Characterizing the Neurochemical Basis of Hypoxia Signaling in the Zebrafish Gill

Abstract

Oxygen sensing and the physiological responses to hypoxia are essential for survival in aquatic environments. In teleost fish, the gills act as the primary site for detecting oxygen levels and initiating compensatory responses. Neuroepithelial cells (NECs) in the gills are key peripheral oxygen sensors in aquatic vertebrates and share morphological features with mammalian oxygen chemoreceptors, such as those in the carotid body. Upon detecting hypoxia, NECs inhibit background K+ channels, leading to membrane depolarization and neurotransmitter release. This cascade activates sensory pathways in the gills projecting to higher order centers, resulting in physiological responses like hyperventilation. Despite the extensive research on vertebrate ventilatory responses to hypoxia, the specific neurotransmitters, receptors, and afferent pathways that mediate these responses in fish remain poorly understood. Using an isolated gill preparation from Tg(elavl3:GCaMP6s) zebrafish, we measured hypoxia-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in both NECs and postsynaptic chain neurons (ChNs). Our results show that both NECs and ChNs exhibit hypoxia-induced increases in [Ca2+]i. Importantly, we found that a functional NEC-ChN synapse is required for the hypoxia-induced increase in [Ca2+]i in ChNs, and that these postsynaptic responses are modulated by presynaptic dopamine through dopamine D2 receptors (D2Rs). Additionally, we show that acetylcholine (ACh) plays a vital excitatory role in activating ChNs and extrabranchial nerves involved in reflex hyperventilation via nicotinic ACh receptors. We further identify two distinct populations of neurosecretory cells in the gill—ACh-positive and 5-HT-positive—that transmit hypoxic signals to different postsynaptic neurons. Our findings provide the first direct evidence of neurotransmission at the NEC-ChN synapse in the gill and suggest that the modulatory role of dopamine and the excitatory role of ACh in oxygen sensing are evolutionarily conserved features that emerged early in vertebrate evolution.