Synaptic nanomodules underlie the organization and plasticity of spine synapses

Abstract

Experience results in long-lasting changes in dendritic spine size, yet how the molecular architecture of the synapse responds to plasticity remains poorly understood. Here a combined approach of multicolor stimulated emission depletion microscopy (STED) and confocal imaging in rat and mouse demonstrates that structural plasticity is linked to the addition of unitary synaptic nanomodules to spines. Spine synapses in vivo and in vitro contain discrete and aligned subdiffraction modules of pre- and postsynaptic proteins whose number scales linearly with spine size. Live-cell time-lapse super-resolution imaging reveals that NMDA receptor-dependent increases in spine size are accompanied both by enhanced mobility of pre- and postsynaptic modules that remain aligned with each other and by a coordinated increase in the number of nanomodules. These findings suggest a simplified model for experience-dependent structural plasticity relying on an unexpectedly modular nanomolecular architecture of synaptic proteins.

Fig. 1. Modular organization of dendritic spine synapses in vitro

(a) Representative high-contrast images of PSD-95 (green) and vGlut1 (red) modules imaged with STED (~80 nm FWHM) in dendritic spines imaged simultaneously in confocal mode (~300 nm FWHM, gray and dashed yellow lines) in tdTomato-transfected DIV21 cortical neurons. Scale bar, 0.8 μm. Schematic (left panel) demonstrating the arrangement of multiple synaptic profiles in individual spines from images in the right panel. (b) Line profiles (white lines in a) of the intensity of PSD-95/vGlut1 labeling in spines from panel a indicate a high degree of apposition (~100 nm) of individual pre- and post-synaptic clusters. (c and f) Quantification of the percentage of spines containing single and multiple (c) PSD-95 (n = 217 spines) and (f) vGlut1 clusters (n = 212 spines, graphs represent mean +/− SEM, dots show percentage of spines from three independent experiments, 9 different neurons). (d and g) Quantification of the average areas of individual (d) PSD-95, and (g) vGlut1 clusters demonstrating no significant size differences between single and multi-cluster spines, one-way ANOVA). (e and h) Quantification of the total area of (e) PSD-95 and (h) vGlut1, one-way ANOVA with Fisher’s LSD post-hoc). (i and j) Plots of the relationship between cluster number and spine size. Positive correlation of (i) PSD-95 (green line, R² = 0.4324, slope = 1.972 ± 0.1559, p<0.0001, F-test, n = 212 spines) and (j) vGlut1 (red line, R² = 0.2795, slope = 1.524 ± 0.1689, p<0.0001, F-test, n = 212 spines) cluster number with areas of individual spines (only spines with both PSD-95 and vGlut1 clusters were included for analysis, dots represent cluster number values of individual spines). Monte Carlo simulation (black line) of the relationship between spine size (500 simulations per spine size, spines without PSD-95 and vGlut1 puncta were not included) and the number of (i) PSD-95 (simulated slope = 0.1783 ± 0.0355, ANCOVA) and (j) vGlut1 (simulated slope = 0.3276 ± 0.0256, ANCOVA) clusters. (k) A plot of the relationship between pre- and post-synaptic nanomodules (R² = 0.4383, slope = 0.6366 ± 0.0497). All experiments were repeated ≥ 3 times. Bar graphs show mean +/− SEM, with numbers of individual spines or clusters represented by dots.

