Fig. 10

Fig. 10. α-Synuclein enhances H₂O₂-induced glutathione oxidation in dopaminergic neurons in vivo. A: Intravital confocal microscopy was used to image the Grx1-roGFP2 biosensor in the ventral diencephalon of live Ctrl; Grx1-roGFP2 (left six images) and α-Syn; Grx1-roGFP2 (right six images) zebrafish. Emission was captured at 515 nm following serial excitation at 405 nm (left column of each set) and 488 nm (center column of each set). The ratiometric image (right column of each set; color scale shown to the right), calculated by dividing each image plane of the 405 nm dataset by the corresponding 488 nm image plane, shows relative glutathione redox potential. The top row of images shows baseline steady-state images; the bottom row shows images of the same zebrafish after application of 3 mM H₂O₂ to the bath. B: Ratiometric histograms showing the frequency distribution of the Grx1-roGFP2 405/488 ratio from every pixel within areas corresponding to DC4 – 6 dopaminergic neurons in 2D images, in Ctrl (top panel) and α-Syn zebrafish (bottom panel), at baseline (gray) and after H₂O₂ exposure (colored). The arrowheads show the mean of each distribution. C: Scatterplot showing Grx1-roGFP2 405/488 ratio calculated using the 3D method, in DC4 – 6 dopaminergic neurons, at baseline and following exposure to H₂O₂. Data points represent individual neurons (Ctrl n = 33; α-Syn n = 51 neurons, combined from 4 replicate zebrafish in each group); bars show mean ± SE; p < 0.01**, 0.0001****, 2-way ANOVA with Tukey multiple comparison test. D: Scatterplot showing fold increase in Grx1-roGFP2 405/488 ratio between baseline and post-H₂O₂ peak for each individual cell from panel C. Bars show mean ± SE; p < 10−10 ****, 2-tailed unpaired t-test. E: Scatterplot showing Grx1-roGFP2 405/488 ratio calculated using the 3D method, in non-dopaminergic neurons following exposure to H₂O₂. Data points represent individual neurons (Ctrl n = 35; α-Syn n = 49 neurons, combined from 4 replicate zebrafish in each group); bars show mean ± SE; p < 0.01**, 0.0001****, 2-way ANOVA with Tukey multiple comparisons test.