Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish

Abstract

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

Aquatic surface respiration and swimming behaviour in adult and developing zebrafish exposed to hypoxia

“…Although these cells may be candidate chemoreceptors for initiation of ASR in early larvae, we were not able to accordingly modify ASR responses in larvae by immersion in 5-HT or ACh before 18 dpf. The reason for this is unclear, since serotonergic receptors participate in hypoxic hyperventilation as early as 7-10 dpf, and cholinergic receptors as early as 12 dpf (Shakarchi et al, 2013). Marginal increases in the frequency of ASR events observed with 5-HT at 7 and 14 dpf suggest that a higher sample size in our experiments might have revealed a more prominent change in behaviour.…”

“…In addition, O 2 -chemoreceptive gill NECs in zebrafish retain 5-HT (Jonz and Nurse, 2003), and cells expressing an ACh transporter were reported in the gills of zebrafish (Shakarchi et al, 2013) and the amphibious fish, Kryptolebias marmoratus (Regan et al, 2011). In zebrafish larvae, 5-HT 2 , 5-HT 3 , and nicotinic receptors of the gills appear to play a role in initiation of the hyperventilatory response to hypoxia (Shakarchi et al, 2013;Jonz et al, 2015) and may be involved in initiating the ASR response. Sensory Merkel-like cells containing 5-HT were described in the oropharyngeal epithelium of zebrafish (Zachar and Jonz, 2012), but these cells did not display any morphological changes following long-term exposure to hypoxia (M.G.J., unpublished observations), as did gill NECs (Jonz et al, 2004), suggesting that the former are not O 2 sensitive.…”

“…Both 5-HT and ACh have been implicated in chemoreceptor control of ventilatory reflexes in developing and adult fish (Burleson and Milsom, 1995a,b;Shakarchi et al, 2013;Jonz et al, 2015), as they have in mammals (Shirahata et al, 2007;Nurse, 2010). In addition, O 2 -chemoreceptive gill NECs in zebrafish retain 5-HT (Jonz and Nurse, 2003), and cells expressing an ACh transporter were reported in the gills of zebrafish (Shakarchi et al, 2013) and the amphibious fish, Kryptolebias marmoratus (Regan et al, 2011). In zebrafish larvae, 5-HT 2 , 5-HT 3 , and nicotinic receptors of the gills appear to play a role in initiation of the hyperventilatory response to hypoxia (Shakarchi et al, 2013;Jonz et al, 2015) and may be involved in initiating the ASR response.…”

“…no. A6625), both of which stimulate gill O 2 chemoreceptors (Burleson and Milsom, 1995a) and induce hyperventilation in fish, including zebrafish (Burleson and Milsom, 1995b;Shakarchi et al, 2013). Both chemicals were obtained from Sigma-Aldrich (Oakville, ON, Canada).…”

“…In addition, cells expressing an acetylcholine (ACh) transporter were reported in the gills of zebrafish (Shakarchi et al, 2013) and the amphibious fish Kryptolebias marmoratus (Regan et al, 2011). Both 5-HT and ACh have been implicated in the chemoreceptor control of ventilatory reflexes in developing and adult fish (Burleson and Milsom, 1995a,b;Shakarchi et al, 2013;Jonz et al, 2015).…”