Ultima 4

A two-photon scanning laser microscope (Ultima IV; Bruker) was utilized to image CX3CR1-GFP transgenic mice expressing GFP in microglia. The components of the microscope included a scan head, OPO laser, non-descanned photomultiplier tubes (Insight DS+; Spectra-Physics), and 16X, 0.8 numerical aperture water immersion objective lens (Nikon). Mice were injected i.p. with sulforhodamine 101 (SR101) as a vascular contrast agent. The microscope laser was tuned to 920 nm to excite both GFP and SR101, and the resulting fluorescence was captured in green and red channels, respectively. ZT-stack images were taken every minute for the first 80 min following implantation for acute mice. In addition, Z-stack images were taken at 2, 3, 4, 5, and 6 h post-implantation for acute mice and 6, 12, 24, 48, and 72 h post-insertion for sub-chronic mice. Mice were stereotaxically secured while anesthetized with 1%–1.5% isoflurane during all imaging sessions. Stacks covered a span of 412.8 by 412.8 μm (1024 by 1024 pixels) in the horizontal plane with depths of around 300 μm. Images were taken above the top shank and/or below the bottom shank depending on variable visibility due to blood vessels or pial surface bleeding. Only microglia outside the outer shanks and in plane with the probes were included for analysis.

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.