Fig. 3. Increased ROS derives from the mitochondrial electron transport chain. A, Effects of mitochondrial agents on ROS levels. Cells were either maintained in NGF-containing medium before treatment (+NGF) with the compounds or were deprived of NGF for 18 hr before treatment (−NGF). ROT, Rotenone (10 μm); ANT, antimycin A (1 μm). There were no significant differences between the CM-H2DCFDA intensities in the ANT-exposed, NGF-maintained cells of the three genotypes (p > 0.27). However, the intensities of all three were significantly higher than in NGF-maintained neurons not exposed to ANT (p < 0.001). There were no significant differences between the CM-H2DCFDA intensities in NGF-deprived, ANT-exposed cells of the three genotypes and in NGF-deprived bax+/+ cells not exposed to ANT (p > 0.14). NGF-deprived cells exposed to ROT had much lower CM-H2DCFDA intensities than NGF-deprived neurons not exposed to ROT (p< 0.001). Compounds were added to the culture medium during the period of CM-H2DCFDA loading. After loading, the dye was washed out, and cultures were bathed in L-15 medium containing the same treatments for confocal microscopy. n = 58–233 neurons. B, Paired phase contrast (left) and fluorescent pseudocolored micrographs (right) of cells stained with the GSH-sensitive dye MCB. Unlike NGF-deprived rat sympathetic neurons (bottom), NGF-deprived mouse neurons (bax+/+) did not exhibit increased MCB staining after NGF withdrawal. The bar indicates relative pseudocolor values for MCB intensity (red,white are most intense and indicate higher GSH concentrations). The top and bottom scale bars are for mouse and rat neurons, respectively. C, GSH levels in the three mouse genotypes (as indicated by MCB staining) were similar and changed little after NGF withdrawal (18 hr). GSH ethyl ester (GSH eth; 10 mm for 18 hr) is a membrane-permeant form of GSH that was used as a positive control for MCB detection of GSH. n = 81–181 neurons.D, ROS levels were linearly related to Bax concentrations (Bax densities on Western blots). Bax data are taken from Figure 1C, and ROS data are from Figure2B. R = 0.99 for each deprivation time.