Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+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 anad hocdesigned 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 ofGjb6(-/-)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 ofPanx1(-/-)withPanx1(+/+)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.