Figure 2.

Effect of Shank3^WT and mutants on dendritic spine induction and maturation. (a) Hippocampal neurons were transiently co-transfected with green fluorescent protein (GFP)–Shank3^WT or mutated forms (green) and red fluorescent protein (red) and processed for immunofluorescence after 4 days (days in vitro 21, DIV21), and the subcellular localization of each construct was analyzed. Neurons are stained with anti-GFP (green) and anti-DsRED (red). Scale bars represent 20 μm. On the bottom side, magnification of 20 μm of the dendrite transfected with each construct. (b) Comparison of spine density in neurons transfected with the different Shank3 constructs (number of spines per 20 μm of dendrite length) in co-transfected neurons. (c) Comparison of the density of filopodia (number of filopodia per 20 μm of dendrite length) in co-transfected neurons. Shank3^WT leads to an increase in the number of spines and a decrease in the number of filopodia. All mutations reduce the ability of Shank3 to induce spines. (^***P<0.001 and ^**P<0.01 compared with the control; °°°P<0.001, °°P<0.01 and °P<0.05 compared with Shank3^WT). (d) Examples of spines from neurons transfected with GFP–C3 or GFP–Shank3^WT. Spine length (green line) and head width (red line) were computed using Volocity software. (e) Truncating mutation causes a decrease in the number of larger spines and an increase in the number of thin spines. Relative abundance of four different spine classes according to spine-head size (0–0.25; 0.25–0.5; 0.5–0.75; and >0.75 μm). (f) Percentage of spine heads >0.5 μm within the same population. Spine heads are significantly larger in GFP–Shank3^WT- and GFP–Shank3^Q321R-transfected neurons compared with the control (^*P<0.05). Spine heads are significantly smaller in GFP–Shank3^STOP. (g) Percentage of spines >1.0 μm within the same population. Spine length is significantly reduced with GFP–Shank3^Q321R and GFP–Shank3^STOP constructs compared with the control (^*P<0.05; n=17–28 neurons and 1500–1800 spines).