Michael G. Jonz

Neuroepithelial oxygen chemoreceptors of the zebrafish gill

In aquatic vertebrates, hypoxia induces physiological changes that arise principally from O2 chemoreceptors of the gill. Neuroepithelial cells (NECs) of the zebrafish gill are morphologically similar to mammalian O2 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 O2 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 O2‐sensitive current (I  KO 2 ) reversed near EK 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 O2 sensing that arose early in vertebrate evolution, and was adopted later in mammalian O2 chemoreceptors.

Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study.

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 O2 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 P2X3‐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 O2 chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation. J. Comp. Neurol. 461:1–17, 2003.

Proton-Mediated Feedback Inhibition of Presynaptic Calcium Channels at the Cone Photoreceptor Synapse

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+ 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+-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.

Development of oxygen sensing in the gills of zebrafish.

SUMMARY Previous studies have described the morphology, innervation and O2-chemoreceptive properties of neuroepithelial cells (NECs) of the zebrafish gill filaments. The present work describes the ontogenesis of these cells, and the formation of functional O2-sensing pathways in developing zebrafish. Confocal immunofluorescence was performed on whole-mount gill preparations using antibodies against serotonin (5-HT) and a zebrafish-derived neuronal marker (zn-12) to identify the appearance and innervation of gill NECs during larval stages. NECs were first expressed in gill filament primordia of larvae at 5 days postfertilization (d.p.f.) and were fully innervated by 7 d.p.f. In vivo ventilation frequency analysis revealed that a behavioural response to hypoxia (11.2±2.8 min–1) developed in embryos as early as 2 d.p.f., and a significant increase (P<0.05) in the ventilatory response to hypoxia (200.8±23.0 min–1) coincided with innervation of NECs of the filaments. In addition, exogenous application of quinidine, a blocker of O2-sensitive background K+ channels in NECs, induced hyperventilation in adults in a dose-dependent manner and revealed the development of a quinidine-sensitive ventilatory response in 7 d.p.f. larvae. This study shows that NEC innervation in the gill filaments may account for the development of a functional O2-sensing pathway and the hyperventilatory response to hypoxia in zebrafish larvae. At earlier stages, however, O2-sensing must occur through another pathway. The possibility that a new type of 5-HT-positive NEC of the gill arches may account for this earlier hypoxic response is discussed.

Comparative study of gill neuroepithelial cells and their innervation in teleosts and Xenopus tadpoles

Peripheral O2 chemoreceptors initiate adaptive cardiorespiratory responses to hypoxia in vertebrates. Morphological and physiological evidence suggests that, in fish, neuroepithelial cells (NECs) of the gill perform this role. We conducted a comparative examination in three species of teleosts (zebrafish, goldfish and trout) and larvae of the amphibian Xenopus laevis, using whole-mount gill preparations and confocal immunofluorescence, to elucidate the distribution, morphology and innervation of gill NECs. Nerve fibres were immunolabelled with the neuronal marker zn-12 and were associated with serotonin-immunoreactive NECs in the gills of all species tested. With the exception of trout, innervated NECs were present on all gill arches in the filaments and respiratory lamellae in fish and on homologous structures in Xenopus (i.e. gill “tufts”, including respiratory terminal branches). Thus, the distribution and innervation of NECs of the internal gills of amphibians and teleosts are relatively well conserved, suggesting an important role for gill NECs as O2 chemoreceptors in aquatic vertebrates. Furthermore, the size and density of gill NECs is variable among teleosts and developmental stages of Xenopus larvae and may be dependent on general gill dimensions or environmental conditions. This report constitutes the first comparative study of gill NECs in fish and amphibians and highlights the significance of gill NECs as an evolutionary model for studying O2 sensing in vertebrates.

Neuroepithelial cells and the hypoxia emersion response in the amphibious fish Kryptolebias marmoratus

SUMMARY Teleost fish have oxygen-sensitive neuroepithelial cells (NECs) in the gills that appear to mediate physiological responses to hypoxia, but little is known about oxygen sensing in amphibious fish. The mangrove rivulus, Kryptolebias marmoratus, is an amphibious fish that respires via the gills and/or the skin. First, we hypothesized that both the skin and gills are sites of oxygen sensing in K. marmoratus. Serotonin-positive NECs were abundant in both gills and skin, as determined by immunohistochemical labelling and fluorescence microscopy. NECs retained synaptic vesicles and were found near nerve fibres labelled with the neuronal marker zn-12. Skin NECs were 42% larger than those of the gill, as estimated by measurement of projection area, and 45% greater in number. Moreover, for both skin and gill NECs, NEC area increased significantly (30–60%) following 7 days of exposure to hypoxia (1.5 mg l–1 dissolved oxygen). Another population of cells containing vesicular acetylcholine transporter (VAChT) proteins were also observed in the skin and gills. The second hypothesis we tested was that K. marmoratus emerse in order to breathe air cutaneously when challenged with severe aquatic hypoxia, and this response will be modulated by neurochemicals associated chemoreceptor activity. Acute exposure to hypoxia induced fish to emerse at 0.2 mg l–1. When K. marmoratus were pre-exposed to serotonin or acetylcholine, they emersed at a significantly higher concentration of oxygen than untreated fish. Pre-exposure to receptor antagonists (ketanserin and hexamethonium) predictably resulted in fish emersing at a lower concentration of oxygen. Taken together, these results suggest that oxygen sensing occurs at the branchial and/or cutaneous surfaces in K. marmoratus and that serotonin and acetylcholine mediate, in part, the emersion response.

Ontogenesis of oxygen chemoreception in aquatic vertebrates.

