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Seminal studies from the early 20th century defined the structural changes associated with development and regeneration of the gills in goldfish at the gross morphological and cellular levels using standard techniques of light and electron microscopy. More recently, investigations using cell lineage tracing, molecular biology, immunohistochemistry and single-cell RNA-sequencing have pushed the field forward and have begun to reveal the cellular and molecular processes that orchestrate cell proliferation and regeneration in the gills. The gill is a multifunctional organ that mediates an array of important physiological functions, including respiration, ion regulation and excretion of waste products. It is comprised of unique cell types, such as pavement cells, ionocytes, chemoreceptors and undifferentiated stem or progenitor cells that regulate growth and replenish cell populations. The gills develop from the embryonic endoderm and are rich in cell types derived from the neural crest. The gills have the capacity to remodel themselves in response to environmental change, such as in the case of ionocytes, chemoreceptors and the interlamellar cell mass, and can completely regenerate gill filaments and lamellae. Both processes of remodeling and regeneration invariably involve cell proliferation. Although gill regeneration has been reported in only a limited number of fish species, the process appears to have many similarities to regeneration of other organs in fish and amphibians. The present article reviews the studies that have described gill development and growth, and that demonstrate a suite of genes, transcription factors and other proteins involved in cell proliferation and regeneration in the gills., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Dopamine is a neurotransmitter involved in oxygen sensing and control of reflex hyperventilation. In aquatic vertebrates, oxygen sensing occurs in the gills via chemoreceptive neuroepithelial cells (NECs), but a mechanism for dopamine in autonomic control of ventilation has not been defined. We used immunohistochemistry and confocal microscopy to map the distribution of tyrosine hydroxylase (TH), an enzyme necessary for dopamine synthesis, in the gills of zebrafish. TH was found in nerve fibers of the gill filaments and respiratory lamellae. We further identified dopamine active transporter (dat) and vesicular monoamine transporter (vmat2) expression in neurons of the gill filaments using transgenic lines. Moreover, TH- and dat-positive nerve fibers innervated NECs. In chemical screening assays, domperidone, a D 2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D 2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D 2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48 h. We localized D 2 receptor immunoreactivity to NECs in the efferent gill filament epithelium, and a novel cell type in the afferent filament epithelium. We provide evidence for the synthesis and storage of dopamine by sensory nerve terminals that innervate NECs. We further suggest that D 2 receptors on presynaptic NECs provide a feedback mechanism that attenuates the chemoreceptor response to hypoxia. Our studies suggest that a fundamental, modulatory role for dopamine in oxygen sensing arose early in vertebrate evolution., (© 2024 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The occurrence of regeneration of the organs involved in respiratory gas exchange amongst vertebrates is heterogeneous. In some species of amphibians and fishes, the gills regenerate completely following resection or amputation, whereas in mammals, only partial, facultative regeneration of lung tissue occurs following injury. Given the homology between gills and lungs, the capacity of gill regeneration in aquatic species is of major interest in determining the underlying molecular or signalling pathways involved in respiratory organ regeneration. In the present study, we used adult zebrafish (Danio rerio) to characterize signalling pathways involved in the early stages of gill regeneration. Regeneration of the gills was induced by resection of gill filaments and observed over a period of up to 10 days. We screened for the effects on regeneration of the drugs SU5402, dorsomorphin and LY411575, which inhibit FGF, BMP or Notch signalling pathways, respectively. Exposure to each drug for 5 days significantly reduced regrowth of filament tips in regenerating tissue, compared with unresected controls. In separate experiments under normal conditions of regeneration, we used reverse transcription quantitative PCR and observed an increased expression of genes encoding for the bone morphogenetic factor, Bmp2b, fibroblast growth factor, Fgf8a, a transcriptional regulator (Her6) involved in Notch signalling, and Sonic Hedgehog (Shha), in regenerating gills at 10 day post-resection, compared with unresected controls. In situ hybridization confirmed that all four genes were expressed in regenerating gill tissue. This study implicates BMP, FGF, Notch and Shh signalling in gill regeneration in zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Vertebrates need oxygen to survive. The central nervous system has an especially high energy demand, so brain and retinal neurons quickly die in anoxia. But fish of the genus Carassius are exceptionally anoxia-tolerant: the crucian carp (C. carassius) can survive months without oxygen in ice-covered ponds, and the common goldfish (C. auratus) can withstand hours of anoxia at room temperature. These fish previously offered insights into anoxia tolerance in the brain, heart, and liver. Here, we advance Carassius spp. as models to study anoxia tolerance in the retina. Electroretinogram and evoked potential recordings show that crucian carp reversibly downregulate their visual systems in anoxia, probably to save ATP. Notably, Carassius suppress their visual systems nearly twice as much as anoxia-tolerant turtles, Trachemys and Chrysemys spp., which are often promoted as the champions of anoxia tolerance. We summarize what is known about anoxia tolerance in the goldfish and crucian carp retinas, including cellular pathways which may protect retinal neurons from excitotoxic cell death. We compare the Carassius retina with two relevant models: natural anoxia tolerance in the turtle brain, and ischemic preconditioning in the rat retina. All three models include mitochondria as oxygen sensors: mitochondria depolarize due to mitochondrial ATP-dependent K + channels, possibly to trigger neuroprotective second messenger cascades. The Carassius retina is an accessible and inexpensive model, with over 70 fruitful years of history in vision research. As a model for anoxia tolerance, it may provide new insights into diseases of the eye (like diabetes, macular degeneration, and eye stroke)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Despite the large body of work describing vertebrate ventilatory responses to hypoxia, remarkably little is known about the receptors and afferent pathways mediating these responses in fishes. In this review, we aim to summarize all receptor types to date implicated in the neurotransmission or neuromodulation associated with O 2 sensing in the gills of fish. This includes serotonergic, cholinergic, purinergic, and dopaminergic receptor subtypes. Recent transcriptomic analysis of the gills of zebrafish using single-cell RNA sequencing has begun to elucidate specific receptor targets in the gill; however, the absence of receptor characterization at the cellular level in the gill remains a major limitation in understanding the neurochemical control of hypoxia signalling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reed and Jonz.)

