Peripheral chemoreceptors responsive to hypoxia have been well characterized in air-breathing vertebrates, but poorly in water-breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O(2) chemoreceptors, and innervation patterns in the zebrafish gill using whole-mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn-12 (a zebrafish-specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5-HT) immunoreactivity (IR), SV2-IR and the purinoceptor P2X(3)-IR. A zn-12-IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2-IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O(2) chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation.

In aquatic vertebrates, hypoxia induces physiological changes that arise principally from O 2 chemoreceptors of the gill. Neuroepithelial cells (NECs) of the zebrafish gill are morphologically similar to mammalian O 2 chemoreceptors (e.g. carotid body), suggesting that they may play a role in initiating the hypoxia response in fish. We describe morphological changes of zebrafish gill NECs following in vivo exposure to chronic hypoxia, and characterize the cellular mechanisms of O 2 sensing in isolated NECs using patch-clamp electrophysiology. Confocal immunofluorescence studies indicated that chronic hypoxia (P O 2 = 35 mmHg, 60 days) induced hypertrophy, proliferation and process extension in NECs immunoreactive for serotonin or synaptic vesicle protein (SV2). Under voltage clamp, NECs responded to hypoxia (P O 2 = 25-140 mmHg) with a dose-dependent decrease in K + current. The current-voltage relationship of the O 2 -sensitive current (I KO 2 ) reversed near E K and displayed open rectification. Pharmacological characterization indicated that I KO 2 was resistant to 20 mM tetraethylammonium (TEA) and 5 mM 4-aminopyridine (4-AP), but was sensitive to 1 mM quinidine. In current-clamp recordings, hypoxia produced membrane depolarization associated with a conductance decrease; this depolarization was blocked by quinidine, but was insensitive to TEA and 4-AP. These biophysical and pharmacological characteristics suggest that hypoxia sensing in zebrafish gill NECs is mediated by inhibition of a background K + conductance, which generates a receptor potential necessary for neurosecretion and activation of sensory pathways in the gill. This appears to be a fundamental mechanism of O 2 sensing that arose early in vertebrate evolution, and was adopted later in mammalian O 2 chemoreceptors.

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Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K ϩ increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K ϩ -induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.