Vocal repertoire expansion in singing mice by co-opting a conserved midbrain circuit node

How neural circuits generate diverse behaviors is a fundamental question in neuroscience. Distinct behavioral outputs may arise from either dedicated motor circuits or shared circuits operating in different functional states. Although multifunctional circuits offer an efficient solution for behavioral flexibility and may drive rapid evolutionary adaptations, their neural mechanisms remain poorly understood, especially in mammals. Here, we leverage the rich vocal repertoire of the singing mouse (Scotinomys teguina) to investigate the organizational logic of multifunctional motor circuits. We developed a behavioral assay (partial acoustic isolation reveals identity [PAIRId]) that enables precise attribution of vocalizations during social interactions. This paradigm revealed two distinct vocal modes: soft, variable, ultrasonic vocalizations (USVs) ancestral to rodents, used for short-range communication, and loud, rhythmic, human-audible songs unique to the singing mouse lineage, used for long-range communication. Despite their substantial acoustic and contextual differences, we found that USVs and songs do not arise from parallel pathways. Instead, they share the same sound production mechanism, phonatory-respiratory coupling, and vocal gating from the midbrain caudolateral periaqueductal gray (clPAG). To understand the mechanism governing song production, we combined mathematical modeling of song rhythm with synaptic silencing of clPAG, which progressively reduced song amplitude and duration. We demonstrate that song duration decreases via a single parameter controlling its termination. Notably, this mechanism also accounts for sexual dimorphism in songs, identifying clPAG as a key locus for driving natural behavioral variability. Our findings reveal how parametric tuning of a central circuit node produces distinct vocal modes, providing a mechanistic basis for rapid behavioral evolution in mammals.