Figure 2.

S478 phosphorylation of TrkB is required for BDNF-induced spine morphogenesis and glutamate-induced spine enlargement. (a) Hippocampal neurons were co-transfected with GFP and vector (mock), wild-type TrkB (WT), or TrkB S478A construct. Neurons were treated with BDNF for 24 h. Representative images are shown. BDNF increased the spine density (middle; ***P < 0.001, Student’s t test) and spine area (bottom; ***P < 0.001, Kolmogorov-Smirnov test) in mock or wild-type TrkB–expressing neurons, but not in neurons that overexpressed the TrkB S478A mutant. We measured 36 dendrites from 12 neurons for each condition. Scale bars represent 10 μm. (b) GFP-transfected hippocampal neurons derived from control (Trkb^WT/WT) or Trkb^S478A/S478A mice were treated with BDNF for 24 h. Representative images are shown. Treatment with BDNF significantly increased the spine density of the control neurons, but not that of neurons lacking TrkB S478 phosphorylation (middle, 9–16 dendrites from 5–10 neurons were measured for each condition; ***P < 0.001, one-way ANOVA, Bonferroni’s multiple comparison test). BDNF also increased the spine area in control neurons, but not in Trkb^S478A/S478A neurons (bottom; **P < 0.01, Kolmogorov-Smirnov test). Scale bars represent 10 μm. (c) GFP-transfected hippocampal neurons (17–18 d in vitro (DIV)) cultured from Trkb^WT/WT or Trkb^S478A/S478A mutant mice were stimulated by uncaging MNI-glutamate, and the changes in spine size at different duration after glutamate receptor activation were monitored by time-lapse confocal microscopy. Representative images are shown (left). Quantification revealed a significant difference in the fold change in spine area between Trkb^WT/WT and Trkb^S478A/S478A neurons in response to uncaged glutamate (right; data were pooled from three experiments, 20 neurons were measured for each genotype; *P < 0.05, Student’s t-test). Scale bar represents 5 μm. Data are expressed as mean ± s.e.m. for the histograms in a–c.