The fish gill is a multifunctional organ involved in numerous physiological processes, such as gas exchange and sensing of hypoxia by respiratory chemoreceptors, called neuroepithelial cells (NECs). Many studies have focused on zebrafish (Danio rerio) to investigate the structure, function and development of the gills, yet the transcriptomic profile of most gill cells remains obscure. We present the results of a comprehensive transcriptomic analysis of the gills of zebrafish using single-cell RNA sequencing (scRNA-seq). Gill cells from ETvmat2:EGFP zebrafish were individually labelled before scRNA-seq library construction using 10× Genomics Chromium technology. 12,819 cells were sequenced with an average depth of over 27,000 reads per cell. We identified a median of 485 genes per cell and 16 cell clusters, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. The identity of NECs was confirmed by expression of slc18a2, encoding the vesicular monoamine transporter, Vmat2. Highly differentially-expressed genes in NECs included tph1a, encoding tryptophan hydroxylase, sv2 (synaptic vesicle protein), and proteins implicated in O 2 sensing (ndufa4l2a, cox8al and epas1a). In addition, NECs and neurons expressed genes encoding transmembrane receptors for serotonergic, cholinergic or dopaminergic neurotransmission. Differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of acclimation to hypoxia. NECs in the hypoxia group showed high expression of genes involved in cell cycle control and proliferation. The present article provides a complete cell atlas for the zebrafish gill and serves as a platform for future studies investigating the molecular biology and physiology of this organ., (© 2022. The Author(s).)

Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system. We compared membrane properties of neurons isolated from the retina of goldfish (Carassius auratus), an organism well adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a twofold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Young's modulus indicated increased membrane-cortical stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (Vrev) and a significantly lower input resistance (Rin) compared with summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in retinal neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of retinal activity., (© 2022. Published by The Company of Biologists Ltd.)

Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and intracellular Ca2+ concentration ([Ca2+]i) increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured [Ca2+]i with Fura-2. Goldfish HCs maintained [Ca2+]i throughout 1 h of hypoxia, whereas [Ca2+]i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K+ channels (mKATP) are necessary to stabilize [Ca2+]i throughout hypoxia. In goldfish HCs, [Ca2+]i increased when mKATP channels were blocked with glibenclamide or 5-hydroxydecanoic acid, whereas the mKATP channel agonist diazoxide prevented [Ca2+]i from increasing in hypoxia in trout HCs. We found that hypoxia protects against increases in [Ca2+]i in goldfish HCs via mKATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca2+]i, which was rescued by hypoxia in a mKATP channel-dependent manner. We found no evidence of plasmalemmal KATP channels in patch-clamp experiments. Instead, we confirmed the involvement of KATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in a mKATP channel-sensitive manner. We conclude that mKATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca2+]i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Transport epithelia maintain the volume, ion concentration and acid-base balance of blood and extracellular fluids. In teleost fish, mitochondrion-rich cells (MRCs) are specialized ionocytes that perform this role. These cells are found in epithelia of the gills and buccal surface of the operculum (the bony structure covering the gills). Proliferation of MRCs in response to changes in water salinity and other environmental stressors is well documented, but the cellular mechanisms underlying MRC proliferation are poorly understood. Recently, regeneration and epithelial cell replacement in the gill filaments was demonstrated in the model vertebrate, zebrafish (Danio rerio), raising the question of whether MRCs are replaced during regrowth of transport epithelia. We chose two anatomical sites where MRCs are found-the gills and the opercular epithelium-to investigate whether MRCs were replaced following surgical resection of these structures. In live imaging experiments, we observed gradual replacement of the branchiostegal valve, an extension of the operculum, in zebrafish over a period of 21 days post-resection (dpr). In regenerating epithelia of both the operculum and gills, we detected MRCs by immunohistochemical localization of the α subunit of plasma membrane Na + /K + -ATPase. In both tissues, MRCs appeared soon after resection, and as early as 1 dpr in the gill filaments. We report regeneration of the operculum and proliferation of MRCs in regenerating tissue in adult zebrafish. These studies may contribute to our understanding of how MRC populations are regulated during the regenerative process, which may occur following exposure to environmental stressors, chemical toxicity or disease., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Proper development of the O 2 -sensing system is essential for survival. Here, we characterized the development of the O 2 -sensing system in the mangrove rivulus (Kryptolebias marmoratus), an amphibious fish that transitions between hypoxic aquatic environments and O 2 -rich terrestrial environments. We found that NECs formed in the gills and skin of K. marmoratus during embryonic development and that both NEC populations are retained from the embryonic stage to adulthood. We also found that the hyperventilatory response to acute hypoxia was present in embryonic K. marmoratus, indicating that functional O 2 -sensing pathways are formed during embryonic development. We then exposed embryos to aquatic normoxia, aquatic hyperoxia, aquatic hypoxia, or terrestrial conditions for the first 30 days of embryonic development and tested the hypothesis that environmental O 2 availability during embryonic development modulates the development of the O 2 -sensing system in amphibious fishes. Surprisingly, we found that O 2 availability during embryonic development had little impact on the density and morphology of NECs in the gills and skin of K. marmoratus. Collectively, our results demonstrate that, unlike the only other species of fish in which NEC development has been studied to date (i.e., zebrafish), NEC development in K. marmoratus is largely unaffected by environmental O 2 levels during the embryonic stage, indicating that there is interspecies variation in O 2 -induced plasticity in the O 2 -sensing system of fishes.

