The dorsomedial hypothalamic nucleus and perifornical area (DMH/PeF), historically identified as the hypothalamic defence area, plays a key role in orchestrating autonomic and respiratory adjustments during threat-related states. To investigate how activation of this region affects upper-airway and respiratory motor circuits, we examined its anatomical and functional interactions with laryngeal-related medullary nuclei using combined immunohistochemical mapping and physiological recordings in spontaneously breathing anaesthetised rats. Neuronal activation was assessed using c-Fos immunohistochemistry (c-Fos-ir) combined with ChAT and FoxP2 labelling to identify motoneurons and non-motoneuronal populations across the compact, semicompact and loose formations of the nucleus ambiguus (nA), as well as within the nucleus retroambiguus (nRA), during DMH/PeF electrical stimulation. Hypothalamic activation produced a clear subdivision-specific recruitment in the nA, with significant ipsilateral increases in c-Fos-ir within the semicompact (motoneurons, p < 0.001; non-motoneurons, p < 0.05) and compact formations (non-motoneurons, p < 0.05). The nRA also exhibited robust ipsilateral activation (non-motoneurons, p < 0.001). FoxP2-positive neurons remained stable across groups, indicating that FoxP2 reflects neuronal phenotype rather than acute activation. A second set of experiments evaluated the functional impact of electrical and chemical (glutamate) DMH/PeF activation on subglottic pressure, an index of laryngeal resistance. Both stimulation modalities produced a marked reduction in subglottic pressure (p < 0.001 and p < 0.01), accompanied by a reproducible autonomic–respiratory pattern characterised by tachypnoea, tachycardia and a pressor response. Overall, our results provide new insight into how hypothalamic circuits regulate subglottic pressure and laryngeal activity, suggesting that DMH/PeF activation is associated with the recruitment of specific nA and nRA domains, which may contribute to the adjustment of upper-airway patency during defensive responses.

