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. 2018 Nov 9;11:405. doi: 10.3389/fnmol.2018.00405

Figure 1.

Figure 1

Increase in synaptic expression of Shank2, Shank3 and VGluT1 during development. (A) Straightened dendrites of young (days in vitro 11, DIV 11) and mature (DIV 18) hippocampal neurons co-immunostained for Shank3 (A488, green), Shank2 (A568, red) and VGluT1 (A647, blue). White puncta in the merge images (bottom) indicate colocalization of all three proteins. Scale bar: 5 μm. (B–D) Three-dimensional analysis (IMFLAN3D) of Shank3 and Shank2 puncta at DIV 11 and 18 quantifying intensity (B), density (C) or volume (D) (mean ± SEM). (E–G) Three-dimensional colocalization analysis of pairwise puncta overlap as measured by the ratio (E), density (F) and volume (G) at DIV 11 and 18 [mean ± SEM; N = 16 dendrites from 10 to 12 neurons from two culture preparations (referred to as culture preps from now on; Mann-Whitney tests. n.s. p ≥ 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001)]. (H) Straightened dendrites of young and mature hippocampal neurons immunostained for Shank3 and Shank2. Scale bar: Image, 5 μm; Corresponding inset, 1 μm. (I) Summary graphs showing quantification of Shank3 and Shank2 relative enrichment in spines vs. in dendritic puncta (DIV 21, N = 18; all others, N = 16 dendrites from 10 to 12 neurons from two cultures preps; Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Dunn’s correction post hoc multiple comparisons. Comparing between Shank2 and Shank3 at the same age: *p < 0.05; ***p < 0.0005. Comparing Shank3 between different ages: ##p < 0.005).