Gill Neuroepithelial Cells Research Articles

Changes in gill neuroepithelial cells and morphology of threespine stickleback (Gasterosteus aculeatus) to hypoxia and simulated ocean acidification.

Coastal marine environments are characterized by daily, seasonal and long-term changes in both O2 and CO2, driven by local biotic and abiotic factors. The neuroepithelial cells (NECs) of fish are thought to be the putative chemoreceptors for sensing oxygen and CO2, and, thus, NECs play a key role in detecting these environmental changes. However, the role of NECs as chemosensors in marine fish remains largely understudied. In this study, the NECs of marine threespine sticklebacks (Gasterosteus aculeatus) were characterized using immunohistochemistry. We then determined if there were changes in NEC size and density, and in gill morphology in response to either mild (10kPa) or moderate (6.8kPa) hypoxia and two levels of elevated CO2 (1,500 and 3,000 µatm). We found that the NECs of stickleback contained synaptic vesicles and were innervated, and were 50-300% larger and 2 to 4 times more abundant than in other similar sized freshwater fishes. NEC size and density were largely unaffected by exposure to hypoxia, but there was a 50% decrease in interlamellar cell mass (ILCM) in response to mild and moderate hypoxia. NECs increased in size, but not abundance in response to elevated CO2. Moreover, fish exposed to moderate or elevated CO2 had 53-78% larger ILCMs compared to control fish. Our results demonstrated that adult marine sticklebacks have NECs that can respond to environmentally relevant pCO2 and likely hypoxia, which highlights the importance of NECs in marine fishes under the heterogeneity of environmental conditions in coastal areas.

Gulf toadfish (Opsanus beta) gill neuroepithelial cells in response to hypoxia exposure.

Neuroepithelial cells (NECs) within the fish gill contain the monoamine neurochemical serotonin (5-HT), sense changes in the partial pressure of oxygen (PO2) in the surrounding water and blood, and initiate the cardiovascular and ventilatory responses to hypoxia. The distribution of neuroepithelial cells (NECs) within the gill is known for some fish species but not for the Gulf toadfish, Opsanus beta, a fish that has always been considered hypoxia tolerant. Furthermore, whether NEC size, number, or distribution changes after chronic exposure to hypoxia, has never been tested. We hypothesize that toadfish NECs will respond to hypoxia with an increase in NEC size, number, and a change in distribution. Juvenile toadfish (N = 24) were exposed to either normoxia (21.4 ± 0.0kPa), mild hypoxia (10.2 ± 0.3kPa), or severe hypoxia (3.1 ± 0.2kPa) for 7 days and NEC size, number, and distribution for each O2 regime were measured. Under normoxic conditions, juvenile toadfish have similar NEC size, number, and distribution as other fish species with NECs along their filaments but not throughout the lamellae. The distribution of NECs did not change with hypoxia exposure. Mild hypoxia exposure had no effect on NEC size or number, but fish exposed to severe hypoxia had a higher NEC density (# per mm filament) compared to mild hypoxia-exposed fish. Fish exposed to severe hypoxia also had longer gill filament lengths that could not be explained by body weight. These results point to signs of phenotypic plasticity in these juvenile, lab-bred fish with no previous exposure to hypoxia and a strategy to deal with hypoxia exposure that differs in toadfish compared to other fish.

The control of breathing in fishes – historical perspectives and the path ahead

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing.

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.

Cardiorespiratory toxicity of environmentally relevant zinc oxide nanoparticles in the freshwater fish Catostomus commersonii

The inhalation of zinc oxide engineered nanomaterials (ENMs) has been linked to cardiorespiratory dysfunction in mammalian models but the effects of aquatic ENM exposure on fish have not been fully investigated. Nano-zinc oxide (nZnO) is widely used in consumer products such as sunscreens and can make its way into aquatic ecosystems from domestic and commercial wastewater. This study examined the impact of an environmentally relevant nZnO formulation on cardiorespiratory function and energy metabolism in the white sucker (Catostomus commersonii), a freshwater teleost fish. Evidence of oxidative and cellular stress was present in gill tissue, including increases in malondialdehyde levels, heat shock protein (HSP) expression, and caspase 3/7 activity. Gill Na+/K+-ATPase activity was also higher by approximately three-fold in nZnO-treated fish, likely in response to increased epithelial permeability or structural remodeling. Despite evidence of toxicity in gill, plasma cortisol and lactate levels did not change in animals exposed to 1.0 mg L−1 nZnO. White suckers also exhibited a 35% decrease in heart rate during nZnO exposure, with no significant changes in resting oxygen consumption or tissue energy stores. Our results suggest that tissue damage or cellular stress resulting from nZnO exposure activates gill neuroepithelial cells, triggering a whole-animal hypoxic response. An increase in parasympathetic nervous signaling will decrease heart rate and may reduce energy demand, even in the face of an environmental toxicant. We have shown that acute exposure to nZnO is toxic to white suckers and that ENMs have the potential to negatively impact cardiorespiratory function in adult fish.

Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish

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.

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

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.

Neuroepithelial cells and the hypoxia emersion response in the amphibious fishKryptolebias marmoratus

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 time domains of the ventilatory responses of bowfin (Amia calva) to prolonged hypoxia

The acute responses of fish to hypoxia are well established, however the time course for the hypoxic ventilatory response in chronic hypoxia is not well understood. In the present study we assessed the ventilatory and cardiovascular responses of the facultative air breather, Amia calva, to severe hypoxia for 3 days. During prolonged exposure to hypoxia, there was an initial increase in gill breathing frequency, amplitude and total gill ventilation over the first three hours of hypoxic exposure, but this was not sustained. After the initial acute response, there was a decrease in total ventilation that lasted until about 24 hours of hypoxic exposure, when a secondary increase in ventilation started taking place. There was also an immediate bradycardia that was not sustained. These changes were accompanied by changes in the size, and density but not distribution of putative gill neuroepithelial cells (homologous to carotid body glomus cells) after 3 days of hypoxic exposure indicating the importance of these cells in the hypoxic ventilatory response. The time domains seen in gill breathing in Amia are similar to those reported in mammals however the time course of these responses is slower with a ventilatory "roll off" developing in hours not minutes. (funded by NSERC of Canada)

Ontogenesis of oxygen chemoreception in aquatic vertebrates

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

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

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.