Visualization of mechanical stress-mediated Ca²⁺ signaling in the gut using intravital imaging

Mechanosensory systems have been implicated in the maintenance of gut homeostasis, but details on the related mechanisms are scarce. Recently, we generated a conditional Ca²⁺ biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca²⁺) intravital imaging system for investigating lymphoid tissues and enteric epithelial cell responses. To validate this gut-sensing system, we visualized responses of enteric nervous system (ENS) cells in Nestin-Cre/YC3.60^flox mice with specific YC3.60 expression. The ENS, including the myenteric (Auerbach's) and submucous (Meissner's) plexuses, could be visualized without staining in this mouse line, indicating that the probe produced sufficient fluorescent intensity. Furthermore, the myenteric plexus exhibited Ca²⁺ signaling during peristalsis without stimulation. Nerve endings on the surface of enteric epithelia also exhibited Ca²⁺ signaling without stimulation. Mechanical stress induced transient salient Ca²⁺ flux in the myenteric plexus and in enteric epithelial cells in the Nestin-Cre/YC3.60 and the CAG-Cre/YC3.60 lines, respectively. Furthermore, the potential TRPM7 inhibitors were shown to attenuate mechanical stress-mediated Ca²⁺ signaling. These data indicate that the present intravital imaging system can be used to visualize mechanosensory Ca²⁺ signaling in ENS cells and enteric epithelial cells.