Visualization of mechanical stress-mediated Ca 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 Ca biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca) 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 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 Ca signaling during peristalsis without stimulation. Nerve endings on the surface of enteric epithelia also exhibited Ca signaling without stimulation. Mechanical stress induced transient salient Ca 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 Ca signaling. These data indicate that the present intravital imaging system can be used to visualize mechanosensory Ca signaling in ENS cells and enteric epithelial cells.