Vagal Nodose Calcium Imaging in Mice

In vivo vagal nodose imaging was performed in Neurod1Cre_Salsa6f (N = 4) mice where GCaMP6f is expressed in vagal neurons. Nodose imaging was performed as previously described^{18 (link)}. Mice were initially anesthetized with urethane (2 mg/g) and maintained with 0-0.5% isoflurane as needed throughout the procedure. The right nodose ganglion was partially excised and placed on a metal platform, stabilized with silicone elastomer (Kwik-Sil), and covered with a 5mm coverslip. A 20-gauge gavage needle attached to a gravity perfusion system was surgically inserted through the stomach wall into the duodenum. A perfusion exit incision was made at the ligament of Treitz. PBS was constantly perfused at <1ml/min for the duration of the recording as a within subject baseline and volume pressure control. Calcium transients were imaged using a multiphoton microscope (Bruker Ultima IV) using a 16X objective. Laser wavelength was set to 920nm, and frames were captured at a rate of 683-ms per frame. Baseline activity was imaged while perfusing PBS. Sucrose [300 mM] and sucralose [15 mM] stimuli were perfused back-to-back and in reverse order to determine whether the same cell or different cells responded to both sugars. In addition, mannitol [300 mM] was used as an osmolarity control. Stimuli were delivered for 60 seconds with 2 minutes of baseline before and after application.