Supplementary Figure 4: Modular organization of dendritic spine synapses in vitro—analysis of non-deconvolved images.
From: Synaptic nanomodules underlie the organization and plasticity of spine synapses
(a) The images from Fig. 1a are shown without deconvolution. PSD-95 (green) and vGlut1 (red) modules were imaged in STED (~80–100 nm FWHM) and tdTomato-labeled dendritic spines were imaged simultaneously in confocal mode (~300 nm FWHM, gray and dashed yellow outlines). Scale bar, 0.8 µm. Schematic (left panels) demonstrating the arrangement of multiple synaptic profiles found in individual spines for the images shown on the right. Similar results were obtained from three independent experiments (See also Fig. 1). (b) Positive correlation of the non-deconvolved PSD-95 clusters with spine size (green line, R2 = 0.4582, slope = 1.940 ± 0.1758, p < 0.0001, F-test, n = 146 spines). Dotted black line represents the deconvolved data from Fig. 1i (deconvolved vs. non-deconvolved, p = 0.9615, ANCOVA). (c) Positive correlation of the non-deconvolved vGlut1 clusters with spine size (red line, R2 = 0.2534, slope = 1.373 ± 0.1964, p < 0.0001, F-test, n = 146 spines). Dotted black line represents the deconvolved data from Fig. 1j (deconvolved vs. non-deconvolved, p = 0.8424, ANCOVA). (d and g) Quantification of the percentage of spines containing single and multiple (d) PSD-95 and (g) vGlut1 clusters (n = 146 spines). (e and h) Quantification of the average areas of individual (e) PSD-95 (233 clusters, n = 85 (single), n = 78 (double), n = 54 (triple), n = 16 (quadruple), p = 0.1533, F(3, 230) = 1.703, one-way ANOVA) and (h) vGlut1 (225 clusters, n = 84 (single), n = 94 (double), n = 39 (triple), n = 8 (quadruple), p = 0.2761, F (3, 221) = 1.031, one-way ANOVA). (f and i) Quantification of the total area of (f) PSD-95 (n = 85 (single), n = 39 (double), n = 18 (triple), n = 4 (quadruple) spines, ***p < 0.0001 all comparisons, F (3, 142) = 55.82), and (i) vGlut1 (n = 84 (single), n = 47 (double), n = 13 (triple), n = 2 (quadruple) spines, 1 vs. 2, 1 vs. 3, ***p < 0.0001; 2 vs. 3, *p = 0.0192, F (3, 142) = 25.22, one-way ANOVA with Fisher’s LSD post hoc). Graphs (d-i) represent mean +/− SEM.
