Figure 6

Wnt-3a stimulates synaptic vesicle release in mature cultured hippocampal neurons. (A) Hippocampal neurons at 21 DIV were treated with control or Wnt-3a-conditioned media for 1 h and the efficiency of activity-dependent vesicle recycling was evaluated by measuring the uptake of the amphiphatic fluorescent dye FM1-43 in response to depolarization with 90 mM KCl. (B-D) Quantification of the number of FM1-43 per neurite length (B), the mean intensity of FM1-43 puncta (C) and the mean area of FM1-43 clusters (D). (E, F) After treatment with recombinant Wnt-3a or vehicle for 3 h, synaptic vesicles were loaded with FM1-43 by depolarization and the decrease in fluorescence intensity induced by 90 mM KCl was measured by confocal time lapse microscopy imaging. (E) Graph of the normalized FM1-43 fluorescence decrease in control and Wnt-3a treated neurons shows that the half-time (t_1/2) of fluorescence decay was higher in the control than in Wnt-3a-treated neurons, indicating that Wnt-3a increased the rate of FM1-43 unloading. Data represent at least 100 sites of vesicle release. (F) pseudocolored images of FM1-43 fluorescence of representative synaptic vesicle exocytosis in control and Wnt-3a treated neurons at different times. The pseudocolor scale is shown at the top right of images. Error bars indicate standard error of the mean. * P < 0.05; *** P < 0.001.