Supplementary Figure 8: Modular organization of spine synapses in brain sections not subjected to CUBIC.

Neurons in mouse cortex were transduced with EGFP at P7 to label spines and dendritic morphology as described in Fig. 4. At P28-35, brain sections were fixed and labeled with antibodies for PSD-95 and vGlut1 (Online Methods). Spines were analyzed for PSD-95 (green) and vGlut1 (red) modules by imaging in 3D STED mode on a Leica SP8 3X instrument. (a) High-contrast images and corresponding 3D reconstruction of one-module (top panels) and two-module (bottom panels) spines. Scale bar, 0.75 µm. Spines in 3D-rendered images are shown as transparent in order to enable the visualization of PSD-95 puncta (green). Scale bars, 0.3 µm (top panel), 0.2 µm (bottom panel). Similar results were obtained using brain sections from three different EGFP-injected animals. (b and e) Quantification of the percentage of spines (n = 3 independent injection experiments/animals) containing single and multiple (b) PSD-95 and (e) vGlut1 modules (n = 156 spines). (c and f) Quantification of the average areas of individual (c) PSD-95 (n = 255 clusters, n = 87 (single), n = 90 (double), n = 54 (triple), n = 24 (quadruple), p = 0.1075, F (3, 251) = 2.050, one-way ANOVA) and (f) vGlut1 (n = 258 clusters, n = 85 (single), n = 90 (double), n = 63 (triple), n = 20 (quadruple), p = 0.0562, F (3, 254) = 2.55, one-way ANOVA) nanomodules. (d and g) Quantification of the total area of (d) PSD-95 (n = 87 (single), n= 45 (double), n = 18 (triple), n = 6 (quadruple), ***p < 0.0001, *p = 0.0414, F (3, 152) = 83.58, one-way ANOVA), and (g) vGlut1 (n = 85 (single), n = 45 (double), n = 21 (triple), n = 5 (quadruple), ***p < 0.0001, **P = 0.0002, *p = 0.0034, F (3, 152) = 53.01, one-way ANOVA with Fisher’s LSD post hoc). (h and i) Plots of the relationship between cluster number and spine size. A positive correlation between (h) PSD-95 (green line, Pearson’s R² = 0.5275, p < 0.0001, n = 156 spines) and (i) vGlut1 (red line, Pearson’s R² = 0.4957, p < 0.0001, n = 156 spines) cluster number and spine size was observed. (j) A plot of the relationship between PSD-95 and vGlut1 nanomodules (Pearson’s R² = 0.6224, p < 0.0001, n = 156 spines). Bar graphs show mean +/− SEM, with numbers of individual spines or clusters represented by dots.