Fig 2: The locations and corresponding phenotypes of epilepsy-related missense GRIN2A mutations. (A) Topological distribution of the GRIN2A missense mutations with different phenotypes. (B) The proportion of GRIN2A mutations with EE in different molecular regions. The proportion of missense mutations with EE around TMD was significantly higher than that in ATD and LBD (Pearson’s chi-square test, *p < 0.05; **p < 0.01). ATD, amino terminal domain; LBD, ligand-binding domain; TMD, transmembrane domains; M1, M3, and M4, transmembrane domains; M2, re-entrant pore loop; CTD, carboxyl-terminal domain; EE, epileptic encephalopathy.

Fig 3: Effects of neonatal Sev exposure on granular neuron number and synapse development in adult hippocampus. (a, b) Double-immunostaining of CC3/NeuN, and western blotting of CC3 in the hippocampus of control and Sev-treated rats at 6 h post last Sev treatment. N = 6 rats per group. (c) Immunostaining and quantification of NeuN in the adult hippocampus of control rats and Sev-treated rats. N = 6 rats per group. (d) Golgi staining and quantification of spines in control rats and Sev-treated rats in adult. N = 6 rats per group. (e) Synaptic ultrastructure in the hippocampus of control and Sev-treated rats in adult. N = 6 rats per group. (f, g) Western blotting and quantification of Synaptophysin, post-synaptic density protein 95 (PSD95), calcium/calmodulin-dependent kinase II (CaMKII), phosphorylation of N-methyl-d-aspartate receptor subunit 2 A (pNR2A), NR2A, and vesicular inhibitory amino acid transporter (vGAT). N = 6 rats per group. Notice that Sev treatment significantly reduced the number of synapse as well as the expression of synaptic proteins. Con, control. Sev, Sevoflurane. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Student’ t-test (b, c, e–g). One-way anova (d). Bar = 50 µm (a–c), 10 µm (d), 200 nm (e).

Fig 4: Differential expression of NMDA receptor subunits in hyperhomocysteinemic mice. mRNA levels for (A) NR1, (B) NR2A, and (C) NR2B in cortical tissues measured by real-time polymerase chain reaction and normalized to 18S rRNA. D, Ratio of NR2A/NR2B mRNA levels. Results are expressed as mean±SEM (n=5–7). E, Representative immunoblot of NR2A, NR2B, and ß-actin protein in lysates of cortical tissues from Cbs+/+, Cbs+/-, and Cbs-/- mice. Protein levels for (F) NR2A (top band) and (G) NR2B quantified by densitometry and normalized to ß-actin. H, Ratio of NR2A/NR2B protein levels. Of technical note, 2 bands were detected using anti-NR2A with both bands showing similar levels of expression between all groups (data not shown for the bottom band which may represent a modified protein, degradation product, or non-specific band). Results are expressed as mean±SEM (n=6). *P<0.05, **P<0.01 vs Cbs+/+ mice, 1-way ANOVA. Cbs, cystathionine beta synthase.

Fig 5: The mRNA expression of Netrin-1 and GluA1 were significantly increased in the hippocampus in MS rats of three different age periods. (A) On PND21, the mRNA expression of Netrin-1 was significantly increased, but its receptor uncoordinated (UNC5D) was significantly decreased. (B) On PND35, the mRNA expression of Netrin-1, and its receptor DCC, neogenin-1 (Neo-1) were significantly increased, but UNC5C was significantly decreased. (C) On PND70, the mRNA expression of Netrin-1 was significantly increased, but its relative receptors showed no significant upregulation. (D–F) GluA1, NR2A, NR2B, and postsynaptic density 95 (PSD95) were significantly increased in all three MS groups. Besides, NR1 was found upregulated in MS rats on PND35 (E) and PND70 (F). On PND21: NC, n = 8; MS, n = 8. On PND35: NC, n = 8; MS, n = 8. On PND70: NC, n = 6; MS, n = 8. All data were given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t-test.