Cell proliferation and regeneration in the gill.

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.

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill.

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 D2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48 h. We localized D2 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 D2 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.

Identification of signalling pathways involved in gill regeneration in zebrafish.

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.

Neurochemical Signalling Associated With Gill Oxygen Sensing and Ventilation: A Receptor Focused Mini-Review.

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 O2 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.

Single-cell transcriptomic analysis of neuroepithelial cells and other cell types of the gills of zebrafish (Danio rerio) exposed to hypoxia.

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 O2 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.

Goldfish and crucian carp are natural models of anoxia tolerance in the retina.

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).

Seasonal changes in membrane structure and excitability in retinal neurons of goldfish (Carassius auratus) under constant environmental conditions.

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.

Mitochondrial KATP channels stabilize intracellular Ca2+ during hypoxia in retinal horizontal cells of goldfish (Carassius auratus).

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.

Replacement of mitochondrion-rich cells during regeneration of the gills and opercular epithelium in zebrafish (Danio rerio).

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.

The development of the O2-sensing system in an amphibious fish: consequences of variation in environmental O2 levels.

Proper development of the O2-sensing system is essential for survival. Here, we characterized the development of the O2-sensing system in the mangrove rivulus (Kryptolebias marmoratus), an amphibious fish that transitions between hypoxic aquatic environments and O2-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 O2-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 O2 availability during embryonic development modulates the development of the O2-sensing system in amphibious fishes. Surprisingly, we found that O2 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 O2 levels during the embryonic stage, indicating that there is interspecies variation in O2-induced plasticity in the O2-sensing system of fishes.

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) 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 (PO2 = 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 O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

Retinal horizontal cells of goldfish (Carassius auratus) display subtype-specific differences in spontaneous action potentials in situ.

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 Ca2+ -based action potentials (APs), yet it is unclear if APs occur in situ, or if all subtypes exhibit APs. We measured intracellular Ca2+ 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 Ca2+ flux). Next, we classified acutely dissociated HCs into subtypes by integrating the ratio of dendritic field size vs. soma size (rd/s ). H1 and H2 subtypes had low rd/s values (<8); H3/H4 had high rd/s (>12). To verify this model, H1s were identified by immunoreactivity for GABA and 95% of these cells had an rd/s < 4. In Ca2+ imaging experiments, as rd/s increased, AP duration and area under the curve increased, while frequency decreased. Our results demonstrate the presence of Ca2+ -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.

A comparative perspective on lung and gill regeneration.

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.

Regeneration of the gill filaments and replacement of serotonergic neuroepithelial cells in adult zebrafish (Danio rerio).

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.

Spontaneous action potentials in retinal horizontal cells of goldfish (Carassius auratus) are dependent upon L-type Ca2+ channels and ryanodine receptors.

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 Ca2+-based action potentials (APs) in isolated goldfish (Carassius auratus) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca2+ concentration ([Ca2+]i) were observed with fura-2 and were abolished by removal of extracellular Ca2+ or by inhibition of Ca2+ channels by 50 µM Cd2+ or 100 µM nifedipine. Inhibition of Ca2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca2+ transients and increased baseline [Ca2+]i. This increased baseline was prevented by blocking L-type Ca2+ channels with nifedipine, suggesting that Ca2+-induced Ca2+ release from stores may be needed to inactivate membrane Ca2+ channels. Caffeine (3 mM) increased the frequency of Ca2+ 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 Ca2+ or in the presence of 5 mM Co2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba2+. Collectively, our data suggest that during APs Ca2+ enters through L-type Ca2+ channels and that Ca2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca2+]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 Ca2+ 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.

Hydrogen sulphide toxicity and the importance of amphibious behaviour in a mangrove fish inhabiting sulphide-rich habitats.

We investigated amphibious behaviour, hydrogen sulphide (H2S) tolerance, and the mechanism of H2S toxicity in the amphibious mangrove rivulus (Kryptolebias marmoratus). We found that fish emersed (left water) in response to acutely elevated [H2S] (~ 130-200 µmol l-1). The emersion response to H2S 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), H2S-rich water, or air and tested the hypotheses that acclimation history influences H2S 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 [H2S] relative to fish acclimated to control or H2S-rich water, but exhibited no change in gill surface area or in NEC density or size in the gills or skin. Despite possessing exceptional H2S tolerance, all fish lost equilibrium when unable to emerse from environments containing extremely elevated [H2S] (2272 ± 46 µmol l-1). Consequently, we tested the hypothesis that impaired blood oxygen transport (i.e., sulphemoglobin formation) causes H2S toxicity in amphibious fishes. In vitro exposure of red blood cells to physiologically relevant [H2S] did not cause a substantial increase in sulphemoglobin formation. We found evidence, however, for an alternative hypothesis that H2S 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 H2S-rich environments as fish experience impaired oxidative phosphorylation when unable to emerse.

Insights into the evolution of polymodal chemoreceptors.

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 O2 uptake and delivery to tissues. These chemoreceptors are sensitive to changes in O2, CO2 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.

Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus).

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 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 Ca2+-dependent K+ current (IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca2+, and was reduced by 100 µM Cd2+ A voltage-dependent inward current in Ba2+ solution, with peak at 0 mV, confirmed the presence of Ca2+ channels. A voltage-dependent K+ current (IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K+ current (IKB) with open rectification. Mean resting membrane potential of -45.2 ± 11.6 mV did not change upon administration of hypoxia (Po2 = 11 mmHg), nor were any of the K+ currents sensitive to changes in Po2 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 Ca2+ concentration and initiated synaptic vesicle activity. 100 µM Cd2+ and 50 µM nifedipine eliminated uptake of FM 1-43. We conclude that Ca2+ influx via L-type Ca2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa 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 Ca2+ channels and three types of K+ channels, one of which was dependent upon intracellular Ca2+ 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 Ca2+ and synaptic vesicle activity.

Purinergic and adenosine receptors contribute to hypoxic hyperventilation in zebrafish (Danio rerio).

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 (IC50=18.9µM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC50=168µM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC50=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.

Distribution and chronotropic effects of serotonin in the zebrafish heart.

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.