Purinergic Signaling Involved in the Cellular Response to Hypoxia in Zebrafish

Abstract

During a hypoxic challenge, the maintenance of cellular function is dependent on an animal’s ability to regulate ventilation. Neurochemical signaling in the zebrafish gill initiates reflex hyperventilatory responses at the whole-animal level. Neuroepithelial cells (NECs) are believed to initiate this response by acting on post-synaptic neurons to facilitate reflex hyperventilation via the hindbrain. A number of excitatory and inhibitory neurotransmitters and their receptors have been implicated in hypoxia signalling in the zebrafish gill; however, the current study will focus on purines (ATP and adenosine) as signalling molecules. In the mammalian carotid body, ATP plays a major excitatory role in hypoxia signalling, whereas adenosine is thought to enhance the hypoxic signal. The transgenic zebrafish line, Tg(elavl3:GCaMP6s), expressing a genetically-encoded Ca²⁺ indicator (GCaMP6s) was used to visualize the NEC post-synaptic junction in isolated gill arches, where this contact remains functional. Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]ᵢ) indicated Ca²⁺ activity and were recorded as a proxy for cell excitation. Exposure to the broad-spectrum P2X agonist, ATPγS, post-synaptic [Ca²⁺]ᵢ increased while exposure to the broad-spectrum antagonist, suramin, caused a decrease in the [Ca²⁺]ᵢ response to hypoxia. Furthermore, the specific P2X2/3 receptor antagonist, A-317491, caused a decrease in [Ca²⁺]ᵢ post-synaptically in the gill as well as at the vagus nerve ganglia. These results suggest purinergic involvement in hypoxia signalling in the zebrafish. By contrast, exposure to the broad spectrum P1 receptor antagonist, caffeine, and the specific A2a receptor antagonist, SCH58261, produced no changes in post-synaptic [Ca²⁺]ᵢ in the gill or at the vagus nerve ganglia. The results of this study suggest that ATP is a major excitatory neurotransmitter acting on post-synaptic P2X2/3 receptors in the initiation of the reflex hyperventilatory response in the zebrafish.