Detecting and Assigning Ultrasonic Vocalizations to Individual Mice during Social Interactions

Kaihang Chen; Claudia J. Collins; Christyana M. Kawar; Devashish Pande; Joel D. Turk; Jessica R. Bogdan; Abby G. Fowler; Juliana C. Pucciarelli; Thomas P. Ring; Joshua P. Neunuebel

doi:10.3791/71121

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Method Article

Detecting and Assigning Ultrasonic Vocalizations to Individual Mice during Social Interactions

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DOI:

10.3791/71121

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July 7th, 2026

Kaihang Chen^*¹ , Claudia J. Collins^*¹^,² , Christyana M. Kawar^*¹^,² , Devashish Pande^*¹^,² , Joel D. Turk^*¹^,³ , Jessica R. Bogdan¹ , Abby G. Fowler¹ , Juliana C. Pucciarelli¹ , Thomas P. Ring¹ , Joshua P. Neunuebel¹^,²^,³

¹Department of Psychological and Brain Sciences, University of Delaware, ²Interdisciplinary Neuroscience Graduate Program, University of Delaware, ³Center for Bioinformatics and Computational Biology, Delaware Biotechnology Institute, University of Delaware

^* These authors contributed equally

Summary

This protocol describes a method for recording synchronized video and audio data during multi-mouse social interactions. The method uses a microphone array and sound source localization to assign ultrasonic vocalizations (USVs) to individual mice, allowing quantitative analysis of vocal behavior across animals and social contexts.

Abstract

Mice (Mus musculus) communicate using ultrasonic vocalizations (USVs) during social interactions. However, because mouse USVs are not associated with clear visual indicators and occur at frequencies outside the human auditory range, it remains challenging to identify individual vocalizing mice within a group. This protocol describes a method for recording synchronized video and ultrasonic audio data during multi-animal social interactions, enabling simultaneous capture of behavior and vocal activity. A computational pipeline is outlined that integrates multi-animal tracking, vocalization detection, sound-source localization, and assignment of vocalizations to individual animals. The validation procedures used to assess tracking accuracy, vocalization extraction, localization precision, and assignment confidence at each stage of the pipeline are further described. Application of this protocol to a four-animal demonstration dataset yields individual-resolved vocalization tracking, spatially precise sound-source estimates, and quantitative measures of vocal output and acoustic features. Together, these approaches provide a robust framework for linking vocal communication to individual behavior and enable downstream analyses of sex differences, individual variability, and social communication in freely interacting mice.

Introduction

Social communication across the animal kingdom is an essential component of behavioral dynamics, aiding in processes such as survival and reproduction¹^,². Among mammals, mice communicate acoustically via USVs, which span frequencies from 35 to 110 kHz³. USVs exhibit complex features, conveying information depending on the behavioral context⁴. Female mice exhibit a preference for male mouse USVs⁵^,⁶, and male and female mice interact vocally during courtship behaviors⁷, indicating th....