Abstract Speech generation critically depends on precise controls of laryngeal muscles and coordination with ongoing respiratory activity. However, the neural mechanisms governing these processes remain unknown. Here, we mapped laryngeal premotor circuitry in adult mice and viral-genetically identified excitatory vocal premotor neurons located in the retroambiguus nucleus (RAm VOC ) as both necessary and sufficient for driving vocal-cord closure and eliciting mouse ultrasonic vocalizations (USVs). The duration of RAm VOC activation determines the lengths of USV syllables and post-inspiration phases. RAm VOC -neurons receive inhibitory inputs from the preBötzinger complex, and inspiration needs can override RAm VOC -mediated vocal-cord closure. Ablating inhibitory synapses in RAm VOC -neurons compromised this inspiration gating of laryngeal adduction, resulting in de-coupling of vocalization and respiration. Our study revealed the hitherto unknown circuits for vocal pattern generation and vocal-respiratory coupling. One-Sentence Summary Identification of RAm VOC neurons as the critical node for vocal pattern generation and vocal-respiratory coupling.

Brainstem premotor mechanisms underlying vocal production and vocal-respiratory coordination