Figure 8.

Digitonin permeabilized the plasma membrane of SCG neurons with little effect on mitochondrial physiology. A, Treatment of SCG neurons in culture with digitonin (5 μg/ml) allowed large molecules to enter the cytoplasm. Top left, Confocal images of NGF-maintained neuronal somas exposed for 20 min to intracellular medium containing polydextran (70 kDa) labeled with the fluorescent dye rhodamine [100 μg/ml dextran/rhodamine (Rhod-Dex)]. No rhodamine labeling occurred in the six neuronal somas shown (●), indicating that the dextran/dye molecules could not cross the plasma membrane. Top right, Confocal images of eight NGF-maintained neuronal somas exposed for 20 min to intracellular media containing rhodamine-labeled polydextran (70 kDa; 100 μg/ml) and digitonin (5 μg/ml). Rhodamine labeling occurred in seven (+) of the eight somas, indicating that the dextran/dye combination had crossed the plasma membrane. Bottom, Time course of increase in somatic rhodamine/dextran (70 kDa; 100 μg/ml) intensity in NGF-maintained neurons exposed to digitonin (5 μg/ml) for 20 min. Data are normalized to the average cellular autofluorescence intensities measured in NGF-maintained cells exposed to intracellular media containing rhodamine/dextran but not receiving digitonin treatment. Note in the micrographs of the nonpermeabilized cells that the autofluorescence intensity was very weak. The average increased intensity reflects both an increase in the number of cells staining and an increase in individual cell intensity. Cells were excited by a 488 nm laser line, and emission was in the red channel of the confocal microscope. n = 15 neurons for each time point. B, Digitonin (Dig) treatment had little effect on Δψ_m. Top, Confocal micrograph of NGF-maintained neurons exposed to TMRM⁺ (10 nm for 20 min at 35°C) after digitonin permeabilization (5 μg/ml for 20 min in intracellular medium containing complex I substrates and ADP). Digitonin was washed out twice with the same intracellular medium not containing digitonin and TMRM⁺. Note punctuate mitochondrial staining. Bottom, Average TMRM⁺ intensities in NGF-replete cells exposed to either TMRM⁺ during digitonin loading (second bar) or TMRM⁺ after load as described for the micrograph (p > 0.01 for with NGF compared with plus NGF and digitonin; p < 0.01 for the postloaded cells compared with either of the other two conditions). TMRM⁺ dye intensities are normalized to the intensity averages of sibling wild-type cultures not receiving apoptotic stimuli (with NGF from time of plating; first bar). n = 193–395 neurons. C, Digitonin treatment did not cause cytochrome c (Cyto c) to be released from mitochondria. Top, Fluorescent micrograph of NGF-deprived (24 h) neurons demonstrating the criteria used to score cytochrome c localization by immunocytochemistry. Note the intense punctate staining in the top cell and the faint staining in the bottom one. The intensely stained neuron is representative of cells that were scored as having retained cytochrome c in mitochondria. The other neuron is representative of cells scored as having released cytochrome c into the cytoplasm in which it appears to be rapidly degraded (Deshmukh and Johnson, 1998; Kirkland and Franklin, 2001; Kirkland et al., 2002, 2007b). Bottom, Twenty minutes of digitonin (5 μg/ml) treatment before fixation did not alter the number of cells scored as having retained cytochrome c in their mitochondria. Digitonin did not affect cytochrome c retention in cells either maintained in or deprived of NGF (24 h; p > 0.01 for both). n = 3–4 cultures with ∼50 cells scored in each. ■ and ● identify conditions that were not statistically different (p > 0.01). All other conditions were different (p < 0.01).