The Journal of Experimental Biology 1269 © 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 1269-1277 doi:10.1242/jeb.098467 ABSTRACT Serotonin containing neuroepithelial cells (NECs) are putative oxygen sensing cells found in different locations within the gills of fish. In this study we wished to determine the effect of sustained internal (blood) hypoxaemia versus external (aquatic) hypoxia on the size and density of NECs in the first gill arch of bowfin (Amia calva), a facultative air breather. We identified five different populations of serotonergic NECs in this species (Types I–V) based on location, presence of synaptic vesicles (SV) that stain for the antibody SV2, innervation and labelling with the neural crest marker HNK-1. Cell Types I–III were innervated, and these cells, which participate in central O 2 chemoreflexes, were studied further. Although there was no change in the density of any cell type in bowfin after exposure to sustained hypoxia (6.0 kPa for 7 days) without access to air, all three of these cell types increased in size. In contrast, only Type II and III cells increased in size in bowfin exposed to sustained hypoxia with access to air. These data support the suggestion that NECs are putative oxygen-sensing cells, that they occur in several locations, and that Type I cells monitor only hypoxaemia, whereas both other cell types monitor hypoxia and hypoxaemia. KEY WORDS: Chronic hypoxia, Time domains, Neuroepithelial cells, Air breathing fish INTRODUCTION In mammals, stimulation of peripheral arterial O 2 chemoreceptors (glomus cells) in the carotid body produces robust cardiovascular and ventilatory responses. Unlike most cells that decrease energy demands during exposure to hypoxia, oxygen chemoreceptors become more metabolically active (Kumar et al., 2009) and increase in size during exposure to sustained hypoxia (Wang et al., 2008). Although the exact mechanism behind this hypertrophy is unknown it is probably related to the increased turnover and synthesis of neurotransmitters associated with the increased activity of the chemoreceptors. In the gills of all fish species studied to date (for review, see Porteus et al., 2012), serotonin-containing neuroepithelial cells (NECs), which are putative peripheral arterial O 2 chemoreceptors, have been found, and evidence suggests that the NECs found in zebrafish (Danio rerio) and mangrove rivulus (Kryptolebias marmoratus) gills also increase in size during exposure to sustained hypoxia (Jonz et al., 2004; Regan et al., 2011). NECs are thought to be located in an ideal position on the gills where they can sense changes in oxygen in both water (external) and RESEARCH ARTICLE 1 Department of Zoology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4. 2 Department of Integrative Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1. *Author for correspondence at present address: College of Life and Environmental Sciences, University of Exeter, Exeter, Devon EX4 4QD, UK (c.s.porteus@exeter.ac.uk) Received 15 October 2013; Accepted 8 January 2014 blood (internal). However, the orientation of the receptors involved in reflex changes in each of the different components of the cardiorespiratory response (breathing frequency, breath amplitude, heart rate, systemic vascular resistance) to hypoxia are highly variable between species of water and air breathing fish (Milsom, 2012). Bowfin, Amia calva Linnaeus 1766, are facultative air breathing fish that use their gills to obtain oxygen from the water, but that can use their gas bladder to supplement oxygen uptake by coming to the water surface to breath air. Bowfin respond to acute hypoxia by increasing air breathing frequency and gill ventilation and reducing heart rate (bradycardia) (Porteus et al., 2014a). Branchial denervation and pseudobranch ablation eliminate the air breathing response and the bradycardia and diminish the gill ventilatory response (McKenzie et al., 1991b). These observations are consistent with the finding that bowfin do not possess central oxygen chemoreceptors (Hedrick et al., 1991) and indicate that the gills are the main location for oxygen sensing in bowfin. Furthermore, studies indicate that the reflex hypoxic bradycardia exhibited by this species is mediated exclusively by externally oriented receptors whereas changes in gill ventilation amplitude and frequency are mediated by both internally and externally oriented receptors (McKenzie et al., 1991a; McKenzie et al., 1991b). Internal injections of NaCN had no effect on air breathing in the bowfin (McKenzie et al., 1991b), whereas external NaCN stimulated air breathing (McKenzie et al., 1991b). These studies indicate that although externally oriented receptors are involved in all responses in this species, the internally oriented receptors have a more restricted role (gill ventilation only). NECs containing serotonin have been described in both the filaments and lamellae of bowfin gills using immunohistochemistry and electron microscopy (Goniakowska-Witalińska et al., 1995). These NECs, however, were not found to be in direct contact with the water in either location. This description of NEC location is inconsistent with the observed reflex responses to hypoxia versus hypoxaemia. The first aim of this study, therefore, was to re- examine the distribution of putative oxygen chemoreceptors in bowfin gills. We hypothesized that there would also be NECs in close contact with the water flowing over the gills. We also hypothesized that there would be NECs with both orientations that were innervated and would contain synaptic vesicles, just like the NECs found in other fish species. A central reflex arc is made up of an afferent neuron transmitting sensory information to either the brain or the spinal cord, integrative interneurons, and efferent neurons sending information from the central nervous system to an effector (muscles involved in ventilation). For NECs to be oxygen chemoreceptors involved in a central reflex response to hypoxia they must be innervated in order for the information to be relayed to the central nervous system for processing, and they must release their neurotransmitters from vesicles into a synapse onto these nerves. A synaptic vesicle marker SV2 and a neuronal marker zn-12 have been previously used to Characterisation of putative oxygen chemoreceptors in bowfin (Amia calva) Cosima S. Porteus 1, *, Patricia A. Wright 2 and William K. Milsom 1