Protocol for wide-field brainstem imaging largely preserving the overlying cerebellum

The nucleus tractus solitarii (NTS) is a brainstem structure that receives information from various internal organs, relaying it to brain networks that regulate emotions and metabolism. Here, we describe a step-by-step experimental protocol for performing minimally invasive in vivo two-photon imaging to record neural population activity in the NTS of anesthetized mice at cellular resolution. This approach is useful for elucidating the neural mechanisms underlying the heterogeneity of NTS neurons. For complete details on the use and execution of this protocol, please refer to Agetsuma et al.¹.