Structure, function and development of oxygen-sensitive neuroepithelial cells of the gills of zebrafish (Danio rerio)

Oxygen (O2) sensing is a fundamental process that is important for animal survival and adaptation to environmental change. In aquatic vertebrates, low O2 (hypoxia) produces physiological adjustments that appear to arise principally from O2 chemoreceptors of the gill. However, such cells have not been definitively identified in these animals. To date, the complete morphological and physiological characterization of O2-sensitive chemoreceptors has been performed only in mammals. The present thesis utilized a multidisciplinary approach to identify and characterize a group of neuroepithelial cells (NECs) located in the gills of the zebrafish (Danio rerio), and demonstrated that these cells exhibit characteristics that define them as O2 chemoreceptors. Using double-label confocal immunofluorescence and a series of intracellular and extracellular markers, several populations of gill NECs were identified in zebrafish. These cells were found to express the neurotransmitter serotonin (5-HT) and/or a conserved synaptic vesicle marker (SV2). They also received a complex pattern of innervation via intrinsic and extrinsic neural pathways, as revealed by a zebrafish-derived neuronal marker (zn-12). Morphometric studies indicated that NECs of the filament epithelium in the gill exhibited proliferation, hypertrophy and extension of neuron-like processes in zebrafish exposed to chronic hypoxia. In patch-clamp electrophysiological experiments, isolated NECs responded to acute hypoxia with inhibition of a background K+ current, as described for O2 chemoreceptors (type I cells) of the mammalian carotid body. This current was sensitive to quinidine, but insensitive to conventional blockers of voltage-dependent K+ channels, i.e. TEA and 4-AP. During hypoxia, inhibition of this current produced membrane depolarization or a receptor potential, which presumably leads to Ca2+-dependent neurosecretion and activation of sensory pathways. In zebrafish larvae, the formation of O2-sensing pathways occurred relatively late in development (<7 days postfertilization), but before complete formation of the gills. Nevertheless, innervation of NECs of the gill filaments of these larvae approximately coincided with an increase in the hyperventilatory response to hypoxia, implicating this sensory pathway as important to the hypoxic response. The present thesis offers the first complete account of the structure, function and development of O2 chemoreceptors in an aquatic vertebrate, and defines a new model by which evolution of the cellular mechanisms of O2 sensing in vertebrates can be studied.