Fig 2: Effect of GluN2A/Rph3A interaction on NMDAR currents.(a) Sample traces showing the effect of intracellular perfusion of the non-permeable 2A-40 or its scramble peptides on pharmacologically isolated NMDAR-mediated EPSCs (+30 mV) recorded from a CA1 pyramidal cell. Traces represent the average of 6–9 responses with a scale of 40 pA over 100 ms. (b) Summary graph illustrating the time course of the effect of 2A-40 or its scramble on the peak amplitude of NMDAR-EPSCs. For comparison, the amplitude of NMDAR-EPSCs in the absence of any peptide in the intracellular solution (open circles) were also plotted (n=5–9). (c,d) Bar graphs summarizing the effect on the amplitude of NMDAR-EPSCs (c; Mann–Whitney test) and decay time (d) of the peptides (n=5–9). (e) Sample traces show the effect of intracellular perfusion of the non-permeable 2A-40 or its scramble control peptides on pharmacologically isolated AMPAR-mediated EPSCs (-70 mV). Traces represent the average of 6–9 responses with a scale of 20 pA over 10 ms. (f) Summary graph illustrating the time course of the effect of 2A-40 or its scramble on the peak amplitude of AMPAR-EPSCs. For comparison, it is also plotted (open circles) the amplitude of AMPAR-EPSCs in the absence of any peptide in the intracellular solution, n=6–7. (g) Sample traces and summary graphs illustrating that transfection of tGFP-shRNA-Rph3A induced a significant reduction in the NMDA/AMPA ratio when compared with cells transfected with tGFP-shRNA-Scramble (tGFP-shRNA-Scramble: 2.03±0.32; n=9; tGFP-shRNA-Rph3A: 0.89±0.29, n=8; Mann–Whitney test). (h) Schematic illustrating paired recordings from neighbouring transfected and non-transfected neurons. (i) Sample traces shown in the inset (not transfected (NT): n=13; tGFP-shRNA-Rph3A: n=13; Mann–Whitney test). (j) Amplitude of AMPAR-mediates EPSCs at SC-CA1 (recorded at -70 mV; NT: n=9; tGFP-shRNA-Rph3A: n=9). (k) Transfection with tGFP-shRNA-Scramble did not modify the amplitude or decay time of NMDAR-mediated EPSCs at SC-CA1 synapse (NT: n=12; tGFP-shRNA-Scramble: n=12). (l) Transfection with tGFP-shRNA-Scramble did not reduce the amplitude of AMPAR-mediated EPSCs at SC-CA1 (NT: n=10; tGFP-shRNA-Scramble: n=10). All data are represented as mean±s.e.m. *P<0.05.

Fig 3: Modulation of GluN2A-containing NMDAR expression at synapses in the developing rat hippocampus.(a) Western blot analysis of GluN2A, GluN2B, PSD-95, Rph3A and tubulin of TIF from treated rat pups hippocampus. (b) Bar graph representing the percentage of tubulin normalized integrated density of GluN2A, GluN2B, Rph3A and PSD-95 WB bands from TIF samples compared with their respective TIF purification TAT-Scr control (n=3–5; unpaired Student's t-test). (c) Sample traces illustrating a decreased NMDA/AMPA in TAT-2A-40-treated animals (blue) compared with TAT-Scr-treated (red) animals at Schaffer collaterals to CA1 pyramidal cell synapses in acute hippocampal slices with 100 pA over 100 ms scale. (d) Bar graph summarizes the significant decrease in NMDA/AMPA in TAT-2A-40-treated animals (blue) compared with TAT-Scr-treated (red) animals (n=9–12; Mann–Whitney test). (e,f) Sample traces (e) and summary bar graph (f) illustrating that the decay time of pharmacologically isolated NMDAR-EPSCs does not differ between TAT-Scr-treated (red) and TAT-2A-40-treated (blue) animals (n=10–14) with 40 pA over 100 ms scale. (g–i) Sample traces (g) and summary graphs illustrating that the amplitude (h) and decay time (i) of the pharmacologically isolated NMDAR-EPSCs are equally modulated by application of TCN 210 in both conditions (n=6–7; Wilcoxon test) with 50 pA over 200 ms scale. (j) Representative images show dendrites of P15 rat pups CA1 neuron either treated with TAT-Scr (red) or TAT-2A-40 (blue), both 3 nmol g-1 s.c. chronically from P6 to P14. Bar graph represents the respective spine densities (n=10–11; unpaired Student's t test). All data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001. Scale bars, 1 µm.

