Cardiac responses to hypercapnia in larval zebrafish (Danio rerio): the links between CO2 chemoreception, catecholamines and carbonic anhydrase.

Abstract

The ontogeny of carbon dioxide (CO2) sensing in zebrafish (Danio rerio) has not been examined. In this study, CO2-mediated increases in heart rate were used to gauge the capacity of zebrafish larvae to sense CO2. CO2 is thought to be detected via neuroepithelial cells (NECs), which are homologous to mammalian carotid body glomus cells. Larvae at 5 days post-fertilization (d.p.f.) exhibited tachycardia when exposed for 30 min to 0.75% CO2 (~5.63 mmHg); at 7 d.p.f., tachycardia was elicited by 0.5% CO2 (~3.75 mmHg). Based on pharmacological evidence using β-adrenergic receptor (β-AR) antagonists, and confirmed by β1-AR translational gene knockdown using morpholinos, the reflex tachycardia accompanying hypercapnia was probably mediated by the interaction of catecholamines with cardiac β1 receptors. Because the cardiac response to hypercapnia was abolished by the ganglionic blocker hexamethonium, it is probable that the reflex cardio-acceleration was mediated by catecholamines derived from sympathetic adrenergic neurons. Owing to its likely role in facilitating intracellular acidification during exposure to hypercapnia, it was hypothesized that carbonic anhydrase (CA) is involved in CO2 sensing, and that inhibition of CA activity would blunt the downstream responses. Indeed, the cardiac response to hypercapnia (0.75% CO2) was reduced in fish at 5 d.p.f. exposed to acetazolamide, a CA inhibitor, and in fish experiencing zCAc (CA2-like a) knockdown. Successful knockdown of zCAc was confirmed by CA activity measurements, western blotting and immunocytochemistry. Co-injection of embryos with zCAc morpholino and mRNA modified at the morpholino binding site restored normal levels of CA activity and protein levels, and restored (rescued) the usual cardiac responses to hypercapnia. These data, combined with the finding that zCAc is expressed in NECs located on the skin, suggest that the afferent limb of the CO2-induced cardiac reflex in zebrafish larvae is initiated by coetaneous CO2-sensing neuroepithelial cells.