TrkB-dependent NO production by astrocytes in response to BDNF triggers neurodegeneration in vitro. (A) Human primary astrocytes loaded with NO dye DAF-FM were stimulated with 5 ng/ml BDNF or 10 ng/ml IL1 or left nontreated (NT). NO-positive cells (green) were counted. Representative fields are shown. (B) Quantification of experiments described in A. The number of NO-producing cells is expressed as percent of total DAPI⁺ cells. (C) Quantification as in B of NO production by nontreated or BDNF-treated astrocytes of TrkBflox/flox and GFAP-cre:TrkBflox/flox mice. (D) NO production in human astrocytes was evaluated as in A and B with the addition of the NO synthase inhibitor L-NAME, where indicated. (E) Analysis of total nitrite/nitrate concentration in human astrocyte supernatants after 24 h of the indicated treatment. (F–I) Assay was performed as in Fig. 5. L-NAME was added to astrocytes (F and G) or neurons (H and I). Representative stainings for β-tubulin, DAPI, and TUNEL in neuronal cultures (F and H) and quantification of β-tubulin MFI and apoptosis (G and I). Changes in β-tubulin MFI were calculated as the percentage of MFI in control cultures exposed to ACM from nontreated (sNT) astrocytes. A concentration of 25 ng/ml BDNF was used in F–I to induce maximal astrocyte activation. Data are shown as mean ± SEM of three (B, C, G, and I) or two (D and E) independent experiments. Bars, 50 µm. ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.