Fig. 4

Fig. 4. Dynamic measurement of cytoplasmic H2O2 flux in dopaminergic neurons in vivo. A: Intravital confocal microscopy was used to image the roGFP2-Orp1 biosensor in the ventral diencephalon of a live α-Syn; roGFP2-Orp1 zebrafish. Emission was captured at 515 nm following serial excitation at 405 nm (left column) and 488 nm (center column). The ratiometric image (right column; 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 integrated H₂O₂ flux. The positions of dopaminergic DC4 – 6 and adjacent non-dopaminergic neuronal groups are shown. The top row of images shows baseline steady-state images after equilibration in the imaging apparatus; the bottom row shows images of the same zebrafish after application of 3 mM H₂O₂ to the bath. B: Unbiased 3D spot detection was used to define regions corresponding to cells within the DC4 – 6 cluster. Within each cell, mean emission following excitation at 405 nm or 488 nm (bottom graph) and the 405/488 ratio (top graph) was calculated in serial images acquired every 4 min for 1 h. Each datapoint shows mean ± SE (n = 9 neurons). After stable baseline data were collected, HH₂O₂ was added to the bath at t = 18 min (shaded area). C: Ratiometric histograms showing the frequency distribution of the roGFP2-Orp1 405/488 ratio from every pixel within areas corresponding to DC4 – 6 dopaminergic neurons in 2D images, at baseline (top panel) and after addition of H₂O₂ to the bath (bottom panel). The arrowhead shows the mean of each distribution. D: Scatterplots showing baseline and peak roGFP2-Orp1 405/488 ratio, calculated using the 3D method, in DC4 – 6 dopaminergic neurons following exposure to H₂O₂. Data points represent individual neurons (Ctrl n = 29; α-Syn n = 45 neurons, combined from 4 replicate zebrafish); bars show mean ± SE; p < 0.0001****, 2-way ANOVA with Tukey multiple comparisons test. E: Scatterplot showing fold increase in roGFP2-Orp1 405/488 ratio between baseline and post-H₂O₂ peak for each individual cell from panel D. Bars show mean ± SE; p = 0.12, 2-tailed unpaired t-test. F: Baseline and peak roGFP2-Orp1 405/488 ratio in non-dopaminergic diencephalic neurons following exposure to H₂O₂. Data points, bars, and analysis identical to panel D (Ctrl n = 34; α-Syn n = 38 neurons). G: Fold increase in roGFP2-Orp1 405/488 ratio between baseline and post-H₂O₂ is shown for each individual cell from panel F. Bars show mean ± SE; p = 0.54, 2-tailed unpaired t-test.