Fig 2: Activation of NR2B-containing NMDA receptors is tightly correlated with the high plasticity in ECMS mice.A. The protein expression levels of NR2B in V1b of Ctrl and ECMS mice of four periods (Early-CP, Peak-CP, Late-CP, Adult). Black column represents Ctrl mouse (n = 6 females and males/group), green hollow column represents ECMS mouse (n = 7 females and males/group). B. The expression of NR2B in the visual cortex of male and female mice in the Ctrl and ECMS groups during development. Relative optical densities are the ratios of band densities at each stage divided by the band densities of Ctrl mice at Early-CP of the sample from the same immunoblot. Error bars, Data represent mean ± SEM. *P < 0.05, Student's t-test. C. The protein expression levels of NR2B, GAD67 and GAD65 in V1b of adult ECMS male mice administrated with estradiol (blue hollow column,n = 7 mice) or vehicle (Veh, black column, n = 6 mice). D. NR2B antagonist Ro 25–6981 blocks the plasticity in adult ECMS females (n = 4 mice/group). Schematic of the experimental procedure (top), the position of the implanted minipump and electrode recording site (left), summary of CBI of ECMS females treated with and without Ro25-6981 (right). The horizontal line represents the average of the data; each symbol represents an animal. E. Cumulative distribution of ODI. P = 0.018, Kolmogorov-Smirnov test. F. Stimulation-evoked firing rates driven by the ipsilateral and contralateral eyes of each group. Contra, contralateral eye; Ipsi, ipsilateral eye. In A, B, F, error bars, data represents mean ± SEM. *P < 0.05, ***P < 0.001, student t-test.

Fig 3: Knockdown of spinal cord TXNIP decreased p-NR2B via NLRP3 inflammasome in spinal cords of DNP rats. The protein levels of p-NR2B, t-NR2B, NLRP3, TXNIP, caspase-1, and IL-1ß were compared among the rats in each group on days 17, 21, and 28 after STZ intraperitoneal injection. (Mean ± standard deviation. n = 3. *P < .05, **P < .01, ***P < .001, ****P < .0001; p-NR2B: DNP group versus C group, **P = .0028 [STZ 17 d] and .0032 [STZ 21 d], ***P = .0008 [STZ 28 d]. DNP group versus PL group, *P = .0103 [STZ 17 d], **P = .0074 [STZ 21 d], ***P = .0006 [STZ 28 d]. DNP group versus DNP + TXNIP siRNA group, **P = .0042 [STZ 17 d] and .0013 [STZ 28 d], *P = .0353 [STZ 21 d]. DNP group versus NC group, P = .9954 [STZ 17 d], .6410 [STZ 28 d], and P > .9999 [STZ 21 d]; t-NR2B: DNP group versus C group, P = .7793 [STZ 17 d], .9439 [STZ 21 d], and .9078 [STZ 28 d]. DNP group versus PL group, P = .5261 [STZ 17 d], .9816 [STZ 21 d], and .9994 [STZ 28 d]. DNP group versus DNP + TXNIP siRNA group, P = .8333 [STZ 17 d], .9854 [STZ 21 d], and .9317 [STZ 28 d]. DNP group versus NC group, P = .4728 [STZ 17 d], .4371 [STZ 21 d], and .9749 [STZ 28 d]; NLRP3: DNP group versus C group, **P = .0010 [STZ 17 d], ***P = .0002 [STZ 21 d]. DNP group versus PL group, ***P = .0008 [STZ 17 d], **P = .0086 [STZ 21 d]. DNP group versus DNP + TXNIP siRNA group, ***P = .0003 [STZ 17 d], **P = .0014 [STZ 21 d]. DNP group versus NC group, P = .2853 [STZ 17 d], .9986 [STZ 21 d], and .9021 [STZ 28 d]; TXNIP: DNP group versus C group, ***P = .0001 [STZ 17 d] and .0001 [STZ 28 d]. DNP group versus PL group, ***P = .0002 [STZ 17 d] and .0002 [STZ 28 d]. DNP group versus DNP + TXNIP siRNA group, ***P = .0008 [STZ 17 d] and .0003 [STZ 28 d]. DNP group versus NC group, P = .9894 [STZ 17 d], .9994 [STZ 21 d], and .9038 [STZ 28 d]; caspase-1: DNP group versus C group, ***P = .0004 [STZ 21 d] and .0001 [STZ 28 d]. DNP group versus PL group, ***P = .0009 [STZ 21 d] and .0001 [STZ 28 d]. DNP group versus DNP + TXNIP siRNA group, ***P = .0002 [STZ 17 d], .0010 [STZ 21 d], and .0006 [STZ 28 d]. DNP group versus NC group, P = .9840 [STZ 17 d], .9607 [STZ 21 d], and .5746 [STZ 28 d]; IL-1ß: DNP group versus C group, ***P = .0003 [STZ 21 d] and .0001 [STZ 28 d]. DNP group versus PL group, ***P = .0007 [STZ 17 d] and .0008 [STZ 28 d], **P = .0017 [STZ 21 d]. DNP group versus DNP + TXNIP siRNA group, ***P = .0009 [STZ 17 d], .0007 [STZ 21 d], and .0002 [STZ 28 d]. DNP group versus NC group, P = .9926 [STZ 17 d], .6838 [STZ 21 d], and .8543 [STZ 28 d]. Tukey post hoc test after 1-way ANOVA.) ANOVA indicates analysis of variance; C group‚ control group; caspase-1, cysteinyl aspartate-specific proteinase-1; DNP group, diabetic neuropathic pain group; IL-1ß, interleukin-1ß; NC group, negative control of siRNA group; NLRP3, NOD-like receptor protein 3; PL group, painless group; p-NR2B, NR2B phosphorylation at Tyr1472; STZ, streptozotocin; t-NR2B, total NR2B; TXNIP, thioredoxin-interacting protein.

