(A) Example gSTED images of Homer1c-ALFA labeled with Cy3, and CLCa-GFP labeled with At647N under basal conditions (upper panel, control), after 5 min of DHPG (100 µM, middle panel) and after 5 min of NMDA (20 µM, lower panel). Scale bar: 5 µm, zoom: 500 nm. (B) Quantification of the number of PSDs associated with multiple CCSs after different timepoints of DHPG and NMDA bath application, normalized to timepoint 0. DHPG significantly increased the number of PSDs associated with multiple CCSs after 5 min (timepoint 0: N = 19, timepoint 5: N = 11, p > 0.01), while NMDA did not alter PSD-EZ association (timepoint 0: N = 13, timepoint 5: N = 7, p > 0.05). (C) Percentage of PSDs associated with multiple CCSs. Five min DHPG significantly increases PSD-EZ association (Control: N = 17, DHPG: N = 13, p < 0.05). MPEP blocked the effect of DHPG (N = 10, p > 0.05). (D) Example gSTED images as described in A, under basal conditions (upper panel) and after cLTP (lower panel). Scale bar: 5 µm, zoom 500 nm. (E) Quantification of the number of PSDs associated with multiple CCSs after cLTP, normalized to timepoint 0 (N = 8–13). (F) Number of CCSs per PSD under basal conditions and after 5 min cLTP followed by 25 min recovery. At timepoint 30, the number of CCSs per PSD was significantly increased (Control: N = 9, cLTP: N = 11, p < 0.05) and the effect was completely blocked in the presence of AP5 (N = 8). (G) Example images of live-cell gSTED on Homer1c-mCherry and Halo-CLCa labeled with JF646. Three sequential images were taken before cLTP (left), directly after (middle left) and after recovery (middle right). Two example spines were selected where reorganization of CLCa was observed (right). Scale bar: 5 µm, zoom: 500 nm. (H) Quantification of a subset of PSDs that increased in size (orange), or remained the same size during imaging (blue). PSDs that were enlarged in response to cLTP were associated with multiple CCSs (N = 4). (I) GFP-CLCa area per spine under basal conditions and after cLTP (Basal: N = 6, cLTP: N = 8). (J) Relative intensity of GFP-CLCa before and after cLTP imaged using live-cell confocal imaging (n = 31).