examine USV acoustic features. When we considered different categories of non-vocal behavior, our analyses revealed that mice in all postnatal age groups produce higher rates of isolation USVs during active non-vocal behaviors than when lying still. Moreover, rates of isolation USVs are correlated with the intensity (i.e., magnitude) of non-vocal body and limb movements within a given trial. In contrast, USVs produced during different categories of non-vocal behaviors and during different intensities of non-vocal movement do not differ substantially in their acoustic features. Our findings suggest that levels of behavioral arousal contribute to within age variability in rates, but not acoustic features, of mouse isolation USVs.
