Fig. 2.

Effect of p85 shRNAs and PI3K activity on GFP-cofilin recruitment into spines during cLTP. (A) Representative images of dendritic branches from CA1 hippocampal neurons expressing GFP-cofilin together with shp85α (L + S), shp85α (L), shp85β or an empty vector (EV, control). Some slices expressing the empty vector were treated with 100 µM APV (NMDAR antagonist). Images are shown for baseline and after cLTP induction (15–25 min). Scale bar: 1 μm. (B) Quantification of GFP fluorescence signal in spines relative to the adjacent dendritic shaft (spine/dendrite ratio) before, during (grey rectangle) and after cLTP induction, from neurons expressing shp85α (L + S) (blue symbols), shp85α (L) (red symbols), shp85β (green symbols) or the empty vector control (black symbols). Some slices expressing GFP-cofilin and the empty vector were treated with 100 µM APV 1 h prior and during the experiment (yellow symbols). Plot represents mean ± SEM. (C) Histogram plot of spine/dendrite ratios from baseline (left; -10–0 min) and after cLTP induction (right; 15–25 min, normalized to baseline values for each condition). Bars show mean ± SEM. n represents number of spines from 3–6 independent experiments in each condition. Statistical differences between conditions were evaluated through Kruskal-Wallis and Mann-Whitney U tests. ****p < 0.0001, ns: not significant. (D) Similar to A, with slices treated with LY294002 (PI3K inhibitor) or the vehicle control (DMSO). (E) Similar to B, with slices treated with 10 µM LY294002 and expressing shp85α (L + S) (pink symbols), shp85α (L) (orange symbols), shp85β (magenta symbols) or the empty vector control (grey symbols). Slices treated with the vehicle control (DMSO) and expressing the empty vector are represented with black symbols. Treatments were for 1 h prior imaging and during the imaging session. Plots represent mean ± SEM. (F) Similar to C, from the values represented in E. Values from neurons expressing shp85α (L + S) (blue symbols), shp85α (L) (red symbols), and shp85β (green symbols) without LY294002 incubation are replotted from panel C, for comparison. Bars show mean ± SEM. n represents number of spines from 4–5 independent experiments in each condition. Statistical differences between conditions were evaluated through Kruskal-Wallis and Mann-Whitney U tests. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns: not significant