Astrocyte BDNF is not the mediator triggering neurodegeneration. (A) Double immunofluorescence for p75NTR or TrkB and β-tubulin in rat primary neurons. Bars, 50 µm. (B and C) Assay performed as in Fig. 5, but BDNF-neutralizing or isotype antibody was added to astrocyte cultures (B) or to neuronal cultures exposed to BDNF (C). In B, stimuli (10 ng/ml IL1 or 25 ng/ml BDNF) or vehicle was given to astrocytes in the presence of BDNF blocking antibody or isotype control for 4 h and then removed. After 24 h, the ACM was collected and added to cultured neurons. In C, astrocytes were treated with stimuli for 4 h. After 24 h, ACM was given to cultured neurons together with BDNF blocking antibody or isotype control. Graphs show quantification of β-tubulin MFI and the percentage of apoptotic cells. All data were obtained in at least two independent experiments and are represented as mean ± SEM. If not indicated, statistics refer to comparisons with relative sNT (nontreated). ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.