Fig. 2. Synaptic vesicle proteins exhibit modular organization at synapses

(a) Representative high-contrast three-color STED images of PSD-95 nanomodules (green, CW STED, FWHM ~80 nm), vGlut1 (red) and Synaptophysin-1 (SYP-1, blue) nanomodules imaged using gated STED (FWHM ~ 50 nm) in EGFP-labeled dendritic spines of DIV21-25 neurons imaged simultaneously in confocal mode (FWHM ~250 nm, gray and dashed white lines). Scale bar, 1 μm. Schematic (left panel) demonstrating the arrangement of synaptic profiles in individual spines from the images in the right panel. Synaptic profiles were determined as an apposition of co-localized vGlut1 and SYP-1 with PSD-95 (white arrows). (b) Quantification of the percentage of spines containing single and multiple PSD-95 (n = 406 spines), vGlut1 (n = 406 spines) and SYP-1 nanomodules (n = 189 spines). Data points indicate replicates from ≥ 3 independent transfection experiments. (c) Quantification of the average areas of individual PSD-95 (n= 648 clusters), vGlut1 (n= 728 clusters) and SYP-1 nanomodules (n= 310 clusters, one-way ANOVA, Tukey’s post hoc). (d) Quantification of the total area of PSD-95, vGlut1 and SYP-1 spines; vGlut1; SYP-1, one-way ANOVA, Tukey’s post hoc). (e) Cumulative probability plots for the data in c (Kruskal-Wallis test). (f-h) Correlation of spine size with (f) PSD-95 nanomodule number (green line, Pearson’s R² = 0.2992, slope = 1.957 ± 0.1490 (g) vGlut1 nanomodule number (red line, Pearson’s R² = 0.2755, slope = 2.212 ± 0.1785) and (h) SYP-1 nanomodule number (blue line, Pearson’s R² = 0.2761, slope = 1.837 ± 0.2131). Monte Carlo simulation (black line) of the relationship between spine size (spines without PSD-95, vGlut1 and SYP-1 puncta were not included) and the number of (f) PSD-95 nanomodules (simulated slope = 0.081 ± 0.02, ANCOVA,), (g) vGlut1 nanomodules (simulated slope = 0.474 ± 0.027, ANCOVA) and (h) SYP-1 nanomodules (simulated slope = 0.2423 ± 0.019, ANCOVA, SYP-1-containing simulated spines). (i-j) A plot of the relationship between PSD-95 nanomodules with (i) vGlut1 nanomodules (Pearson’s R² = 0.7945) and (j) SYP-1 nanomodules (Pearson’s R² = 0.8449). Graphs in b-d represent mean +/− SEM, dots show (b) percentage of spines, (c) individual nanomodules and (d) areas from at least three independent experiments, at least 11 different neurons.

Fig. 3. Synaptic vesicle and active zone markers colocalize at spines as synaptic nanomodules

(a) Representative high-contrast three-color STED images of PSD-95 nanomodules (green, CW STED, FWHM ~80 nm), vGlut1 (red) and Bassoon (blue) nanomodules imaged using gated STED (FWHM ~50 nm) in EGFP-labeled dendritic spines of DIV21-25 neurons imaged simultaneously in confocal mode (FWHM ~250 nm, gray and dashed white lines). Scale bar, 1 μm. Schematic (left panel) demonstrating the arrangement of synaptic profiles in individual spines from the images in the right panel. Synaptic profiles were determined as an apposition of colocalized vGlut1 and Bassoon with PSD-95 (white arrows). Similar results were obtained from three independent transfection experiments in spines from a total of 10 neurons. (b-d) Quantification of the percentage of spines containing single and multiple PSD-95 (n = 217 spines also shown as part of Fig. 2b), vGlut1 (n = 217 spines also shown as part of Fig. 2b) and Bassoon nanomodules (n = 217 spines). Data points represent percent spines with the indicated number of nanomodules in three independent transfection experiments. (e) Quantification of the average areas of individual Bassoon nanomodules (n= 379 clusters, one-way ANOVA, Tukey’s post hoc). (f) Quantification of the total Bassoon area at spines (one-way ANOVA, Tukey’s post hoc). Graphs in b-f represent mean +/− SEM. (g) Cumulative probability plots for the data in e (Kruskal-Wallis test). (h) Positive correlation of spine size with the number of Bassoon nanomodules (gray line, Pearson’s R² = 0.2524, slope = 2.065 ± 0.2430). Monte Carlo simulation (black line) of the relationship between spine size and the number of Bassoon nanomodules (spines without Bassoon puncta were not included, simulated slope = 0.2018 ± 0.018, ANCOVA). (i) A plot of the relationship between PSD-95 and Bassoon nanomodules (Pearson’s R² = 0.8549).

