Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice

•New transgenic mice co-express G-CaMP6 and mCherry in ENS neurons.•Intestinal movement stabilization by suction enables imaging of ENS activity in living mice.•Spontaneous and evoked activity can be imaged at cellular and subcellular resolutions. Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease.