Fig. 1.

Induction of structural and functional AZ maturation by NL1 and BDNF. A, B Representative images and quantification of the number of bassoon puncta per 10 μm dendrite on DIV5 cultured hippocampal neurons transfected with membrane-targeted EGFP (farnesylated EGFP, EGFP-F) or GFP-tagged NL1, with and without prior LatA treatment and immunostained for bassoon (magenta). For separation of the three fluorescent channels, see Additional File 1: Fig. S1 A. Virtually all bassoon puncta formed on NL1-expressing cells are LatA resistant. 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)=67.49; ***p< 0.0001 for GFP control vs. GFP + LatA, ***p< 0.0001 for GFP control vs. NL1 control, p> 0.05 for NL1 control vs. NL1 + LatA. C, D Representative images and quantification of the number of bassoon puncta per 10 μm dendrite on cultured hippocampal neurons treated with BDNF or buffer (not treated, NT) on DIV4, with and without LatA treatment, and fixed on DIV5 and immunostained for bassoon (magenta). Mean + SEM; N=3 experiments, n=10 cells per condition and experiment; two-way ANOVA with post hoc Sidak tests: interaction is significant, ***p< 0.001, F(1, 116)=67.49; ***p< 0.0001 for NT control vs. NT + LatA, ***p< 0.0001 for GFP control vs. NL1 control, p> 0.05 for NL1 control vs. NL1 + LatA. E, F Overexpression of NL1 together with a BDNF treatment does not lead to an additive effect. Representative images and quantification of the number of bassoon puncta per 10 μm dendrite on DIV5 DIV5 cultured hippocampal neurons transfected with NL1, treated with BDNF or buffer, treated with LatA or DMSO, and immunostained for bassoon (red) and MAP2 (blue). For separation of the three fluorescent channels, see Additional File 1: Fig. S1 B. Mean + SEM; N=3 experiments, n=10 cells per experiment and condition; two-way ANOVA with post hoc Sidak tests: p> 0.05 for all comparisons. Scale bar is 10 μm