As to how these heterogeneous neurons cooperate together to process sensory information. Monitoring the pattern of neuronal activity of a large ensemble of SDH neurons would provide data for a comprehensive view about SDH circuits. Since central terminals of primary sensory neurons are spatially organized according to their response profile and their peripheral projection field, analysis of the GDC-0879 global distribution pattern of SDH neuronal activity in response to sensory stimulation would uncover how sensory information of primary afferents propagates to the surrounding area by SDH circuits. In vivo calcium imaging is a promising technique to monitor activities of many neurons in a single animal, as it overcomes technical limitations of electrophysiological studies. Several investigators have reported in vivo calcium imaging of SDH neurons. Helmchen’s group and Cote’s group recently devised the way to minimize motion artifacts of the SDH during calcium imaging by mechanical stabilization, ratiometric imaging, and the movement compensation device, allowing stable measurement of neuronal activity. However these studies did not focus on the distribution of the recorded neurons. In the present study, we performed in vivo calcium imaging of SDH neurons by using a two-photon microscope to analyze the global distribution pattern of SDH neuronal activity in response to sensory stimulation. For introduction of calcium indicator proteins, we took advantage of in utero electroporation, which enables stable expression of calcium indicators in the SDH along a wide area across the rostrocaudal axis. Moreover, the usage of a fluorescence resonance energy transfer -based ratiometric calcium indicator protein drastically decreases motion artifacts during calcium recordings. Based on these technological backgrounds, we succeeded in monitoring the activities of multiple SDH neurons at a single cell resolution across a wide region localized 1.4 mm along the rostrocaudal axis and 150 mm in depth. Moreover, we determined the three-dimensional localization of the recorded neurons and analyzed its relationship with their response profile. Since previous electrophysiological studies have examined sensory stimulation-evoked neuronal activity of very few SDH neurons in a single animal, neuronal activity pattern of ensemble of SDH neurons remains unclear. By imaging a large population of SDH neurons in vivo, we for the first time clarified the threedimensional neuronal activity map of SDH neurons in response to cutaneous sensory stimulation.