Horizontal cells (HCs) are neurons of the outer retina, which provide inhibitory feedback onto photoreceptors and contribute to image processing. HCs in teleosts are classified into four subtypes (H1-H4), each having different roles: H1-H3 feed back onto different sets of cones, H4 feed back onto rods, and only H1 store and release the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Dissociated HCs exhibit spontaneous Ca 2+ -based action potentials (APs), yet it is unclear if APs occur in situ, or if all subtypes exhibit APs. We measured intracellular Ca 2+ and report APs in slice preparations of the goldfish retina. In HCs furthest from photoreceptors (i.e., H3/H4), APs were less frequent, with greater duration and area under the curve (a measure of Ca 2+ flux). Next, we classified acutely dissociated HCs into subtypes by integrating the ratio of dendritic field size vs. soma size (r d/s ). H1 and H2 subtypes had low r d/s values (<8); H3/H4 had high r d/s (>12). To verify this model, H1s were identified by immunoreactivity for GABA and 95% of these cells had an r d/s < 4. In Ca 2+ imaging experiments, as r d/s increased, AP duration and area under the curve increased, while frequency decreased. Our results demonstrate the presence of Ca 2+ -based APs in the goldfish retina in situ and show that HC subtypes H1 through H4 exhibit progressively longer and less frequent spontaneous APs. These results suggest that APs may play an important role in inhibitory feedback, and may have implications for understanding the relative contributions of HC subtypes in the outer retina., (© 2020 Wiley Periodicals LLC.)

In teleost fish, specialized oxygen (O 2 ) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2 weeks of severe hypoxia (PO 2  = 35 mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O 2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

The ability to continuously grow and regenerate the gills throughout life is a remarkable property of fish and amphibians. Considering that gill regeneration was first described over one century ago, it is surprising that the underlying mechanisms of cell and tissue replacement in the gills remain poorly understood. By contrast, the mammalian lung is a largely quiescent organ in adults but is capable of facultative regeneration following injury. In the course of the past decade, it has been recognized that lungs contain a population of stem or progenitor cells with an extensive ability to restore tissue; however, despite recent advances in regenerative biology of the lung, the signaling pathways that underlie regeneration are poorly understood. In this Review, we discuss the common evolutionary and embryological origins shared by gills and mammalian lungs. These are evident in homologies in tissue structure, cell populations, cellular function and genetic pathways. An integration of the literature on gill and lung regeneration in vertebrates is presented using a comparative approach in order to outline the challenges that remain in these areas, and to highlight the importance of using aquatic vertebrates as model organisms. The study of gill regeneration in fish and amphibians, which have a high regenerative potential and for which genetic tools are widely available, represents a unique opportunity to uncover common signaling mechanisms that may be important for regeneration of respiratory organs in all vertebrates. This may lead to new advances in tissue repair following lung disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Respiratory epithelia and chemoreceptors of the gills and mammalian lung derive from the same embryonic structures. While the lung is limited to facultative regeneration, the regenerative capacity of the gill has not been adequately explored. We report regeneration of gill filaments and respiratory lamellae in adult zebrafish (Danio rerio). Gill filaments retained a constitutive population of mitotic cells identified by the proliferating cell nuclear antigen (PCNA). Within 24 h of resection, a new mass of PCNA-positive cells appeared at the filament tip. At 40 days post-resection, approximately half of resected tissue was replaced; and at 160 days post-resection, regeneration was nearly complete. Chemoreceptive neuroepithelial cells, identified by serotonin immunohistochemistry, were present in regenerates and established innervation by nerve fibres. Use of the transgenic zebrafish line Tg(fli1a:EGFP), in which the gill vasculature was labelled with enhanced green fluorescent protein, indicated that angiogenesis occurred during the regenerative process. Thus, the zebrafish is capable of substantive gill regeneration and replacement of respiratory chemoreceptors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Horizontal cells (HCs) are interneurons of the outer retina that undergo graded changes in membrane potential during the light response and provide feedback to photoreceptors. We characterized spontaneous Ca 2+ -based action potentials (APs) in isolated goldfish ( Carassius auratus ) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) were observed with fura-2 and were abolished by removal of extracellular Ca 2+ or by inhibition of Ca 2+ channels by 50 µM Cd 2+ or 100 µM nifedipine. Inhibition of Ca 2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca 2+ transients and increased baseline [Ca 2+ ] i . This increased baseline was prevented by blocking L-type Ca 2+ channels with nifedipine, suggesting that Ca 2+ -induced Ca 2+ release from stores may be needed to inactivate membrane Ca 2+ channels. Caffeine (3 mM) increased the frequency of Ca 2+ transients, and the store-operated channel antagonist 2-aminoethyldiphenylborinate (100 μM) counteracted this effect. APs were detected with voltage-sensitive dye imaging (FluoVolt) and current-clamp electrophysiology. In current-clamp recordings, regenerative APs were abolished by removal of extracellular Ca 2+ or in the presence of 5 mM Co 2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba 2+ . Collectively, our data suggest that during APs Ca 2+ enters through L-type Ca 2+ channels and that Ca 2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca 2+ ] i . This work may lead to further understanding of the possible role APs have in vision, such as transitioning from light to darkness or modulating feedback from HCs to photoreceptors. NEW & NOTEWORTHY Horizontal cells (HCs) are interneurons of the outer retina that provide inhibitory feedback onto photoreceptors. HCs respond to light via graded changes in membrane potential. We characterized spontaneous action potentials in HCs from goldfish and linked action potential generation to a rise in intracellular Ca 2+ via plasma membrane channels and ryanodine receptors. Action potentials may play a role in vision, such as transitioning from light to darkness, or in modulating feedback from HCs to photoreceptors.

We investigated amphibious behaviour, hydrogen sulphide (H 2 S) tolerance, and the mechanism of H 2 S toxicity in the amphibious mangrove rivulus (Kryptolebias marmoratus). We found that fish emersed (left water) in response to acutely elevated [H 2 S] (~ 130-200 µmol l -1 ). The emersion response to H 2 S may be influenced by prior acclimation history due to acclimation-induced alterations in gill morphology and/or the density and size of neuroepithelial cells (NECs) on the gills and skin. Thus, we acclimated fish to water (control), H 2 S-rich water, or air and tested the hypotheses that acclimation history influences H 2 S sensitivity due to acclimation-induced changes in (i) gill surface area and/or (ii) NEC density and/or size. Air-acclimated fish emersed at significantly lower [H 2 S] relative to fish acclimated to control or H 2 S-rich water, but exhibited no change in gill surface area or in NEC density or size in the gills or skin. Despite possessing exceptional H 2 S tolerance, all fish lost equilibrium when unable to emerse from environments containing extremely elevated [H 2 S] (2272 ± 46 µmol l -1 ). Consequently, we tested the hypothesis that impaired blood oxygen transport (i.e., sulphemoglobin formation) causes H 2 S toxicity in amphibious fishes. In vitro exposure of red blood cells to physiologically relevant [H 2 S] did not cause a substantial increase in sulphemoglobin formation. We found evidence, however, for an alternative hypothesis that H 2 S toxicity is caused by impaired oxidative phosphorylation (i.e., cytochrome c oxidase inhibition). Collectively, our results show that amphibious behaviour is critical for the survival of K. marmoratus in H 2 S-rich environments as fish experience impaired oxidative phosphorylation when unable to emerse.

Respiratory chemoreceptors in vertebrates are specialized cells that detect chemical changes in the environment or arterial blood supply and initiate autonomic responses, such as hyperventilation or changes in heart rate, to improve O 2 uptake and delivery to tissues. These chemoreceptors are sensitive to changes in O 2 , CO 2 and/or H + . In fish and mammals, respiratory chemoreceptors may be additionally sensitive to ammonia, hypoglycemia, and numerous other stimuli. Thus, chemoreceptors that affect respiration respond to different types of stimuli (or modalities) and are considered to be "polymodal". This review discusses the polymodal nature of respiratory chemoreceptors in vertebrates with a particular emphasis on chemoreceptors of the carotid body and pulmonary epithelium in mammals, and on neuroepithelial cells in water- and air-breathing fish. A major goal will be to examine the evidence for putative polymodal chemoreceptors in fish within the context of studies on mammalian models, for which polymodal chemoreceptors are well described, in order to improve our understanding of the evolution of polymodal chemoreceptors in vertebrates, and to aid in future studies that aim to identify putative receptors in air- and water-breathing fish., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The chemoreceptors involved in oxygen sensing in teleost fish are neuroepithelial cells (NECs) in the gills, and are analogous to glomus cells in the mammalian carotid body. Purinergic signalling mechanisms involving the neurotransmitters, ATP and adenosine, have been identified in mediating hypoxic signalling in the carotid body, but these pathways are not well understood in the fish gill. The present study used a behavioural assay to screen for the effects of drugs, that target purinergic and adenosine receptors, on the hyperventilatory response to hypoxia in larval zebrafish (Danio rerio) in order to determine if the receptors on which these drugs act may be involved in hypoxic signalling. The purinergic receptor antagonist, PPADS, targets purinergic P2X2/3 receptors and inhibited the hyperventilatory response to hypoxia (IC 50 =18.9μM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC 50 =168μM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC 50 =220nM). These results suggest that P2X2/3 and A2a receptors are candidates for mediating hypoxic hyperventilation in zebrafish. This study highlights the potential of applying chemical screening to ventilatory behaviour in zebrafish to further our understanding of the pathways involved in signalling by gill NECs and oxygen sensing in vertebrates., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The neuroepithelial cell (NEC) of the fish gill is an important model for O 2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O 2 chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca 2+ -dependent K + current ( I KCa ) peaked at ~20 mV, was potentiated by increased intracellular Ca 2+ , and was reduced by 100 μM Cd 2+ A voltage-dependent inward current in Ba 2+ solution, with peak at 0 mV, confirmed the presence of Ca 2+ channels. A voltage-dependent K + current ( I KV ) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K + current ( I KB ) with open rectification. Mean resting membrane potential of -45.2 ± 11.6 mV did not change upon administration of hypoxia (Po 2  = 11 mmHg), nor were any of the K + currents sensitive to changes in Po 2 during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca 2+ concentration and initiated synaptic vesicle activity. 100 μM Cd 2+ and 50 μM nifedipine eliminated uptake of FM 1-43. We conclude that Ca 2+ influx via L-type Ca 2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of I KCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia. NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca 2+ channels and three types of K + channels, one of which was dependent upon intracellular Ca 2+ Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca 2+ and synaptic vesicle activity., (Copyright © 2017 the American Physiological Society.)

Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5-HT in autonomic cardiac control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In zebrafish, cutaneous neuroepithelial cells (NECs) contain serotonin (5-HT) and are believed to initiate physiological and behavioral responses to hypoxia during embryonic and early larval development, when mature gills and O 2 chemoreceptors are not yet present. The number of skin NECs rapidly declines as embryos develop into larvae, but acclimation to hypoxia leads to retention of a greater number of these cells. We hypothesized that reduction of the partial pressure of oxygen (P O2 ) in water would stimulate mitosis in cutaneous NECs in zebrafish. Zebrafish were exposed to 5-bromo-2'-deoxyuridine (BrdU) and immunolabeled with antibodies against serotonin and BrdU to identify mitotic skin cells, including NECs. Cells were imaged and quantified using confocal microscopy. From embryonic to larval stages, we observed an overall increase in the number of BrdU-positive cells in the skin, but a decrease in BrdU-positive serotonergic NECs. Exposure of larvae to hypoxia (P O2  = 30 mmHg) in vivo for 24 h produced a 1.7-fold increase in the number of NECs labeled with BrdU. We conclude that under normal environmental P O2 the population of cutaneous NECs declines due to a decrease in mitotic activity. During environmental hypoxia, the number of NECs undergoing cell division in the skin is increased, and this promotes retention of NECs under these conditions. These data demonstrate the direct action of hypoxia upon the cell cycle of cutaneous NECs in developing zebrafish, and support the notion that cutaneous NECs are embryonic O 2 chemoreceptors. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 789-801, 2017., (© 2017 Wiley Periodicals, Inc.)

