Fig. 6.

Neurons treated with the combination of camptothecin and general caspase inhibitors die in a delayed manner in the absence of the classical nuclear manifestations of apoptosis. Rat E18 cortical neurons were plated in poly-d-lysine-coated 35 mm dishes and treated 2 d later with media alone (A) or 10 μm camptothecin alone (B) or together with 100 μmzVAD-FMK (C). Twenty hours later, the cells were fixed and stained with Hoechst dye 33342 (1 μg/ml; Sigma), and nuclei were visualized by fluorescence microscopy (magnification, 100×). Note the presence of multiple classical apoptotic profiles inB (one of which is highlighted by anarrowhead). An abnormally condensed nucleus is shown inC (arrow). Such nuclei were seen in similar numbers in control cultures. Some of the other nuclei inC are shrunken but do not demonstrate apoptotic morphology (an example of which is shown by thearrowhead). Scale bar, 10 μm. InD, the percentage of apoptotic nuclei for each condition was assessed and reported as mean ± SEM (three separate fields of 100 nuclei each). VAD, zVAD-FMK; Flavo, flavopiridol.