Figure 7. Released enteric alpha-synuclein can be up-taken and retrogradely transported by sympathetic neurons.
Scale bars are 20 μm in all images. (A) Fluorescence microscope images of co-cultured ENS cells expressing mCherry-alpha-synuclein (red) and sympathetic neurons stained with a rabbit anti-TH antibody (green) (n = 3). Sympathetic neurons (arrows in the upper left image in A are in contact with mCherry-alpha-synuclein enteric neurons (*). Upper right image in panel A shows another image of the sympathetic neuron marked with an arrow in the upper left image highlighting the somal accumulation of mCherry-alpha-synuclein (arrow). Lower left image in panel A shows a zoomed in region of TH+ neurites in contact with mCherry-alpha-synuclein expressing enteric neurons framed in the upper left image . mCherry-alpha-synuclein can be observed inside sympathetic neurites (arrowheads). (B) Schematics of the Campenot chamber with sympathetic neurons in green and enteric neurons in red. (C) Montages of microscopy low magnification images from Campenot chambers corresponding to the region underneath the divider and the proximal neurite compartment. Sympathetic neurons were stained with rabbit anti-TH antibody (green) and enteric neurons express cherry-alpha-synuclein (red). Each row of images represents different channels (from top to bottom: cherry-alpha-synuclein, TH, bright-field (BF)) and a merge. Sympathetic TH+ neurons can be observed under the divider and forming a dense network around cherry-alpha-synuclein expressing enteric neurons. (D) Zoomed in region of the sympathetic neurites framed in (C). Cherry-alpha-synuclein can be observed along sympathetic neurites under the divider. (E) Confocal images showing cherry-alpha-synuclein (red) accumulating inside sympathetic TH+ neurons in grey (BF) and green in F.