Horizontal cells (HCs) are inhibitory interneurons of the vertebrate retina. Unlike typical neurons, HCs are chronically depolarized in the dark, leading to a constant influx of Ca 2+ Therefore, mechanisms of Ca 2+ homeostasis in HCs must differ from neurons elsewhere in the central nervous system, which undergo excitotoxicity when they are chronically depolarized or stressed with Ca 2+ HCs are especially well characterized in teleost fish and have been used to unlock mysteries of the vertebrate retina for over one century. More recently, mammalian models of the retina have been increasingly informative for HC physiology. We draw from both teleost and mammalian models in this review, using a comparative approach to examine what is known about Ca 2+ pathways in vertebrate HCs. We begin with a survey of Ca 2+ -permeable ion channels, exchangers, and pumps and summarize Ca 2+ influx and efflux pathways, buffering, and intracellular stores. This includes evidence for Ca 2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors and for voltage-gated Ca 2+ channels. Special attention is given to interactions between ion channels, to differences among species, and in which subtypes of HCs these channels have been found. We then discuss a number of unresolved issues pertaining to Ca 2+ dynamics in HCs, including a potential role for Ca 2+ in feedback to photoreceptors, the role for Ca 2+ -induced Ca 2+ release, and the properties and functions of Ca 2+ -based action potentials. This review aims to highlight the unique Ca 2+ dynamics in HCs, as these are inextricably tied to retinal function., (Copyright © 2017 the American Physiological Society.)

Acetylcholine is an excitatory neurotransmitter important for oxygen sensing in mammals. A cholinergic mechanism in the fish gill has been implicated in the hyperventilatory response to acute hypoxia; however, the identity and distribution of acetylcholine-containing cells in the gills is poorly defined. We test the hypothesis that cholinergic cells are present in the gill filament epithelium in zebrafish (Danio rerio), a model vertebrate for which oxygen chemoreceptors are well characterized, and that these cells would receive nervous innervation. Using immunohistochemistry and confocal microscopy, we observed 10.2 ± 0.6 cells immunoreactive for the vesicular acetylcholine transporter (VAChT) on the efferent aspect of each gill filament, where a high density of serotonergic oxygen-sensitive neuroepithelial cells (NECs) were located. VAChT-positive cells of the efferent epithelium were positioned within 10 μm of NECs. On the afferent aspect of the gill filaments, VAChT-positive cells were greater in number (30.8 ± 3.1 per filament). On the efferent and afferent filament aspects, VAChT-positive cells did not contain serotonin, but did express choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, and were often closely apposed to nerve fibers labeled with the neuronal marker, zn-12. We conclude that cholinergic cells in the zebrafish gills were present in the primary epithelium of gill filaments, and formed contacts with nerve fibers. These studies provide morphological evidence for the presence of a cholinergic system in the zebrafish gill. Such a pathway may contribute to the reflex hyperventilatory response during hypoxia.

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 μM (EC50 = 24.5 μM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced fV at 50 μM (EC50 = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Surviving hypoxia is one of the most critical challenges faced by vertebrates. Most species have adapted to changing levels of oxygen in their environment with specialized organs that sense hypoxia, while only few have been uniquely adapted to survive prolonged periods of anoxia. The goal of this review is to present the most recent research on oxygen sensing, adaptation to hypoxia, and mechanisms of anoxia tolerance in nonmammalian vertebrates. We discuss the respiratory structures in fish, including the skin, gills, and air-breathing organs, and recent evidence for chemosensory neuroepithelial cells (NECs) in these tissues that initiate reflex responses to hypoxia. The use of the zebrafish as a genetic and developmental model has allowed observation of the ontogenesis of respiratory and chemosensory systems, demonstration of a putative intracellular O2 sensor in chemoreceptors that may initiate transduction of the hypoxia signal, and investigation into the effects of extreme hypoxia on cardiorespiratory development. Other organisms, such as goldfish and freshwater turtles, display a high degree of anoxia tolerance, and these models are revealing important adaptations at the cellular level, such as the regulation of glutamatergic and GABAergic neurotransmission in defense of homeostasis in central neurons., (© 2015 New York Academy of Sciences.)

Aquatic hypercapnia may have helped to drive ancestral vertebrate invasion of land. We tested the hypothesis that amphibious fishes sense and respond to elevated aquatic PCO2 by behavioural avoidance mechanisms, and by morphological changes at the chemoreceptor level. Mangrove rivulus (Kryptolebias marmoratus) were exposed to 1 week of normocapnic control water (pH 8), air, hypercapnia (5% CO2, pH 6.8) or isocapnic acidosis (pH 6.8). We found that the density of CO2/H(+) chemoreceptive neuroepithelial cells (NECs) was increased in hypercapnia or isocapnic acidosis-exposed fish. Projection area (a measure of cell size) was unchanged. Acute exposure to progressive hypercapnia induced the fish to emerse (leave water) at water pH values ∼6.1, whereas addition of HCl to water caused a more variable response with a lower pH threshold (∼pH 5.5). These results support our hypothesis and suggest that aquatic hypercapnia provides an adequate stimulus for extant amphibious fishes to temporarily transition from aquatic to terrestrial habitats., (© 2015. Published by The Company of Biologists Ltd.)

