(A) Schematic representation of experimental design (top). Hippocampal neurons 14 div, transfected at 10 div with: GluN1-GFP (donor) or GluN1-GFP plus GluN1-mCherry (acceptor, bottom). Both conditions were co-transfected with GluN2B-Flag. Scale: 10 µm. (B) Example of GluN1-GFP fluorescent (left) and FLIM image (right). NMDAR clusters (spines clusters: arrows; shaft clusters: arrow heads, top) lifetime in nanoseconds (ns) quantification (bottom). Scale: 10 µm, insert 1 µm. Schematic representation of lifetime decay of a donor-only (full line) and of the donor in the presence of the acceptor (dashed line, bottom left). Comparison between GluN1-GFP lifetime alone (donor-only), co-transfected with GluN1-mcherry with a ratio 1:3 or 1:2 (donor-acceptor pair) and co-transfected with GluN2B-mCherry (negative control). GluN1-GFP: n = 70, GluN1-GFP + GluN1-mCherry (1:3): n = 88, GluN1-GFP + GluN1-mCherry (1:2): n = 44, GluN1-GFP + GluN2B-mCherry: n = 60 spines and shaft clusters; p<0.0001, One-way analysis of variance, followed by Dunnett's Multiple Comparison Test, ***p<0.0001 (bottom right). (c) Example of FLIM image of GluN1-GFP/GluN1-mCherry clusters after addition of tyrode (control) or NMDA (left). Quantification of GluN1-GFP lifetime (right). Tyrode: n = 291, NMDA: n = 124; p=0.0165, F = 1.719, Unpaired t test, one-tail. (D) Example of FLIM image of GluN1-GFP/GluN1-mCherry clusters after addition of D-serine or glycine (top) in tyrode only or in the presence of NMDA (bottom). Quantification of GluN1-GFP lifetime change (lifetime after minus the lifetime before co-agonist addition, right). Tyrode: n = 291, glycine: n = 110, D-serine: n = 103, glycine in NMDA: n = 268, D-serine in NMDA: n = 233 spine and shaft clusters; tyrode p=0.4234, r2 = 0.0001, glycine p=0.3650, r2 = 0.0011, D-serine p=0.0255, r2 = 0.0368, glycine in NMDA p=0.0949, r2 = 0.0065, D-serine in NMDA p=0.4348, r2 = 0.0001, Paired t-test, one-tail, before and after, *p<0.05. Data are represented as mean ± s.e.m. (E) Schematic representation of the c-terminus tails of the NMDAR in basal conditions (tyrode, (i) or in the presence of NMDA (ii), the co-agonists alone (iii, iv) or in activating conditions (co-agonists together with NMDAR, (v, vi).