The role of endogenous carbon monoxide (CO) in the control of breathing in zebrafish (Danio rerio)

24 Carbon monoxide (CO) is a gaseous signalling molecule and is produced in vivo from the 25 intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study 26 we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio 27 rerio. Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive 28 neuroepithelial cells (NECs) of larvae (4 days post fertilization) and adults indicating the 29 potential for endogenous CO production in the NECs. Hypoxia (20 min, water PO2 of 30 mm 30 Hg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. 31 Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX 32 (ZnPPIX) treatment in adults) exhibited increased ventilation frequency (Vf) under normoxic but 33 not hypoxic conditions. The addition of exogenous CO restored resting Vf in fish with 34 diminished CO production and in some cases (e.g. hypoxic sham larvae) CO modestly reduced 35 Vf below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the 36 potential confounding effects of CO-haemoglobin interactions which might influence ventilation. 37 PHZ treatment did not cause changes in Vf of normoxic larvae and the addition of CO to PHZ38 exposed larvae resulted in a significant decrease in sham and HO-1 deficient fish under normoxic 39 conditions. This study demonstrates for the first time that CO plays an inhibitory role in the 40 control of breathing in larval and adult zebrafish. 41