The story of control of cardiorespiratory reflexes by peripheral chemoreceptors includes a chapter on evolution in large part because of the work of Prof. William K. Milsom. Bill has reminded us to think comparatively about O2 and CO2/H(+) sensing. We present a brief review of the fish gill and O2 chemoreceptors, as well as recent results from our laboratory, that were discussed at a symposium in honour of Prof. Milsom's extensive career. In a series of papers from the Milsom laboratory from 1986 to 1995, it was demonstrated that the fish gill is a major site of chemosensory discharge during hypoxia, and that this response is sensitive to multiple neurochemicals involved in chemosensing. These and other more recent studies by Bill et al. are now fundamental and have helped to shape the field as it is today. At the cellular level, we have shown that chemosensitive neuroepithelial cells (NECs) of the gills may possess unique adaptations compared to their mammalian homologues. In addition, we used injection of the styryl dye, FM1-43, to identify gill NECs in zebrafish and demonstrate increased vesicular activity in NECs in vitro during acute stimulation. In vivo, we have identified 5-HT2, 5-HT3, dopaminergic and nicotinic receptor activity involved in the hyperventilatory response in developing zebrafish. With this model we have also traced the fate of mitotic cells in the gills, and demonstrated the regeneration of resected gill filaments and replacement of O2-sensitive NECs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Severe hypoxia elicits aquatic surface respiration (ASR) behaviour in many species of fish, where ventilation of the gills at the air-water interface improves O2 uptake and survival. ASR is an important adaptation that may have given rise to air breathing in vertebrates. The neural substrate of this behaviour, however, is not defined. We characterized ASR in developing and adult zebrafish (Danio rerio) to ascertain a potential role for peripheral chemoreceptors in initiation or modulation of this response. Adult zebrafish exposed to acute, progressive hypoxia (PO2 from 158 to 15 mmHg) performed ASR with a threshold of 30 mmHg, and spent more time at the surface as PO2 decreased. Acclimation to hypoxia attenuated ASR responses. In larvae, ASR behaviour was observed between 5 and 21 days postfertilization with a threshold of 16 mmHg. Zebrafish decreased swimming behaviour (i.e. distance, velocity and acceleration) as PO2 was decreased, with a secondary increase in behaviour near or below threshold PO2 . In adults that underwent a 10-day intraperitoneal injection regime of 10 μg g(-1) serotonin (5-HT) or 20 μg g(-1) acetylcholine (ACh), an acute bout of hypoxia (15 mmHg) increased the time engaged in ASR by 5.5 and 4.9 times, respectively, compared with controls. Larvae previously immersed in 10 μmol l(-1) 5-HT or ACh also displayed an increased ASR response. Our results support the notion that ASR is a behavioural response that is reliant upon input from peripheral O2 chemoreceptors. We discuss implications for the role of chemoreceptors in the evolution of air breathing., (© 2015. Published by The Company of Biologists Ltd.)

Neuroepithelial cells (NECs) of the fish gill are respiratory chemoreceptors that detect changes in O2 and CO2/H(+) and are homologous to type I cells of the mammalian carotid body. In zebrafish (Danio rerio), stimulation of NECs by hypoxia or hypercapnia initiates inhibition of K(+) channels and subsequent membrane depolarisation. The goal of the present study was to further elucidate, in zebrafish NECs, the signalling pathways that underlie CO2/H(+) sensing and generate intracellular Ca(2+) ([Ca(2+)]i) signals. Breathing frequency was elevated maximally in fish exposed to 5 % CO2 (~37.5 mmHg). Measurement of [Ca(2+)]i in isolated NECs using Fura-2 imaging indicated that [Ca(2+)]i increased in response to acidic hypercapnia (5 % CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5 % CO2, pH 7.6). Measurement of intracellular pH (pHi) using BCECF demonstrated a rapid decrease in pHi in response to acidic and isohydric hypercapnia, while isocapnic acidosis produced a smaller change in pHi. Intracellular acidification was reduced by the carbonic anhydrase inhibitor, acetazolamide, without affecting [Ca(2+)]i responses. Moreover, intracellular acidification using acetate (at constant extracellular pH) was without effect on [Ca(2+)]i. The acid-induced increase in [Ca(2+)]i persisted in the absence of extracellular Ca(2+) and was unaffected by Ca(2+) channel blockers (Cd(2+), Ni(2+) or nifedipine). The results of this study demonstrate that, unlike type I cells, extracellular H(+) is critical to the hypercapnia-induced increase in [Ca(2+)]i in NECs. The increase in [Ca(2+)]i occurs independently of pHi and appears to originate primarily from Ca(2+) derived from intracellular stores.

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish., (Copyright © 2013 Wiley Periodicals, Inc.)

The chemosensory roles of gill neuroepithelial cells (NECs) in mediating the hyperventilatory response to hypoxia are not clearly defined in fish. While serotonin (5-HT) is the predominant neurotransmitter in O(2)-sensitive gill NECs, acetylcholine (ACh) plays a more prominent role in O(2) sensing in terrestrial vertebrates. The present study characterized the developmental chronology of potential serotonergic and cholinergic chemosensory pathways of the gill in the model vertebrate, the zebrafish (Danio rerio). In immunolabelled whole gills from larvae, serotonergic NECs were observed in epithelia of the gill filaments and gill arches, while non-serotonergic NECs were found primarily in the gill arches. Acclimation of developing zebrafish to hypoxia (P(O2)=75 mmHg) reduced the number of serotonergic NECs observed at 7 days post-fertilization (d.p.f.), and this effect was absent at 10 d.p.f. In vivo administration of 5-HT mimicked hypoxia by increasing ventilation frequency (f(V)) in early stage (7-10 d.p.f.) and late stage larvae (14-21 d.p.f.), while ACh increased f(V) only in late stage larvae. In time course experiments, application of ketanserin inhibited the hyperventilatory response to acute hypoxia (P(O2)=25 mmHg) at 10 d.p.f., while hexamethonium did not have this effect until 12 d.p.f. Cells immunoreactive for the vesicular acetylcholine transporter (VAChT) began to appear in the gill filaments by 14 d.p.f. Characterization in adult gills revealed that VAChT-positive cells were a separate population of neurosecretory cells of the gill filaments. These studies suggest that serotonergic and cholinergic pathways in the zebrafish gill develop at different times and contribute to the hyperventilatory response to hypoxia.