Fig. 4. Modular organization of dendritic spine synapses in vivo

(a) Schematic representation of the experiment. (b) 3D Imaris reconstruction (left panel) of the dendritic section, spines and corresponding synaptic modules of a layer 3 neuron shown on the right. PSD-95 and vGlut1 puncta not colocalized with spines were removed for clarity. Scale bar, 2 μm. A representative maximum intensity projection image (right panel) from a layer 3 neuron showing an EGFP-labeled apical dendrite (gray) imaged in confocal mode (~300 nm FWHM). Spines were analyzed for PSD-95 (green) and vGlut1 (red) modules by imaging in 3D STED mode (90 nm XY, 200 nm Z FWHM). Scale bar, 2 μm. (c) High-resolution image and corresponding 3D reconstruction of a one-module spine. (d) High-resolution image and corresponding 3D reconstruction of a three-module spine. Scale bar, 1 μm for images in c and d. Spines in 3D rendered images in c and d were made transparent in order to visualize PSD-95 puncta (green) inside these structures. Scale bars, 0.5 μm. Similar results were obtained from brain sections of three EGFP injected animals. (e and f) Quantification of the percentage of spines (n = 3 independent injection experiments) containing single and multiple (e) PSD-95 and (f) vGlut1 modules (one-way ANOVA, Fisher’s LSD post hoc, graphs represent mean +/− SEM, dots show percentage of spines from three independent experiments). (g and h) Quantification of the average areas of individual (g) PSD-95 (n = 171 clusters) and (h) vGlut1 (n = 167 clusters, one-way ANOVA) nanomodules. (i and j) Plots of the relationship between cluster number and spine size. A positive correlation of (i) PSD-95 (green line, Pearson’s R² = 0.4695) and (j) vGlut1 (red line, Pearson’s R² = 0.5680, n = 84 spines, dots represent cluster number values of individual spines) nanomodules numbers with areas of individual spines was observed. Monte Carlo simulation (black line) of the relationship between spine size (500 simulations per spine size, black lines, spines without puncta were removed) and the number of (i) PSD-95 (simulated slope = 0.2152 ± 0.0151, measured slope 2.385 ± 0.2766, ANCOVA) and (j) vGlut1 (8011 total simulations, simulated slope = 0.5695 ± 0.019, measured slope = 2.427 ± 0.2309,, ANCOVA) clusters. Simulated spines without PSD-95 or vGlut1 clusters were not included in the analysis. (k) A plot of the relationship between pre- and post-synaptic nanomodules (Pearson’s R² = 0.7265, n= number of apposed vGlut1/PSD-95 pairs per spine from 84 spines). Bar graphs show mean +/− SEM, with numbers of individual spines or clusters represented by dots.

Fig. 5. Structural plasticity associated with cLTP is linked to synaptic module number

(a-d) Representative three-hour time-lapse images (top panels, confocal resolution) and retrospective high-contrast STED images (bottom panels) of the same dendritic spines (white squares) of tdTomato-transfected DIV21-25 cortical neurons. Spines (gray and yellow dashed outlines) were imaged at confocal resolution with simultaneous STED imaging of endogenous PSD-95 (green, arrowheads) and vGlut1 (red, arrows). Scale bars corresponding to all images a-d: top panel, 2 μm, inset, 1 um; lower panel, 2 μm, inset, 0.75 μm. (e) Schematic of the experiment showing initial cLTP live-cell imaging of spine morphology followed by retrospective STED imaging of endogenous proteins at the same spines. (f) Quantification of percent change in spine head area during the three-hour live-cell imaging experiment after a three-minute treatment with glycine (200 μM). Potentiated spines were defined by a sustained increase in spine head area of >10% over baseline (green traces, n = 30 spines). Spines that did not increase in size were defined as non-responsive (red traces, n = 21 spines). Spine enlargement was blocked by treatment with the NMDAR blockers 50 μM APV and 10 μM MK-801 (gray traces, n = 34 spines). Control spines were not subjected to glycine treatment (black traces, n = 56 spines). Graph represents mean +/− SEM. (g and h) Quantification of the average number of (g) PSD-95 and (h) vGlut1 module, one-way ANOVA with Fisher’s LSD post hoc) in spines from the indicated conditions following retrospective STED imaging. (i and j) Distributions of spines with single and multiple modules in the indicated conditions, binned based on the number of PSD-95 and vGlut1 clusters they contained. (k and l) Quantification of the average area of individual PSD- and vGlut1 clusters per spine in the indicated conditions, one-way ANOVA, Fisher’s LSD. (m and n) Quantification of the average area of individual PSD-95 and vGlut1 modules in single, two and three-module containing potentiated spines (One-way ANOVA). All experiments were repeated ≥ 3 times. Bar graphs show mean +/− SEM, with the numbers of individual spines or clusters represented by dots.

