Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca 2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca 2+ imaging, we monitored the propagation of intercellular Ca 2+ waves in the GER of COCs and ATP-dependent Ca 2+ responses in overlying ATP-WCBs. Ca 2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca 2+ signals were strongly depressed in the presence of Gjb6 −/− COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca 2+ signals were not affected by replacement of Panx1 −/− with Panx1 +/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca 2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods. Using microfluidics, ATP biosensors, multiphoton microscopy and genetically targeted mice, we show that ATP release through connexin hemichannels, and not pannexin 1 channels, underlies spontaneous Ca 2+ wave propagation in the greater epithelial ridge of the developing cochlea.