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bioRxiv · 10.1101/2025.05.15.654070

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

Reed, M.·Mennigen, J. A.·Jonz, M. G.
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Abstract

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 nicotinic antagonist, hexamethonium. Using immunohistochemistry and RT-qPCR, we identified a novel population of ACh-containing NECs associated with sensory neurons expressing the 2 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 the first physiological evidence that gill chemoreceptors may be homologues of both pulmonary and carotid body chemoreceptors in mammals. Significance statementThe 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.

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Reed, M., Mennigen, J. A., Jonz, M. G.. 2025-05-19. Dual cholinergic and serotonergic excitatory pathways mediate oxygen sensing in the zebrafish gill. https://doi.org/10.1101/2025.05.15.654070

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