Fig. 6. Rapid remodeling of aligned pre- and post-synaptic modules underlies cLTP structural plasticity

(a-d) Representative images of time-lapse dual-color live-cell STED of PSD-95-EGFP (green) and mTurquoise-2-Synaptophysin-1 (SYP-mTurq2, red). Gray shows cell morphology with cell-filling tdTomato in confocal mode. Green and red arrows indicate the appearance of new PSD-95-EGFP and SYP-mTurq2 modules, respectively. Lower ‘Track’ panels indicate the movement of PSD-95-EGFP (green dots and lines) and SYP-mTurq2 (red dots and lines) modules over the course of imaging (3 hours). Chemical LTP was induced by application of glycine (200 μM, 3 minutes, black arrow). Scale bar corresponding to all images a-d, 500 nm. (e and f) Quantification of the number of (e) PSD-95-EGFP and (f) SYP-mTurq2 nanomodules per spine over the course of 3 hours. Measurements were performed at each time point (one-way ANOVA with Fisher’s LSD post hoc, “#” = significant differences between all conditions, “*” = significant differences only between Potentiated and Non-responsive conditions). Graphs show mean +/− SEM at each time point). (g and h) Quantification of the total distance moved over three hours for (g) PSD-95-EGFP (Control, n = 23; Potentiated, n = 34, Non-responsive; n = 19; APV+MK-801, n = 20 modules) and (h) SYP-mTurq2 (Control, n = 21; Potentiated, n = 18; Non-responsive, n = 14; APV+MK-801, n = 23 modules; one-way ANOVA with Fisher’s LSD post hoc). (i) Schematic representation of the method used to determine the distance and alignment between PSD-95-EGFP and SYP-mTurq2 nanomodules (see Methods). (j) Quantification of the average distance between the centers of PSD-95-EGFP and SYP-mTurq2 (Control, n = 18; Potentiated, n = 13; Non-responsive, n = 14; APV+MK-801, n = 20 module pairs, one-way ANOVA). (k) Quantification of the relative alignment as described in panel i (Control, n = 18; Potentiated, n = 14; Non-responsive, n = 14; APV+MK-801, n = 20 aligned pairs; one-way ANOVA) for the indicated conditions. Treatment with the NMDAR blockers APV+MK-801 resulted in significantly better alignment between PSD-95-EGFP and SYP-mTurq2 (two-tailed Student’s t-test). All experiments were repeated ≥ 3 times. Bar graphs represent mean +/− SEM with numbers of individual pre- and post-synaptic clusters (g and h) and numbers of aligned pairs of clusters (j and k) indicated by dots.

Fig. 7. NMDAR-dependent plasticity is associated with fast modification of synaptic nano-architecture

(a-b) Representative images of time-lapse dual-color live-cell STED of DIV21-25 neurons transfected with PSD-95-EGFP (green) and mTurquoise-2-Synaptophysin-1 (SYP-mTurq2, red). Gray shows cell morphology visualized by cell-filling tdTomato in confocal mode. Lower ‘Track’ panels indicate the movement of PSD-95-EGFP (green dots and lines) and SYP-mTurq2 (red dots and lines) modules over the course of imaging (1 hour). Arrows indicate the appearance/disappearance of PSD-95-EGFP and SYP-mTurq2 modules during imaging. Chemical LTP was induced by application of glycine (200 μM, 3 minutes, black arrow) and images were acquired every 12.5 minutes for one hour. Scale bar, 1 μm. (c) Quantification of the number of PSD-95-EGFP per spine (Control, n = 14; Potentiated, n = 16; Non-responsive, n = 17 spines) over the course of 1 hour. Measurements were performed at each time point (one-way ANOVA with Fisher’s LSD post hoc). (d) Quantification of the number of SYP-mTurq2 modules per spine (one-way ANOVA with Fisher’s LSD post hoc, graphs in c and d show mean +/− SEM at each time point). (e) Schematic for testing the relationship between PSD-95-EGFP nanomodule increase and PSD-95-EGFP turnover immediately following cLTP. (f) Representative STED and time-lapse FRAP images of DIV21-25 control, enlarged and unenlarged spines in neurons transfected with cell-filling tdTomato and PSD-95-EGFP. Scale bars, STED and FRAP images, 1 μm. (g) Quantification of PSD-95-EGFP recovery under basal condition and following cLTP. PSD-95-EGFP recovery rapidly increases in enlarged spines following glycine treatment and is significantly higher than in control and unenlarged spines (*p = 0.0119, Kruskal-Wallis, Dunn’s post-hoc, Control, n = 21 spines; Potentiated, n = 12 spines; Non-responsive, n =12 spines). (h) Quantification of PSD-95-EGFP mobile fractions (calculated from the average of three time points) before glycine (-5 min) and following glycine treatment (one-way ANOVA, Fisher’s LSD post hoc, n = number of spines as designated in g). Graphs in h represent mean +/− SEM. All experiments were repeated ≥ 3 times