The effect of BDNF on VGLUT1 and VGLUT2 protein levels is dependent on gene expression.

<p>(A–B) Hippocampal neurons were stimulated with BDNF (100 ng/ml) for the indicated periods of time, in the presence or in the absence of the transcription inhibitors α-amanitin (1.5 µM) or actinomycin-D (1.5 µM), and VGLUT1 (A) and VGLUT2 (B) protein levels were determined by western blot. (C) The variation of <i>Slc17a7</i> (VGLUT1) and <i>Slc17a6</i> (VGLUT2) mRNA levels was assayed by real-time PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053793#s2" target="_blank">methods</a> section. The neurons were stimulated with 100 ng/ml BDNF during 30 minutes (grey) or 3 hours (red). (A–C) Quantification of 4–5 experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons), and normalized to <i>18S</i> reference gene. Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001).