INVOLVEMENT OF NTS PHOX2B-POSITIVE NEURONS IN OPTOGENETICALLY EVOKED HICCUP-LIKE ACTIVITY

Hiccups are involuntary rhythmic diaphragmatic contractions, but the central rhythmogenic mechanism remains unclear. We previously showed that blue-light stimulation evokes sucking-, hiccup-, and swallowing-like behaviors in transgenic neonatal rats expressing the channelrhodopsin variant ChRFR (C167A) in Phox2b-positive neurons (Journal of Physiological Sciences, 74 Suppl 2: 131, 2024). Here, using an isolated brainstem–spinal cord preparation, we examined whether Phox2b-positive neurons in the dorsal vagal complex (DVC; nucleus tractus solitarii [NTS], dorsal motor nucleus of the vagus [DMV], and area postrema [AP]) contribute to hiccup-like motor output. Photoactivation of Phox2b-positive neurons in the dorsal medulla including the DVC elicited rhythmic motor bursts in the C4 ventral root (containing phrenic motor axons), phrenic nerve, hypoglossal nerve, and trigeminal nerve. Bursts overlapped across these motor nerves, consistent with a hiccup-like pattern. Because Phox2b-positive neurons in NTS/AP are predominantly glutamatergic whereas those in DMV are predominantly cholinergic, we tested receptor antagonists. Cholinergic blockade (10 µM atropine and 50 µM mecamylamine) did not affect the photoevoked hiccup-like rhythm. In contrast, glutamatergic blockade abolished it: the NMDA receptor antagonist AP5 (50 µM) and the AMPA/kainate receptor antagonist CNQX (2 µM) suppressed and ultimately eliminated rhythmic bursting, with decreasing burst amplitude and prolonged cycle period. Bilateral electrocoagulation of the NTS abolished hiccup-like activity, whereas AP ablation did not. These results indicate that bilateral NTS circuitry, involving Phox2b-positive neurons and glutamatergic transmission, is critical for optogenetically evoked hiccup-like activity, whereas cholinergic DMV output is not essential.