Zebrafish (Danio rerio) gill neuroepithelial cells are sensitive chemoreceptors for environmental CO2

Abstract

Adult zebrafish exhibit hyperventilatory responses to absolute environmental CO(2) levels as low as 0.13% ( mmHg), more than an order of magnitude lower than the typical arterial levels (40 mmHg) monitored by the mammalian carotid body. The sensory basis underlying the ability of fish to detect and respond to low ambient CO(2) levels is not clear. Here, we show that the neuroepithelial cells (NECs) of the zebrafish gill, known to sense O(2) levels, also respond to low levels of CO(2). An electrophysiological characterization of this response using both current and voltage clamp protocols revealed that for increasing CO(2) levels, a background K(+) channel was inhibited, resulting in a partial pressure-dependent depolarization of the NEC. To elucidate the signalling pathway underlying K(+) channel inhibition, we used immunocytochemistry to show that these NECs express carbonic anhydrase (CA), an enzyme involved in CO(2) sensing in the mammalian carotid body. Further, the NEC response to CO(2) (magnitude of membrane depolarization and time required to achieve maximal response), under conditions of constant pH, was reduced by 50% by the CA-inhibitor acetazolamide. This suggests that the CO(2) detection mechanism involves an intracellular sensor that is responsive to the rate of acidification associated with the hydration of CO(2) and which does not require a change of extracellular pH. Because some cells that were responsive to increasing also responded to hypoxia with membrane depolarization, the present results demonstrate that a subset of the NECs in the zebrafish gill are bimodal sensors of CO(2) and O(2).

Figure 4. Current–voltage (I–V) relationship for a CO₂-sensitive current in isolated cultured gill neuroepithelial cells (NECs) of zebrafish (Danio rerio)

A, mean current–voltage (I–V) relationships during exposure to normoxia (Nox) or hypercapnia (Hpc) from 12 cells. Currents were evoked by changing the voltage from −90 mV to +60 mV following a ramp protocol from a holding potential of −60 mV (inset). Inset also shows representative current traces from a single NEC. B, mean I–V relationship of the CO₂-sensitive difference current (open symbols, Nox-Hpc) from 12 cells. The CO₂-sensitive difference current reverses near E_K (calculated using bath and pipette K⁺ concentrations) and fits the corresponding Goldman–Hodgkin–Katz current equation (GHK, continuous curve; Hille, 2001), suggesting that it is predominantly carried by K⁺ ions.

Figure 1. Effects of hypercapnia or hypoxia on membrane potential in isolated cultured gill neuroepithelial cells (NECs) of zebrafish (Danio rerio)

A and B, typical current clamp recordings showing membrane depolarization during perfusion with hypercapnic (Hpc, 1% CO₂= 7.5 mmHg, continuous line) or hypoxic solutions (Hox, mmHg, dashed line), respectively. C, current clamp recording showing depolarization during perfusion with hypercapnic (filled arrow bar) or hypoxic solution (dashed bar) measured on the same cell. D, mean ±s.e.m. (n= 18 cells) membrane potential (V_m) of NECs during perfusion with control (Ctrl) or hypercapnic (Hpc) solutions, and subsequent recovery (Rec). V_m was significantly reduced by 26 mV during hypercapnia (paired t test, *P < 0.001). E, mean ±s.e.m. (n= 6) V_m of NECs for sequential stages of control, hypercapnia, hypoxia (Hox) and recovery during recording from the same cell. V_m was significantly and reversibly reduced in the presence of hypercapnia and hypoxia relative to controls (one-way repeated measures ANOVA, *P < 0.001).

Figure 2. Dose ()-dependent effects of hypercapnia on membrane potential in isolated cultured neuroepithelial cells (NECs) of zebrafish (Danio rerio)

A–C, representative traces of reversible depolarization of the same NEC during perfusion (bar) with hypercapnic solutions, 0.25% ( mmHg), 0.5% ( mmHg) or 1% ( mmHg) CO₂. Scale bar = 10 mV/10 s in C (applies to A and B as well). D, mean ±s.e.m. values of membrane potential (V_m) during hypercapnia. Continuous line and filled squares show the dose-dependent trend of V_m during hypercapnia on the same set of NECs (n= 4; filled triangle for recovery n= 3; one-way repeated measures ANOVA versus control, *P < 0.05). Open circles represent data from NECs using control and 1% CO₂ alone (n= 21).

