Labeling and Imaging Scheme.

a, Schematic of the sample preparation 59. 200 µm acute brain slices fromThy1-YFPH mice are live-stained with CAM2-Alexa Fluor 647 followed by fixation and immunostaining for PSD and cryosectioning (Materials and Methods) b. Schematic of the microscope setup. Imaging in refractive index matched medium allowed reducing light scattering. Additional aberrations were corrected using a deformable mirror and in-situ PSF retrieval (INSPR). Real-time stage drift correction allowed for coarse drift correction, which was corrected further using cross-correlation during image processing.

Nanoscale Organization of native surface AMPARs in mouse brain slices.

a, Diffraction limited image of a YFP neuron (yellow); AMPAR (cyan) labeled with CAM2-Alexa647 and PSD-95 (magenta) labeled with a nanobody conjugated to CF568. b, Super-resolution dSTORM reconstruction of AMPAR and PSD-95 from b. c, Zoomed in ROIs on individual YFP spines (white boxes) in a. d, Zoomed in ROIs on individual YFP spines (white boxes) in b. e, AMPAR and PSD-95 clusters from the zoomed in ROIs in d. f, AMPAR nanodomains (navy blue) within the clusters in e. g, Diffraction limited image of a YFP neuron (yellow); AMPAR (cyan) labeled with CAM2-Alexa647 and Homer-1 (magenta). h. Super-resolution dSTORM reconstruction of AMPAR and PSD-95 from g. i, Zoomed in ROIs of individual YFP spines (white boxes) from g. j. Zoomed in ROIs of individual YFP spines (white boxes) from h. k, Distance between AMPAR-PSD-95 clusters (n=499 cluster pairs from the cortex from three 6-month-old Thy1-YFP-H mice). The peak of the fitted Gaussian function is at 14 nm with standard deviation of 30 nm (R-square=0.95). l, Distance between AMPAR-Homer-1 clusters (n=158 cluster pairs from one 6-month-old Thy1-YFP-H mouse. The peak of the fitted Gaussian function is at 64 nm with a standard deviation of 51 nm (R-square=0.90). m, Paired correlation function of AMPAR and PSD-95 localizations within a cluster pair (n= 499 pairs). Red: experimental data, Blue: simulated randomized distribution. n, AMPAR nanodomain size. The peak of the fitted Gaussian function is at 69 nm with a standard deviation of 22 nm (R-square=0.90). o, Distance of AMPAR localization from the nearest PSD-95 cluster (n=15 YFP cortical neurons from three 6-month-old Thy1-YFP-H mice). p, Schematic of the post-synaptic organization of AMPA receptor, PSD-95 and Homer.

AMPAR distribution in hippocampus vs Cortex.

a, Thy1-YFP mouse brain coronal section image using confocal microscopy. Super-resolution dSTORM imaging was performed in the regions highlighted for cortex (blue box) and hippocampus (red box) (Scale bar: 400 µm). b, Surface AMPAR distribution (cyan) along a YFP neuron (yellow) in the cortex of a 6-months old Thy1-YFP-H mouse, along with PSD-95 (magenta). (Scale bar: 2 µm) c, Zoomed in ROIs from b (Scale bar: 0.5 µm). d, Surface AMPAR distribution (cyan) along a YFP neuron (yellow) in the CA1 region of the hippocampus of a 6-months old Thy1-YFP-H mouse, along with PSD-95 (magenta). (Scale bar: 2 µm) e, Zoomed in ROIs from d (Scale bar: 0.5 µm). f, Total AMPAR localization density per imaging field of view (FoV) (p=0.042). g, Fraction of AMPARs in the whole FoV found in clusters (p=0.004). h, Fraction of AMPARs along a YFP neuron found in clusters (p=0.0004). i, Cumulative frequency distribution of the distance between each AMPAR localization and its nearest PSD-95 cluster in the cortex. j, Cumulative frequency distribution of the distance between each AMPAR localization and its nearest PSD-95 cluster in the hippocampus. k, Fraction of AMPARs lying within 140 nm of a PSD-95 cluster (p=0.003). l, Number of AMPAR clusters in the whole FoV (p=0.441). m, Number of AMPAR clusters on a YFP neuron (p=0.506). n, AMPAR localizations per cluster in the whole FoV (p=0.263). o, AMPAR localizations per cluster on a YFP neuron (p=0.226). p, Histograms of PSD-95 cluster size distribution for cortex (red) and hippocampus (blue). All error bars depict SE. For f-h and l-o: n=3 mice, at least 6 FoVs per mouse per brain region. Each point represents average value per mouse. For i,j,p: Averaged over 3 mice, at least 3 FoVs per mouse. For k: n=3 mice, at least 3 FoVs per mouse per brain region. Each point represents average value per mouse. ***:P<0.001, as determined using paired sample t-test; **: p<0.01, as determined using paired sample t-test; *: p<0.05, as determined using paired sample t-test; n.s.: p>0.05 as determined using paired sample t-test

AMPAR organization in PS19 mice.

Surface AMPAR distribution (cyan) along a YFP neuron (yellow), along with PSD-95 (magenta) in the hippocampus of a 6-months old a, WT and b, PS19 mouse (Scale bar: 2 µm). c, Total AMPAR localization density per imaging FoV (p=0.084, p=0.023). d, Fraction of AMPARs in the whole FoV found in clusters (p=0.024). e, Fraction of AMPARs on a YFP neuron found in clusters (p=0.035). f, Fraction of AMPARs lying within 140 nm of a PSD-95 cluster (p=0.009). g, AMPAR localizations per cluster over whole FOV (p=0.056). h, AMPAR localizations per cluster along a YFP neuron (p=0.048). i, Fraction of AMPARs in a cluster over the whole FoV found in nanodomains (p=0.001). j, Fraction of AMPARs in a cluster on a YFP neuron found in nanodomains (p=0.002). k, Number of AMPARs per nanodomain over the whole FoV (p=0.051). l, Number of AMPARs per nanodomain in a YFP neuron (p=0.027). m, Histograms of PSD-95 cluster size distribution for WT hippocampus (red) and PS19 hippocampus (blue). All error bars depict SE. For c-e and g-l: n=3 mice each for WT and PS19, at least 6 FoVs per mouse per brain region. Each point represents average value per mouse. For f, m: n=3 mice each for WT and PS19, at least 3 FoVs per mouse. Each point represents average value per mouse. **: p<0.01, as determined using two-sample t-test (Welch Correction); *p<0.05, as determined using two-sample t-test (Welch Correction); n.s.: p>0.05 as determined using two-sample t-test (Welch Correction).