Transient or sustained TrkB activation and Erk signaling induced by acute or gradual BDNF stimulation.
(a) Protocols for application of BDNF in acute and gradual modes. Hippocampal neurons were cultured for 14 days. In the acute protocol, BDNF concentration in the culture increased immediately from 0 to 1 nM (25 ng/ml). Neuronal proteins were extracted at 0, 0.25, 0.5, 1, 2, 4, 8 hours after BDNF application. In the gradual protocol, BDNF concentration increased tenfold in every 30 min, starting from 10−4 nM (2.5 pg/ml) until it reached 1 nM (25 ng/ml; total 2 hours). Protein extraction started at 30 min after 10−2 nM (0.25 ng/ml) BDNF application (1.5 hr after the first BDNF application, indicated by arrow). When BDNF concentration reached 1 nM (25 ng/ml), protein extraction followed the same time course. (b) Transient and sustained activations of TrkB and Erk induced by acute and gradual stimulation with BDNF, respectively. Representative Western blots are shown on top of the quantitative plots. The pTrkB and pErk signals were measured by densitometry, normalized to total TrkB and Erk respectively, and data from 6 independent experiments (N = 6) were averaged and plotted. CON: vehicle control. Saline: 5 consecutive times of saline treatment with 30 min interval as a negative control. Data obtained from acute (blue line) and gradual (pink line) experiments in Fig. 1b, and were compared by paired t-test. In this and all other figures, data are presented as mean ± SEM, *: p < 0.05, **: p < 0.01. (c) Biotinylation assay of surface TrkB at the indicated time points after acute and gradual BDNF treatment. Data were compared to control by ANOVA.