Figure 2

Neurotoxicity induced by GT1b in neuron-enriched mesencephalic cultures. (A-D) TUNEL reaction showing DNA fragmentation in the absence (A) or presence (C) of 20 μg/ml GT1b for 24 h in neuron-enriched mesencephalic cultures. B and D, Hoechst 33258 counterstaining of A and C, respectively. Inset, highly magnified cell indicated by arrow. Scale bar, 50 μm. (E) TUNEL (+) cells were counted and expressed as a percentage of Hoechst 33258(+) total cells in control cultures (opened circles) or in cultures treated with 20 μg/ml GT1b (filled circles) at indicated time points. The values represent the mean ± SEM of triplicate cultures in three separate samples; *P < 0.001, significantly different from control cultures (ANOVA and Student-Newman-Keuls analyses). (F-I) Electron microscopic observations showed morphology of neuronal cell death in the absence (F and H) or presence (G and I) of 20 μg/ml GT1b for 24 h. To distinguish DA neurons from non-DA neurons, cultures were immunostained with TH antibody and then electron micrographs taken. Nucleus was well-preserved with fairly intact cell membrane in the control cultures of DA (F) or non-DA (H) neurons. Note that following GT1b, fragmented nuclei were clearly visible in DA (G) or non-DA (I) neurons. CM, cell membrane; N, nucleus; NM, nuclear membrane. Scale bar, 5 μm.