Fig 1: Pharmacological suppression of GluN2A- and GluN2B-NMDARs regulates tonic inhibition and α5-GABAAR internalization(A) Experimental design. Hippocampal neurons at DIV14 were treated with NVP (GluN2A-preferring antagonist NVP-AAM077, 100 nM), Ifen (GluN2B-preferring antagonist ifenprodil, 5 μM), or APV (broad-spectrum NMDAR antagonist, 100 μM) for 24 h and then recorded for tonic currents at DIV15.(B) Ifen treatment increased tonic currents, whereas NVP treatment decreased tonic currents. n = 10–11 for each group, one-way ANOVA test, F(3,38) = 13.14, p < 0.0001 with Dunnett’s multiple comparisons test, Ctrl versus NVP, p = 0.031; Ctrl versus Ifen, p = 0.0023.(C) Ifen treatment increased surface α5 expression, whereas NVP treatment decreased surface α5 expression. n = 33–43 for each group, Kruskal-Wallis test with Dunnett’s multiple comparisons test, Ctrl versus NVP, p = 0.0041; Ctrl versus Ifen, p = 0.0003.(D) Endocytosis assay of α5-GABAARs in cultures. Surface α5-GABAARs (Sα5) were labeled in green, and internalized α5-GABAARs (Iα5) were in red. Bar graphs in the right showing that 24-h NVP treatment increased α5 internalization, whereas 24-h Ifen treatment decreased α5 internalization. n = 15–16 for each group, one-way ANOVA test, F(3,56) = 22.96, p < 0.0001 with Dunnett’s multiple comparisons test, Ctrl versus NVP: p = 0.0044; Ctrl versus Ifen: p < 0.0001.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All data are presented as mean ± SEM.See also Figures S2 and S3.
Fig 2: 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 α5-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 3: 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 α5 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.
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