Fig. 4

Anterograde labeling of caudal DMN efferents can be tracked directly to the spleen. A Schematic drawing of the plasmids separately packaged into AAV9 and used to direct neuronal expression of tdTomato for tract tracing. B Representative photomicrographs showing tdTomato expression 30 days after virus injection in the mouse caudal DMN. The continuity of tdTomato signal could be observed in the vagus efferents present at the C cervical and D mediastinal (between the lungs) regions. E No tdTomato signal (or RFP immunoreactivity) was detected in the celiac ganglia, the source of the splenic nerve. F tdTomato immunopositive (detected using anti-RFP) vagal efferents were seen in the duodenum (a site of known vagal innervation). G Montage image showing that tdTomato could be tracked from the peritoneum near the upper medial tip of the spleen, emerging into the parenchyma. Inset, High magnification (100 µm width). H Representative images showing nerves containing tdTomato- and CARTp-positive (and tdTomato- and ChAT-positive) fibers along the boundary of the white pulp of spleen. I When viral injection was targeted to the rostral DMN, tdTomato could be detected in the rostral DMN, but not the caudal DMN or the spleen