Dual cholinergic and serotonergic excitatory pathways mediate oxygen sensing in the zebrafish gill

The evolution of oxygen sensing included a transition from a diffuse distribution of respiratory chemoreceptors in the gills of water-breathing vertebrates to chemoreceptor clusters confined to the pulmonary epithelium and carotid body in air-breathers. Since the excitatory neurotransmitters mediating oxygen sensing in anamniotes have never been confirmed, the origins of oxygen sensing in vertebrates have remained controversial. In gills isolated from Tg(elavl3:GCaMP6s) zebrafish expressing a genetically encoded reporter of intracellular Ca²⁺ concentration ([Ca²⁺]_i), we demonstrate that acetylcholine (ACh) and nicotine induced a dose-dependent increase in [Ca²⁺]_i in postsynaptic sensory neurons innervating oxygen-chemoreceptive neuroepithelial cells (NECs). Hypoxic stimulation of NECs evoked a similar rise in neuronal [Ca²⁺]_i that was abolished by the nicotinic antagonist hexamethonium. Using immunohistochemistry and RT-qPCR, we identified a novel population of ACh-containing NECs associated with sensory neurons expressing the α2 subunit of nicotinic ACh receptors. In vivo whole-larva Ca²⁺ imaging showed that cholinergic and hypoxic activation of the gills generated Ca²⁺ activity in neurons of vagal sensory ganglia with time-dependent characteristics of neurotransmission towards the hindbrain. We identified a second source of hypoxic activity in vagal sensory ganglia operating exclusively through 5-HT₃ receptors and dependent upon vesicular monoamine transport (VMAT2) in the gill. We traced expression of 5-HT₃ receptors to nerve terminals surrounding a separate population of serotonergic VMAT2-positive NECs. Our investigation reveals independent cholinergic and serotonergic autonomic pathways of oxygen sensing in zebrafish and provides novel physiological evidence consistent with the idea that gill chemoreceptors are homologues of both pulmonary and carotid body chemoreceptors in mammals. KEY POINTS: The excitatory neurotransmitters mediating oxygen sensing in anamniotes have never been confirmed. Thus, the origins of oxygen sensing in vertebrates have remained controversial. In transgenic zebrafish expressing a genetically encoded reporter of intracellular Ca²⁺ concentration, we identified two independent pathways of oxygen sensing in the gill: one involving acetylcholine and interneurons intrinsic to the gill, and the other via serotonin acting directly upon ganglionic neurons. Both pathways resulted in excitation of vagal sensory ganglia that receive hypoxic inputs from the gills and innervate the hindbrain. We argue that gill chemoreceptors are homologues of both pulmonary and carotid body chemoreceptors in mammals.