Fig. 1: NMDA and ATP application triggers a rapid and long-lasting nanoscale reorganization of AMPAR at synapses associated to a long-term synaptic current depression.

A Example of super-resolution intensity images of a piece of dendrite obtained using dSTORM technique on live stained neurons for endogenous GluA2 containing AMPARs at basal state (t0) or 30 min (t30) following NMDA application (30 µM, 3 min). Enlarged synapses are shown on the right. B Cumulative distribution of nanodomain AMPAR content (n = 275, 159 and 152 for t0, t10 and t30 respectively), and in the inset, the mean per cell. The number of AMPARs per nanodomain was estimated 0, 10 and 30 min following NMDA treatment as explained in Nair et al. 2013 (mean ± SEM, n = 17, 14 and 14 respectively, one-way ANOVA, p < 0.0001 and Dunnett’s post-test found significant differences between t10 or t30 and t0, p < 0.0001). Nanodomain content is significantly decreased 10 and 30 min following NMDA treatment compared to non-treated cells. C Diameter of AMPAR synaptic nanodomains. Nanodomain sizes were measured by anisotropic Gaussian fitting of pre-segmented clusters obtained on dSTORM images. Nanodomain diameter (mean ± SEM) 0, 10 and 30 min following NMDA treatment are plotted (n = 191, 127 and 100 respectively, one-way ANOVA, p = 0.2487). Nanodomain size is not affected by NMDA application. D Left panel: example of miniature EPSC traces recorded on cultured neurons in basal condition (dark trace) or 30 min after NMDA treatment (blue trace). Right panel: Superposition of a mean trace of AMPAR mEPSC in basal (dark) and 30 min post-NMDA treatment (blue). E and F Average of the mESPC amplitude recorded on neurons 0, 10 or 30 min (E) and 180 min (F) after NMDA treatment. Miniature EPSC amplitudes are significantly depressed 10 and 30 min after NMDA treatment (E, n = 13, 13 and 10 respectively, one-way ANOVA p < 0.0001 and Dunnett’s post-test found significant differences p < 0.0001 and p = 0.0003 between t0 and t10, and t0 and t30 respectively), and this depression stays for at least 3 h (F, n = 12 and 11 respectively, t-test p = 0.0003). G Example of super-resolution intensity images of a piece of dendrite obtained using dSTORM technique on neurons live stained for endogenous GluA2 containing AMPARs at basal state (t0) or 30 min (t30) following ATP (100 µM, 1 min). Enlarged synapses are shown on the right. H Cumulative distribution of nanodomain AMPAR content (n = 158, 120 and 115 for t0, t10 and t30 respectively), and in the inset, the mean per cell. The number of AMPARs per nanodomains was estimated 0, 10 and 30 min following ATP treatment (mean ± SEM, n = 7, 6 and 7 respectively, one-way ANOVA, p = 0.0006 and Dunnett’s post-test found significant differences between t10 or t30 and t0, p = 0.0004 and p = 0.0063 respectively). Nanodomain content is decreased 10 and 30 min following ATP treatment compared to non-treated cells. I Measure of nanodomain diameter is not affected 0, 10 and 30 min following ATP treatment (n = 130, 112 and 91 respectively, one-way ANOVA, p = 0.6391). J Left panel: example of miniature EPSC traces recorded on cultured neurons in basal condition (dark trace) or 30 min after ATP treatment (red trace). Right panel: Superposition of a mean trace of AMPAR mEPSC in basal (dark) and 30 min post-ATP treatment (red). K and L Average of the mESPC amplitudes recorded on neurons 0, 10 or 30 min (K) and 180 min (L) after ATP treatment (100 µM, 1 min). Synaptic transmission (mEPSCs) is significantly depressed 10 and 30 min after ATP treatment (mean ± SEM, K, n = 15, 14 and 14 respectively, one-way ANOVA p = 0.0124 and Dunnett’s post-test found significant differences p = 0.0214 and p = 0.0172 between t0 and t10, and t0 and t30 respectively), and this depression stays for at least 3 h (L, n = 12 and 10 respectively, t-test p = 0.0485). Scale bars (A and G) left images = 2 µm, zoom on synapses (left panels) = 500 nm.