SUMMARYThe chemosensory roles of gill neuroepithelial cells (NECs) in mediating the hyperventilatory response to hypoxia are not clearly defined in fish. While serotonin (5-HT) is the predominant neurotransmitter in O 2 -sensitive gill NECs, acetylcholine (ACh) plays a more prominent role in O 2 sensing in terrestrial vertebrates. The present study characterized the developmental chronology of potential serotonergic and cholinergic chemosensory pathways of the gill in the model vertebrate, the zebrafish (Danio rerio). In immunolabelled whole gills from larvae, serotonergic NECs were observed in epithelia of the gill filaments and gill arches, while non-serotonergic NECs were found primarily in the gill arches. Acclimation of developing zebrafish to hypoxia (P O2 =75mmHg) reduced the number of serotonergic NECs observed at 7days post-fertilization (d.p.f.), and this effect was absent at 10d.p.f. In vivo administration of 5-HT mimicked hypoxia by increasing ventilation frequency (f V ) in early stage (7-10d.p.f.) and late stage larvae (14-21d.p.f.), while ACh increased f V only in late stage larvae. In time course experiments, application of ketanserin inhibited the hyperventilatory response to acute hypoxia (P O2 =25mmHg) at 10d.p.f., while hexamethonium did not have this effect until 12d.p.f. Cells immunoreactive for the vesicular acetylcholine transporter (VAChT) began to appear in the gill filaments by 14d.p.f. Characterization in adult gills revealed that VAChT-positive cells were a separate population of neurosecretory cells of the gill filaments. These studies suggest that serotonergic and cholinergic pathways in the zebrafish gill develop at different times and contribute to the hyperventilatory response to hypoxia. Supplementary material available online at