Effects of 25-OH Chol on ChAT expression in 2-week nBM brain slices (assay 2). Slices were incubated for 2 weeks with 10 ng/ml NGF, then for 3 days without NGF and then with (NGF, filled circles) or without (−) NGF, or 0.02, 0.2, 2 μg/ml 25-OH Chol (filled triangles). After 4 days brain slices were fixed and stained for ChAT. The number of ChAT-positive neurons was counted in whole slices. Statistical analysis was performed by one-way ANOVA with a Fisher PLSD post hoc test. ***p < 0.001; **p < 0.01; NS = not significant.