MINIMALLY INVASIVE, WIDE-FIELD TWO-PHOTON IMAGING OF THE BRAINSTEM AND NUCLEUS TRACTUS SOLITARII 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 its deep location below the cerebellum and proximity to vital brain regions, the specific mechanisms by which the NTS integrates and processes VNS or other multisensory inputs from peripheral organs for emotion regulation remain unclear. To address these questions, here we developed an in vivo two-photon imaging technique using a prism-based optical interface (Double-Prism based brainStem imaging under Cerebellar Architecture and Neural circuits, "D-PSCAN"). The D-PSCAN method enables the observation of neural population activity at single-cell resolution in the wide brainstem areas including the NTS while maintaining the cerebellum and surrounding neural connections. It distinguishes NTS activity from descending vagus neurons located ventrally. The recorded neural activity data were used to evaluate the physiological characteristics of NTS neurons, e.g., their responsiveness to VNS. We also applied this technique to study responses to cholecystokinin-8 (CCK), a gastrointestinal hormone that serves as a more physiological stimulus than VNS. Our results suggest the applicability of this method for detailed understanding of the physiological mechanism of the NTS and future research on emotional regulation through the vagus-NTS pathway.