Female C57BL/6 J mice perform distinctive urination behaviour accompanied by ultrasonic vocalisation sequences with a stereotyped temporal organisation.

Ultrasonic vocalisations (USVs) are widely studied in mice as a marker of social communication. Typically, USVs are recorded during brief social encounters in unfamiliar test cages. In the present study, we explored how freely interacting pairs of C57BL/6 J adult female mice spontaneously use USVs during long-term monitoring. We discovered that these mice display a previously undescribed behaviour: they emit specific USV sequences while depositing a large volume of urine in a corner of the cage. The most striking feature of USVs accompanying this vocalised urination behaviour was the stereotyped duration of the intervals between acoustically simple USVs. The frequency of this behaviour was highly variable between pairs. Interestingly, when urination was accompanied by the specific USV sequence, it was associated with a significant increase in locomotor activity in both the emitter and the cage mate, compared with urination without USVs. Altogether, these observations and the description of this vocalised urination behaviour highlight the importance of exploring mouse vocalisations at the sequence level to better understand the functions of USVs in different behavioural contexts.