Figure 3. Time-series effects of hypercapnia-induced current in cultured gill neuroepithelial cells (NECs) of zebrafish (Danio rerio) under voltage-clamp (V_clamp) recording

A, representative whole cell current traces evoked every 10 s using voltage steps from −60 to 30 mV before, at times 10–30 s of hypercapnia and during recovery, respectively. B, mean ±s.e.m. (n= 6) percentage of steady state current inhibition before, at times 10–30 s of hypercapnia and during recovery (continuous line with filled circles). For comparison, dashed line and triangles show mean ±s.e.m. values of percentage of membrane potential (V_m) depolarization (data taken from Fig. 2D) during bath perfusion with different levels of hypercapnia under current clamp (one-way repeated measures ANOVA versus control, *P < 0.05).

Figure 5. Pharmacological characteristics of CO₂ sensing in isolated cultured gill neuroepithelial cells (NECs) of zebrafish (Danio rerio)

A, average current–voltage (I–V) relationship of whole-cell voltage clamp recordings from five CO₂-sensitive NECs during exposure to normoxia (Nox), hypercapnia (Hpc), 4-AP and 4-AP+Hpc showing the persistence of the hypercapnic effects in the presence of 4-AP. Currents were evoked by changing the voltage from −100 mV to +60 mV following a ramp protocol (1 s) from a holding potential of −60 mV. B, typical current clamp recording of a reversible hypercapnic depolarization following application of 2.5 mm 4-AP. C, average current–voltage (I–V) relationship of whole-cell voltage clamp recordings from six CO₂-sensitive NECs during exposure to Nox, Hpc, quinidine (Quid) and Quid+Hpc; a ramp protocol was used (as in A). Hypercapnia (Hpc) reduced currents over a range of potentials, but had little effect in the presence of 0.5 mm quinidine (Quid+Hpc). D, typical current clamp recording showing the reversible depolarization following bath application of 0.5 mm quinidine, but subsequent hypercapnia having no additional affect. E, mean ±s.e.m. inhibition of whole-cell current at 50 mV using the data points in Fig. 4 as well as in panels A and C, showing hypercapnia effects alone (Hpc), hypercapnia effects in the presence of 4-AP (4AP, 4AP + Hpc, paired t test, *P < 0.05) and quinidine (Quid, Quid+Hpc) respectively.

Figure 6. Effects of acetazolamide (AZ) on the CO₂-sensitive membrane potential in isolated cultured gill neuroepithelial cells (NECs) of zebrafish (Danio rerio)

A, representative image of 5HT-immunoreactive (IR; red) and/or CA-IR (green) NECs in primary cell culture. The cell nuclei appear as blue; yellow/orange represents co-localization of 5HT and CA. Note that all 5HT-IR cells exhibit CA-IR. In addition, some cells appeared to exclusively express CA, whereas other cells appeared to lack both 5HT and CA. Inset: confocal image of a cell colocalizing 5HT and CA. B, preabsorption of the CA antibody with excess CA followed by application of 5HT and CA antibodies yielded only 5HT-IR NECs. Scale bars = 20 μm in A (applies to B also). C, typical current-clamp recording of a reversible depolarization during perfusion with hypercapnic solution (upper trace) as well as a reduced and delayed depolarization after the administration of 50 μm acetazolamide (AZ) on the same cell (lower trace). D, mean ±s.e.m. (n= 6) membrane potential changes (ΔV_m) and time to peak depolarization (ΔT) during hypercapnia alone (ctrl; filled bars) and in the presence of 50 μm acetazolamide (AZ; open bars; paired t test, *P < 0.05).