Fig 4: NMDA-induced superoxide production is mediated through the C-terminal cytoplasmic domain of GluN2B and is associated with PI3K binding. (a) Selective deletion of GluN2B but not GluN2A subunits prevents superoxide formation induced by NMDA. Cultures were treated with NMDA as in Fig. 3, with results from each of n = 3 experiments expressed relative to the NMDA treated condition; *p < 0.05 vs control. (b) In neurons lacking both endogenous GluN2A and GluN2B, NMDA-induced superoxide production was observed in cultures transfected with either the GluN2B subunit (GluN2B wt) or the GluN2A subunit fused to a GluN2B C-terminal domain (GluN2A-ct2B), but not in cultures transfected with GluN2A wt or GluN2B-ct2A. Results from each of 3 experiments are expressed relative to signal obtained in cultures transfected with the empty vector GFP (Ev-GFP). The reduced magnitude of the NMDA effect in the GluN2B - transfected neurons (b) relative to that observed in Wt neurons (a) is consistent with the transfection efficiency of approximately 10% achieved with each of the GluN2 constructs. Data are means + s.e.m.; *p < 0.05 v Ev-GFP. (c) Immunoblots (IB) of PI3K p85 and GluN2B in lysates immunoprecipitated (IP) with antibody to GluN2B. IP with normal mouse IgG served as negative control. The p85 band is at ~90 kD and the GluN2B band at ~200 kD. An aliquot of the cell lysate was used as an input control to verify the presence of detected protein in the original samples. Full-length blots are presented in Supplementary Fig. S4. (d) Quantified results of p85 binding to GluN2B normalized to the control values; n = 3; *p < 0.05 vs control.

Fig 5: Western blot analysis of the N-methyl-D-aspartate receptor (NMDA-R) subunit 2B (GluN2B) in the inferior colliculus (IC). (A) The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein among the four groups. (B) The expression of the GluN2B protein was significantly higher in the SS group compared to the control group. In addition, GluN2B protein expression was significantly lower in the EGb+SS group, as compared to the SS group. (C) Relative protein expression of GluN2B in the IC. The expression of GluN2B protein was significantly higher in the SS group and EGb+SS group compared to the control and EGb groups. Notably, the expression of GluN2B protein was significantly decreased in EGb+SS group following EGb pretreatment compared to that in SS group. SS, sodium salicylate; EGb, EGb 761 Ginkgo biloba extract. *P<0.05 (the SS group vs. EGb+SS group). **P<0.01 (the control group vs. SS group).