Minimally invasive, wide-field two-photon imaging of the brainstem at cellular resolution

Brain-viscera communication is crucial for regulating mental health, with the vagus nerve being a key structure mediating this interaction. Clinically, artificial vagus nerve stimulation (VNS) is used to treat various neuropsychiatric disorders, highlighting the importance of vagal afferent fibers in emotion regulation. The nucleus tractus solitarii (NTS) is a brainstem structure proposed to receive signals from vagal afferents and relay them to brain networks for emotion regulation. However, due to the anatomical complexity and difficulty in accessing the deep-brain NTS region in vivo, its underlying mechanisms remain unclear. Here, we developed a wide-field and deep-brain two-photon imaging method using a double-prism optical interface. This approach enables cellular-resolution imaging to specifically detect NTS neural activity while largely preserving the overlying cerebellum, a region also implicated in emotion regulation. We evaluated NTS neuronal responses to VNS and a gastrointestinal hormone, demonstrating the method's utility for investigating the vagus-NTS pathway in vivo.