In aquatic vertebrates, peripheral O(2) chemoreceptors initiate compensatory physiological and behavioural responses to hypoxia, beginning at very early stages of development, to maintain sufficient gas exchange across the skin or gills. This review highlights the morphological and physiological studies, particularly those of zebrafish, that have contributed to the current understanding of the development of O(2) chemoreception and the response to hypoxic challenges in embryonic and larval stages of fish and amphibians. The gills appear to be the primary site of O(2) chemoreception in developing aquatic vertebrates and initiate ventilatory changes, and adult-like O(2)-sensitive neuroepithelial cells (NECs) are found in the gills in larval stages of zebrafish and Xenopus laevis. However, evidence from zebrafish studies indicates that extrabranchial O(2) chemoreceptors appear before gill NECs and regulate responses to hypoxia that develop earlier. The developmental and evolutionary significance of the internal migration of O(2)-chemoreceptive sites with changes in respiratory organs is also discussed.

Neuroepithelial cells of the gill and their role in oxygen sensing

A highly sensitive oxygen (O(2)) sensing mechanism is critical for the survival of all vertebrate species. In fish, this requirement is fullfilled by the neuroepithelial cells (NECs) of the gill. NECs are neurotransmitter-containing chemosensory cells that are diffusely distributed within a thin epithelial layer of the filaments and respiratory lamellae of all gill arches, and are innervated by afferent fibers from the central nervous system. In acute cell culture, NECs respond immediately, and in a dose-dependent manner, to acute changes in O(2) tension. Thus, hypoxic stimulation of gill NECs appears to initiate the production of adaptive, cardiorespiratory reflexes that contribute to the maintenance of O(2) uptake in order to meet metabolic demands. This review covers the current evidence for the status of NECs as the primary peripheral O(2) sensors in fish. We have included an overview of the phylogeny of O(2) sensing structures among vertebrate groups, and morphological and physiological evidence for the importance of NECs in O(2) sensing.

Ammonia sensing by neuroepithelial cells and ventilatory responses to ammonia in rainbow trout

SUMMARY Ammonia, the third respiratory gas in teleost fish, acts as an acute stimulant to ventilation in ammoniotelic rainbow trout. We investigated whether this sensitivity is maintained in trout chronically exposed (1+ months) to high environmental ammonia [HEA, 250 μmol l–1 (NH4)2SO4] in the water, and whether gill neuroepithelial cells (NECs) are involved in ammonia sensing. Hyperventilation was induced both by acute external (NH4)2SO4 exposure [250 or 500 μmol l–1 (NH4)2SO4] and by intra-arterial (NH4)2SO4 injection (580 μmol kg–1 of ammonia) in control trout, but these responses were abolished in chronic HEA animals. Hyperventilation in response to acute ammonia exposure persisted after bilateral removal of each of the four gill arch pairs separately or after combined removal of arches III and IV, but was delayed by removal of gill arch I, and eliminated by combined removal of arches I and II. NECs, identified by immunolabeling against 5-HT, were mainly organized in two lines along the filament epithelium in all four gill arches. In control trout, NECs were slightly smaller but more abundant on arches I and II than on arches III and IV. Chronic HEA exposure reduced the density of the NECs on all four arches, and their size on arches I and II only. Fura-2 fluorescence imaging was used to measure intracellular free calcium ion concentration ([Ca2+]i) responses in single NECs in short-term (24–48 h) culture in vitro. [Ca2+]i was elevated to a comparable extent by perfusion of 30 mmol l–1 KCl and 1 mmol l–1 NH4Cl, and these [Ca2+]i responses presented in two different forms, suggesting that ammonia may be sensed by multiple mechanisms. The [Ca2+]i responses to high ammonia were attenuated in NECs isolated from trout chronically exposed to HEA, especially in ones from gill arch I, but responses to high K+ were unchanged. We conclude that the hyperventilatory response to ammonia is lost after chronic waterborne HEA exposure, and that NECs, especially the ones located in gill arches I and II, are probably ammonia chemoreceptors that participate in ventilatory modulation in trout.

Epithelial mitochondria-rich cells and associated innervation in adult and developing zebrafish.

Studies of ion regulation by mitochondria‐rich cells (MRCs) of transport epithelia in fish have revealed many processes by which ion homeostasis is achieved. However, the control of these mechanisms and, particularly, the extent of nervous system involvement are not completely understood. We characterized the potential innervation of MRCs in various gill and extrabranchial tissues involved in ion transport in the model vertebrate the zebrafish. Confocal and conventional microscopy of whole‐mount preparations were combined with immunofluorescence techniques to label MRCs with antibodies against a subunit of the enzyme Na+/K+‐ATPase and nerve fibers with a zebrafish neuronal marker, zn‐12. MRCs of the gill filaments were identified by their morphology and migration out to the lamellae in response to ion‐poor water acclimation. Gill MRCs were intimately associated with nerve fibers originating from outside the filaments. MRCs of the opercular epithelium resembled those of the gill and were also located adjacent to nerve fibers. Mitochondria‐rich “pseudobranch cells” were identified in the pseudobranch by immunofluorescence and labeling of dissociated cells with the mitochondrial marker DASPEI. Pseudobranch MRCs resembled gill MRCs and received innervation from a dense network of nerve fibers. In larvae, MRCs were distributed across the surface of the skin. These cells were situated among a dense network of varicose nerve fibers, and some MRCs of the skin displayed extensive cytoplasmic processes. Evidence is presented suggestive of widespread association of MRCs with the nervous system in transport epithelia and the neural control of MRC‐mediated ion regulation in teleost fish. J. Comp. Neurol. 497:817–832, 2006.