Fig. 5.

BDNF signaling participates in NL1 action. A, B DIV5 cultured hippocampal neurons transfected with farnesylated EGFP (EGFP-F) or NL1-EGFP (NL1) (green) grown in the absence or presence of TrkB-Fc and immunostained for bassoon (magenta), to determine the number of AZs. Mean + SEM are shown; N = 3 experiments, n = 10 cells per condition and experiment; two-way ANOVA with post hoc Sidak tests: effects of TrkB-Fc treatment (***p< 0.0001, F(1, 116)=24.09) and construct used (***p< 0.0001, F(1, 116)=708.9) are significant; ***p< 0.0001 for GFP control vs. GFP + TrkB-Fc; p> 0.05 for NL1 control vs. NL1 + TrkB-Fc. C, D TrkB-Fc-treated cultures with and without LatA treatment immunostained for bassoon, to determine the number of LatA-resistant AZs. Mean + SEM are shown; N = 3 experiments, n = 10 cells per condition and experiment; two-way ANOVA with post hoc Sidak tests: interaction is significant (***p< 0.0001, F(1, 116)=70,19); ***p< 0.0001 for NL1 + TrkB-Fc vs. NL1 + TrkB-Fc + LatA; p> 0.05 for GFP + TrkB-Fc vs. GFP + TrkB-Fc + LatA. E, F BDNF signaling is required for NL1-mediated functional presynaptic maturation. Representative images and quantification of cultured hippocampal neurons transfected on DIV2 with EGFP-F or NL1-EGFP (NL1) (green), grown in the absence or presence of TrkB-Fc, and stimulated in the presence of antibodies directed against the lumenal domain of synaptotagmin-1 (Syt1) to label recycling synaptic vesicles. After washing, the cells were fixed and immunolabeled with secondary antibodies against the Syt1 antibody (red) and with MAP2 antibodies (blue). For separation of the three fluorescent channels, see Additional File 2: Fig. S2. Mean ± SEM; N = 3 experiments, n = 10 cells per condition and experiment; two-way ANOVA with post hoc Sidak tests: interaction is significant (***p< 0.0001, F(1, 116)=66.60); ***p< 0.0001 for GFP control vs. GFP + TrkB-Fc and for NL1 control vs. NL1 + TrkB-Fc. Scale bar is 10 μm