Mitochondrial respiration is mediated by a set of multisubunit assemblies of proteins that are embedded in the mitochondrial inner membranes. Respiratory complexes do not only contain central catalytic subunits essential for the bioenergetic transformation, but also many short trans-membrane subunits (sTMs) that are implicated in the proper assembly of complexes. Defects in sTMs have been discovered in some human neurodegenerative diseases. Here we identify a new subunit that we named Stmp1 and have characterized its function using both computational and experimental approaches. Stmp1 is a short trans-membrane protein, and sequence/structure analysis revealed that it shares common features like the small size, presence of a single or two TM region, and a COOH-terminal charged region, as many typical sTMs of respiratory complexes. In situ hybridization and RT-PCR assays showed that the Stmp1 expression is ubiquitous throughout zebrafish embryogenesis. In adults, Stmp1 expression was highest in the brain compared with muscle and liver. In zebrafish larvae (3-5 days postfertilization), antisense morpholino oligonucleotide-mediated knockdown of the Stmp1 gene (Stmp1-MO) resulted in a series of mild morphological defects, including abnormal shape of head and jaw and cardiac edema. Larvae injected with the Stmp1-MO had negligible responses to touch stimuli. By ventilation frequency analysis we found that Stmp1-MO-injected zebrafish displayed a severe dysfunction of ventilatory activities when exposed to hypoxic conditions, suggesting a defective mitochondrial activity induced by the loss of Stmp1. Phylogenetic profiling of known respiratory sTMs compared with Stmp1 revealed that all defined sTMs from four respiratory complexes have restricted or variable phyletic distribution, indicating that they are products of evolutionary innovations to fulfill lineage-related functional requirements for respiratory complexes. Thus, being present in animals, filasterea, choanoflagellida, amoebozoa, and plants, Stmp1 may have evolved to confer a new or complementary regulation of respiratory activities.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In teleost fish, O(2) chemoreceptors of the gills (neuroepithelial cells or NECs) initiate cardiorespiratory reflexes during hypoxia. In developing zebrafish, hyperventilatory and behavioural responses to hypoxia are observed before development of gill NECs, indicating that extrabranchial chemoreceptors mediate these responses in embryos. We have characterised a population of cells of the skin in developing zebrafish that resemble O(2)-chemoreceptive gill NECs. Skin NECs were identified by serotonin immunolabelling and were distributed over the entire skin surface. These cells contained synaptic vesicles and were associated with nerve fibres. Skin NECs were first evident in embryos 24-26 h post-fertilisation (h.p.f.), and embryos developed a behavioural response to hypoxia between 24 and 48 h.p.f. The total number of NECs declined with age from approximately 300 cells per larva at 3 days post-fertilisation (d.p.f.) to ~120 cells at 7 d.p.f., and were rarely observed in adults. Acclimation to hypoxia (30 mmHg) or hyperoxia (300 mmHg) resulted in delayed or accelerated development, respectively, of peak resting ventilatory frequency and produced changes in the ventilatory response to hypoxia. In hypoxia-acclimated larvae, the temporal pattern of skin NECs was altered such that the number of cells did not decrease with age. By contrast, hyperoxia produced a more rapid decline in NEC number. The neurotoxin 6-hydroxydopamine degraded catecholaminergic nerve terminals that made contact with skin NECs and eliminated the hyperventilatory response to hypoxia. These results indicate that skin NECs are sensitive to changes in O(2) and suggest that they may play a role in initiating responses to hypoxia in developing zebrafish.

The oropharyngeal cavity in fish supports a range of sensory modalities, including detection of chemical and mechanical stimuli. Taste buds are found throughout this tissue and may participate in both processes. We used confocal microscopy and immunohistochemistry to characterize the morphology of Merkel-like cells and their association with other cell types and nerve fibers of the taste bud in the vertebrate model, the zebrafish. In addition, we document procedures for the observation of these structures in whole-tissue preparations from larvae and adults using zebrafish-specific and monoclonal antibodies. A single microvillus Merkel-like cell was found in each taste bud regardless of age or location. Merkel-like cells were neurosecretory, as indicated by labelling with the styryl dye, FM1-43, and the synaptic vesicle marker, SV2. Merkel-like cells were associated with SV2- and calretinin-positive taste receptor cells, received innervation from discoid aggregations of nerve fibers, and retained serotonin-filled synaptic vesicles oriented within the cytoplasm toward adjacent innervation. Moreover, a ring-like formation of nerve endings was identified with the neuronal marker, zn-12 that circumscribed the taste receptor area, surrounding calretinin-immunoreactive taste cell microvilli, and appeared to associate with the nerve plexus adjacent to Merkel-like cells. We suggest that these nerve fibers are somatosensory, perhaps associated with mechanoreception or the common chemical sense., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

In the zebrafish, O(2)-chemoreceptive neuroepithelial cells (NECs) of the gill arches detect changes in PO(2) and are believed to initiate cardiorespiratory responses to hypoxia. Goldfish have gill NECs of similar morphology and innervation, yet these animals are naturally tolerant to prolonged periods of anoxia. Whole-cell, voltage-clamp experiments indicated that goldfish NECs express a variety of membrane ion channels, including background and Ca(2+)-activated K(+) channels. Our initial studies suggest that goldfish NECs do not respond equally to hypoxia (N(2), 25 mmHg) as do those of zebrafish; however, current-clamp recordings indicated that anoxia produced membrane depolarization. In addition, we found that cyanide, which mimics hypoxia, depolarized NECs. Our goal is to further characterize these membrane conductances and determine their potential contribution to O(2) sensing in NECs of the anoxia-tolerant goldfish.

