Fig 2: PSD-95 deficiency causes an increase in NMDAR/AMPAR-current ratio during development in layer V pyramidal neurons. (A) Schematic of layer II/III stimulation and layer V whole-cell patch clamp recordings of pyramidal neurons in the mPFC. (B) Example traces of AMPAR eEPSCs recorded at -60 mV and NMDAR eEPSCs recorded at +60 mV in the presence of picrotoxin from control and PSD-95-/-mice at postnatal days 21–25. NMDAR eEPSC analysis was measured 50-ms post-stimulus as illustrated by the yellow circle. (C & D) Graphs show mean peak amplitudes of AMPAR-eEPSCs (p = 0.77, n = 10) and NMDAR-eEPSCs (p = 0.31, n = 10) at P21-25. (E,F) Graphs represent AMPAR and NMDAR decay time constant (t) ± s.e.m. in ms (AMPAR, p = 0.31; NMDAR, p = 0.44, n = 10). (G) Example traces of AMPAR eEPSCs recorded at -60 mV and NMDAR eEPSCs recorded at +60 mV in the presence of picrotoxin from control and PSD-95-/-mice at postnatal days 35–39. (H,I) Graphs show mean peak amplitudes of AMPAR-eEPSCs and NMDAR-eEPSCs (AMPAR, p = 0.03; NMDAR; p = 0.03, Con, n = 16, KO, n = 14). (J & K) Graphs represent AMPAR and NMDAR decay time constant (AMPAR, p = 0.96; NMDAR, p = 0.04 Con, n = 16, KO, n = 14). (L) Summary of mean NMDAR/AMPAR-current ratio ± s.e.m, ANOVA, F(1,46) = 23.457,p = 1 × 10-5; paired t tests, P21-25, p = 0.02; P35-P39, p = 7 × 10-5, Con, n = 16, KO, n = 14. (M) Example traces of NMDAR eEPSCs recorded at +60 mV in the presence of CNQX and picrotoxin from control and PSD-95-/-mice at postnatal day 35–39. Ro25 was then bath applied to block GluN2B-containing NMDARs. (N) Quantifications show normalized NMDAR-eEPSCs (p = 0.043, Con, n = 7, KO, n = 8). *p < 0.05, ****p < 0.0001, n.s., not significant.

Fig 3: Native supermolecular assembly of NMDAR subunits in the mammalian brain.(a) Schematic showing the higher-order assembly from individual proteins to complexes and supercomplexes (complexes of complexes). (b) Native protein complexes of GluN1, GluN2A and GluN2B detected in BNP immunoblot screen of mouse forebrain extracts. Approximately 0.8 and ~1.5 MDa complexes indicated by filled and open arrowheads, respectively (hereafter used to label all figures). The expected size of each protein in monomeric form indicated with pink rectangle. On left side, non-denaturing molecular mass indicated in mega-Daltons (MDa). (c) BNP GluN1 immunoblot of fractions from glycerol gradient (10–30%) ultracentrifugation. ‘IN', forebrain extract supernatant. On right side, non-denaturing molecular mass indicated in MDa. (d) BNP GluN1 immunoblot of fresh human cortical biopsy samples from the inferior frontal (inf. front.), inferior temporal (inf. temp.) and superior frontal (sup. front.) lobes. Mouse forebrain extract supernatant shown for comparison. These data show the ~0.8 and ~1.5 MDa NMDA receptor complexes (1.5-NR and 0.8-NR) were conserved between mouse and humans.

Fig 4: Dentate gyrus: (A,B) Analysis of Arc immunoreactivity and mRNA expression during CPA and extinction test. (C,D). Analysis of GluN1 immunoreactivity and mRNA expression during CPA and extinction test. (E,F). Analysis of Homer1 immunoreactivity and mRNA expression during CPA and extinction test. (G) Correlation between relative mRNA levels of Arc and the CPA score in morphine dependent rats. (H) Correlation between GluN1 immunoreactivity and the difference between the time spent in the naloxone chamber during extinction test and CPA test in placebo rats that underwent extinction protocol. (I) Correlation between Homer1 immunoreactivity and the difference between the time spent in the naloxone chamber during extinction test and CPA test in morphine-treated rats that underwent extinction protocol and between Arc immunoreactivity and same time parameter. (J) Representative epifluorescence images showing triple immunostaining of Arc (green), GluN1 (red) and Homer1 (pink) during CPA and extinction test. * p < 0.05, ** p < 0.01 vs. P-CPA. Each bar corresponds to mean ± SEM.

Fig 5: Quantification of the GluN1/PSD95 relative abundance in vivo gave a 17 : 1 molar ratio of PSD95 over GluN1. (a) GluN1 and PSD95 were detected by sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (lower panel) and triplicate dot blot (DB) Flag immunoblot (upper panel) of Glun1 TAP / TAP, Psd95 TAP / TAP mouse forebrains respectively. The TAP-GluN1 and PSD95-TAP SDS-PAGE bands are similar to their counterparts in singularly TAP-tagged mice (Psd95 TAP / TAP and Glun1 TAP / TAP), which indicates equal loading. (b) The molar ratio of Flag from Psd95 TAP / TAP and Glun1 TAP / TAP was quantified densitometrically using a dilution series, in which Psd95 TAP / TAP forebrain extracts were diluted with that of wildtype. Densitometry of dilution series indicated TAP-PSD95 was 17 ± 3-fold (mean ± SD) more concentrated than TAP-GluN1.