Fig. 2: ARC POMC neurons innervate the liver via DMV cholinergic neurons.

a, b Schematic diagram of the experimental configuration. AAV8-WGA-Cre viruses were injected into the liver of floxed-stop Rosa26-GFP mice (a). b Images of confocal fluorescence microscopy showing co-expression of GFP (left panel) and ChAT (middle panel) in a subset of DMV cholinergic neurons (right panel, white arrows). GFP-positive cells in the DMV represent retrogradely identified neurons. Bottom panel: higher magnification view of neurons co-expressing GFP (left) and ChAT (middle) (right panel, white arrowhead). Scale bar: 30 µm. CC central canal. c, d Schematic illustration of our experimental configuration (c). d Images of confocal fluorescence microscopy showing Cre-mediated GFP expression in the ARC. A subset of GFP-positive cells (left panel) co-expressed POMC (red, middle panel) (right panel, white arrows). Bottom panel: higher magnification view of neurons co-expressing GFP and POMC (right panel, white arrow; green, GFP; red, POMC; blue, DAPI). Scale bar: 30 µm. e, f Schematic diagram of our experimental configuration. AAV8-WGA-Cre viruses were injected into the liver prior to injection of AAV5-nPE-DIO-ChR2-eYFP viruses into the ARC of C57BL/6J mice (e). f Images of confocal fluorescence microscopy showing that a subset of POMC (middle panel) neurons expressed ChR2 (right panel, white arrows). Green, YFP staining. Scale bar: 20 μm. g, h Images of confocal fluorescence microscopy showing double immunostaining with anti-YFP (green) and anti-ChAT (red) antibodies. DMV ChAT neurons received YFP-positive axon terminals from the ARC (right panel), indicating that ARC POMC neurons innervate liver through DMV cholinergic neurons. Bottom panel: higher magnification view of DMV cholinergic neurons (red) receiving YFP-positive fibers (green) and terminals. Scale bar: 25 µm.