Fig 1. In vitro experiments.

Ammocoete brainstems were isolated and exposed to normocapnia (1.5% CO₂) and hypercapnia (5% CO₂) to investigate the “cough” CRG response. Activity was recorded from intact cranial nerve roots of the in vitro brainstem, which normally innervate structures generating rhythmic pharyngeal ventilation (A; CN V, VII and X illustrated). With normocapnia, cranial nerve discharge (CN VII shown) exhibited regular and rhythmic burst patterns representative of pharyngeal pouch ventilation (top), with irregular, periodic and distinct “cough” (bottom; Hsia et al., 2013; Martel et al., 2007; Rovainen, 1977) indicated by arrows (B). Data were analyzed for event frequency in 10-minute periods immediately prior to (NC) and after (HC) the gas change, and with recovery following a return to NC (RNC; C). Elevating CO₂ increased the frequency of “cough” (n = 5; P = 0.02; NC vs. HC P=0.031, t=3.32; HC vs. RNC, P=0.037, t =2.937, 1-way RMANOVA).