Fig. 6.
From: Neuroligin-1 mediates presynaptic maturation through brain-derived neurotrophic factor signaling
BDNF is required for endogenous and NL1-induced presynaptic maturation. Cultures of BDNFlox/lox hippocampal neurons with Cre-recombinase viral transduction (green) to reduce BDNF levels compared to non-transduced cultures. A, B Representative images and quantification of the number of bassoon puncta per 10 μm dendrite on on DIV15 neurons, with and without prior 18-h LatA treatment, immunostained for bassoon (magenta) to determine the number of AZs. 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)=30.82); ***p< 0.0001 for BDNF depleted vs. BDNF depleted + LatA; p> 0.05 for NT vs. NT + LatA. C Representative images of cultures of BDNFlox/lox hippocampal neurons with Cre-recombinase viral transduction (green) to reduce BDNF levels compared to non-transduced cultures, stimulated in the presence of antibodies directed against the lumenal domain of Syt1 to label recycling synaptic vesicles. After washing, the cells were fixed and immunolabeled with secondary antibodies against the Syt1 antibody (magenta). D Quantification of the fluorescence intensity of the Syt1 label. Mean + SEM; N=3 experiments, n=10 cells per condition and experiment; Student’s t test: ***p< 0.0001. E, F Representative images and quantification of the number of bassoon puncta per 10 μm dendrite on DIV6 BDNFlox/lox cultures and Cre-recombinase transduced cultures transfected with mOrange or NL1-mOrange (NL1) (red) and immunostained for bassoon (blue). For separation of the three fluorescent channels, see Additional File 3: Fig. S3 A. 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(3, 232)=98.00; ***p< 0.0001 for mOrange control vs. mOrange control + LatA, for mOrange control/control + LatA vs. mOrange + Cre and for NL1 + Cre vs. NL1 + Cre + LatA; p> 0.05 for mOrange + Cre vs. mOrange + Cre + LatA, for NL1 control vs. NL1 control + LatA, and for NL1 control/control + LatA vs. NL1 + Cre. G, H Representative images and quantification of cultured hippocampal neurons from BDNFlox/lox mice with or without transduction with Cre-recombinase (green) transfected with mOrange or NL1–IRES–mOrange (NL1) (red) on DIV4. On DIV6 cultures were stimulated in the presence of antibodies against the lumenal domain of Syt1 to label recycling synaptic vesicles. After washing, the cells were fixed and immunolabeled with secondary antibodies against the Syt1 antibody (blue). For separation of the three fluorescent channels, see Additional File 3: Fig. S3 B. 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)=29.33; ***p< 0.0001 for mOrange vs. mOrange + Cre and for NL1 vs. NL1 + Cre. Scale bar is 10 μm