Among the vertebrates, peripheral chemoreceptors have evolved to play a key role in matching oxygen delivery to the metabolic needs of the body cells and tissues. Specialized neuroepithelial cells (NECs) distributed within the gill filaments and/or lamellae of water-breathers appear to subserve this function by initiating an increase in ventilation in response to lowering of blood or water PO(2) (hypoxia). It is only recently, however, that these cells have become amenable for detailed investigations using electrophysiological tools. By contrast, the well-studied specialized neuroendocrine cells (i.e. glomus or type I cells) located principally in the carotid body of air-breathers initiate a similar reflex ventilatory response to hypoxia so as to maintain blood PO(2) homeostasis. In some species, however, the carotid body is immature and relatively insensitive to hypoxia at birth; it is during this period that their sympathoadrenal counterparts in the adrenal medulla act as key PO(2) receptors, critical for the proper transition to air-breathing life. It is becoming increasingly clear that in general these chemoreceptors act as polymodal receptors, i.e. capable of detecting several sensory modalities including high CO(2)/H(+) or acid hypercapnia. Given the phylogenetic and ontogenetic evidence pointing to homology between the mammalian carotid artery and the first gill arch of teleosts, the question arises whether the mechanisms of chemosensing are conserved among these cell types. This review examines some of the anatomical and functional similarities among these peripheral chemoreceptors, while raising the possibility that the fundamental mechanisms of O(2) and CO(2)/H(+) sensing arose first in water-breathers and are conserved among the vertebrates.

Adult zebrafish, Danio rerio, exhibit hyperventilatory responses to absolute environmental CO2 levels as low as 1.0 mmHg. The ability of zebrafish to detect and respond to low ambient CO2 appears to be mediated by chemosensory neuroepithelial cells (NECs) of the gill filaments. Recent electrophysiological characterization of this response revealed that the partial pressure-dependent depolarization of NECs in response to a hypercapnic stimulus is dependent on the rate of acidification associated with the hydration of CO2 and the inhibition of background K(+) channels. In order to further elucidate the signaling pathway underlying CO2 chemotransduction in NECs, we used microfluorimetric techniques to study intracellular changes in pH (pHi) and calcium ([Ca(2+)]i). Using the ratiometric indicators BCECF-AM and fura-2-AM, we found that a hypercapnic stimulus evoked a decrease in pHi and an increase in [Ca(2+)]i.

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) (NH(4))(2)SO(4)] in the water, and whether gill neuroepithelial cells (NECs) are involved in ammonia sensing. Hyperventilation was induced both by acute external (NH(4))(2)SO(4) exposure [250 or 500 μmol l(-1) (NH(4))(2)SO(4)] and by intra-arterial (NH(4))(2)SO(4) 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 ([Ca(2+)](i)) responses in single NECs in short-term (24-48 h) culture in vitro. [Ca(2+)](i) was elevated to a comparable extent by perfusion of 30 mmol l(-1) KCl and 1 mmol l(-1) NH(4)Cl, and these [Ca(2+)](i) responses presented in two different forms, suggesting that ammonia may be sensed by multiple mechanisms. The [Ca(2+)](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.

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.

The fish gill is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervation from both afferent (sensory) and efferent (motor) fibres. Innervation from the latter source includes autonomic nerve fibres of spinal (sympathetic) and cranial (parasympathetic) origin whose primary role is to induce vasomotor changes within the respiratory or nonrespiratory pathways of the gill vasculature. Autonomic control of the gill occurs by nerve fibres identified as adrenergic, cholinergic, and more recent evidence indicates that nonadrenergic-noncholinergic (NANC) nerve fibres, such as those that express amines, peptides, or nitric oxide, may also play an important role. The distribution and physiological function of NANC nerve fibres, however, is less clear. This review primarily discusses histochemical studies that have characterized the nervous innervation and autonomic control of the gill vasculature. In addition, supporting evidence from recent studies for the efferent control, or modulation, of other homeostatic processes in the gill is examined.

The fish gill is a highly specialized and complex organ that performs a variety of important physiological functions. In this article, we briefly review the innervation of important structures of the branchial region, such as the gill filaments, respiratory lamellae and pseudobranch, and discuss the physiological significance of this innervation within the context of homeostatic functions of the gill, such as oxygen sensing and ion regulation. Studies in zebrafish utilizing techniques of confocal microscopy and immunolabelling, with specific antibodies against neuronal markers, have recently led to the characterization of innervation patterns in the gills not attained with traditional techniques of histochemistry and electron microscopy. We will discuss the association of putative sensory nerve fibres with O2-chemoreceptive neuroepithelial cells and the implications of dual sensory pathways for cardiorespiratory and vascular control. In addition, the idea of the neural control of ion regulation in the gill based on the apparent innervation of mitochondria-rich cells, and the role of innervation in the pseudobranch, will be presented.

Transient changes in extracellular pH (pH(o)) occur in the retina and may have profound effects on neurotransmission and visual processing due to the pH sensitivity of ion channels. The present study characterized the effects of acidification on the activity of membrane ion channels in isolated horizontal cells (HCs) of the goldfish retina using whole-cell patch-clamp recording. Currents recorded from HCs were characterized by prominent inward rectification at potentials negative to -80 mV, a negative slope conductance between -70 and -40 mV, a sustained inward current, and outward rectification positive to 40 mV. Inward currents were identified as those of inward rectifier K(+) (Kir) channels and Ca(2+) channels by their sensitivity to 10 mM Cs(+) or 20 microm Cd(2+), respectively. Both of these currents were reduced when pH(o) decreased from 7.8 to 6.8. Glutamate (1 mM)-activated currents were also identified, as were hemichannel currents that were enhanced by removal of extracellular Ca(2+) and application of 1 mM quinidine. Both glutamate-activated and hemichannel currents were suppressed by a similar reduction of pH(o). When all of these H(+)-inhibited currents were blocked, a small, sustained inward current at -60 mV increased following a decrease in pH(o) from 7.8 to 6.8. In addition, slope conductance between -70 and -20 mV increased during this acidification. Suppression of this H(+)-activated current by removal of extracellular Na(+), and an extrapolated E(rev) near E(Na), indicated that this current was carried predominantly by Na(+) ions.

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.

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.

Peripheral O(2) 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 O(2) 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 O(2) sensing in vertebrates.

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.

Previous studies have described the morphology, innervation and O(2)-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 O(2)-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 O(2)-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 O(2)-sensing pathway and the hyperventilatory response to hypoxia in zebrafish larvae. At earlier stages, however, O(2)-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.

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.

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., (Copyright 2003 Wiley-Liss, Inc.)