Role of endogenous carbon monoxide in the control of breathing in zebraﬁsh ( Danio in the

Role control of breathing in zebraﬁsh monoxide (CO) is a gaseous signaling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebraﬁsh, Danio rerio . Immunohistochemistry revealed the presence of HO-1 in the chemorecep-tive neuroepithelial cells (NECs) of larvae (4 days postfertilization) and adults, indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water P O 2 of 30 mmHg) caused a signiﬁcant increase in HO-1 activity in whole larvae and in the gills of adult ﬁsh. Zebraﬁsh with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX treatment in adults) exhibited increased ventilation frequency ( V f ) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting V f in ﬁsh with diminished CO production, and in some cases (e.g., hypoxic sham larvae) CO modestly reduced V f below resting levels. Larval ﬁsh were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-hemoglobin interactions that might inﬂuence ventilation. PHZ treatment did not cause changes in V f of normoxic larvae, and the addition of CO to PHZ-exposed larvae resulted in a signiﬁcant decrease in sham and HO-1-deﬁcient ﬁsh under normoxic conditions. This study demonstrates for the ﬁrst time that CO plays an inhibitory role in the control of breathing in larval and adult zebraﬁsh. HO-1 (UniProt accession no. B0UXS0–1) and prevent translation of the HO-1 protein. Embryos were injected at the one-cell developmental stage, which lasts 15–30 min postfertilization at 28.5°C (26). The working stock morpholino was diluted before injection in 1 (cid:3) Danieu buffer [(in mM/l) 58 NaCl, 0.7 KCl, 0.4 Ca(NO ) , and 5.0 HEPES (pH 7.6)] and 0.05% Phenol The working concentration of the morpholino was 4 ng/nl. Approximately 1 nl was injected at the border between the yolk sack and the cell of each embryo to facilitate entry of the morpholino in the cell. To control for any effects of injection or nucleotide addition, matching concentrations of a control morpholino with no known target (5 = -CCTCTTACCTCAGTTACAATTTATA-3 = ; Gene Tools) were administered to sham embryos. Injected embryos were transferred to 30-ml Petri dishes containing system water with 0.03% methylene blue and incubated at 28°C for 4 days at which time experiments were performed. Immunohistochemistry Larvae. The immunohistochemical observations were based on ﬁve sham and ﬁve HO-1 morphant larvae. A sequential staining procedure was used for antibodies raised in the same host (rabbit). We used sequential labeling for larvae because we did not have a working anti-5-HT primary antibody raised in a different host species at the time the immunohistochemistry was performed. This technique has been shown to successfully label cell types in previous studies performed on zebraﬁsh larvae (23, 36). The primary antibodies were omitted in a separate group of larvae, which served as a negative control. No immunoﬂuorescence was detected in sham or HO-1 morphant larvae. Zebraﬁsh larvae were killed by an overdose of MS-222, and whole larvae were placed in 4% PFA prepared in PBS containing (in mmol/l): 137 NaCl, 15.2 Na 2 HPO 4 , 2.7 KCl, and 1.5 KH 2 PO 4 buffered to pH 7.8 with 1 mol/l NaOH overnight at 4°C (24). The larvae were then rinsed with PBST (0.1% Tween), gradually dehydrated to 100% MeOH, and incubated at (cid:8) 20°C for at least 1 h. After incubation, the larvae were rehydrated gradually by ﬁrst replac- ing the 100% MeOH with 50% MeOH and incubating the larvae for 5 min followed by another 5-min incubation in 100% PBST. The PBST was replaced with 1 ml Tris·HCl (150 mM, pH 9) and left to incubate at room temperature for 10 min and then at 65°C for 15 min. The larvae were washed ﬁve times with PBST and placed in blocking solution (2% BSA, 2% goat serum, in PBS with 0.8% Triton X) for 1 h at room temperature. The blocking solution was removed, and the larvae were incubated in an anti-5-HT primary antibody raised in rabbit to label the NECs (1:250; catalog no. S5545; Sigma Aldrich) on a shaker overnight at 4°C. The anti-5-HT