Fig 4: Pharmacological suppression of GluN2A- and GluN2B-NMDARs regulates tonic inhibition in the KA-induced seizure model(A–C) Representative western blots and summary graphs from cell-surface biotinylation assays showing that surface and total GluN2A (A, n = 3 independent experiments, one-way ANOVA test, F(2,6) = 5.928, p = 0.0379 with Dunnett’s multiple comparisons test, p = 0.0321), GluN2B (B, n = 3 independent experiments, one-way ANOVA test, F(2,6) = 10.80, p = 0.0103 with Dunnett’s multiple comparisons test, p = 0.0283), and a5-GABAAR (C, n = 4 independent experiments, one-way ANOVA test, F(2,9) = 34.75, p < 0.0001 with Dunnett’s multiple comparisons test, saline 1 h versus KA 1 h: p = 0.0098; saline 1 h versus KA 24 h: p = 0.0022) expression in the KA-induced seizure model.(D and E) Tonic currents in hippocampal CA3 neurons were decreased 1 h after KA injection, whereas increased 24 h after KA injection. Ifen or NVP treatment 1 h prior to KA injection, respectively, restored the decreased or increased tonic currents at corresponding time point after KA injection. (D) n = 10 for each group, one-way ANOVA test, F(3,36) = 11.42, p < 0.0001 with Dunnett’s multiple comparisons test, saline 1 h versus KA 1 h: p = 0.0014; KA 1 h versus KA + Ifen: p = 0.0001. (E) n = 10 for each group, one-way ANOVA test, F(3,36) = 8.925, p = 0.0001 with Dunnett’s multiple comparisons test, saline 24 h versus KA 24 h: p = 0.0065; KA 24 h versus KA+NVP: p = 0.0047.*p < 0.05, **p < 0.01, and ***p < 0.001. All data are presented as mean ± SEM.See also Figure S4.

Fig 5: Differential regulation of tonic inhibition by GluN2 subunits(A) Overexpression experiment design. Neurons were transfected at 11 days in vitro (DIV11) for 72 h and then recorded for tonic inhibitory currents at DIV14.(B) GluN2B, but not GluN2A, overexpression decreased tonic currents in cultured neurons. n = 10–13 for each group, one-way ANOVA test, F(2,31) = 5.029, p = 0.0128 with Dunnett’s multiple comparisons test. Ctrl versus GluN2B: p = 0.0093.(C) GluN2B, but not GluN2A, overexpression decreased surface a5 expression in cultured neurons. n = 28–31 for each group, one-way ANOVA test, F(2,87) = 6.369, p = 0.0026 with Dunnett’s multiple comparisons test, Ctrl versus GluN2B: p = 0.0071.(D) Knockout (KO) experiment design. Neurons were transfected at DIV3–DIV4 and then recorded for tonic currents at DIV16–DIV17.(E) GluN2A KO decreased tonic currents, whereas GluN2B KO increased tonic currents. The changes in tonic currents induced by either GluN2A or GluN2B KO were restored back to the control level by co-expression of corresponding sgRNA-resistant constructs, respectively. n = 8–10 for each group, one-way ANOVA, F(4,39) = 13.12, p < 0.0001 with Tukey’s multiple comparisons test, Ctrl versus GluN2A sgRNA: p = 0.0039; Ctrl versus GluN2B sgRNA: p = 0.0224: GluN2A sgRNA versus GluN2A sgRNA + sgRNA-resistant GluN2A: p = 0.0091; GluN2B sgRNA versus GluN2B sgRNA + sgRNA-resistant GluN2B: p = 0.0202.*p < 0.05 and **p < 0.01. All data are presented as mean ± SEM.See also Figure S1.