Vocalizations provide a means of communication with high fidelity and information rate for many species. Male mice emit ultrasonic vocalizations (USVs) during female courtship. Diencephalon and brainstem neural circuits have been shown to control the production of USVs, however, the role of cortical circuits in this process is debatable. We show that male mice produced USVs following intracortical microstimulation (ICMS) in a specific location of their anterior cingulate cortex (ACC). Moreover, ACC Ca2+-imaging showed an increase in Ca2+ dynamics preceding USV initiation. Optogenetically suppressing ACC activity caused mice to emit fewer USVs during courtship. Neuropixel electrophysiological recordings in head-restrained male mice indicated a differential increase in neural activity in response to female social exposure (SE). The firing rate in SE trials where USVs were emitted was significantly higher when compared to SE leading to silence. Taken together, these results indicate that the ACC is a key node in the neuronal circuits controlling USV production.
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