Fig 1: The relative expression of genes of NMDA and AMPA receptor subunits in the dorsal hippocampus 7 days after pilocarpine-induced SE. Subunit genes: Grin1–GluN1, Grin2a–GluN2a, Grin2b–GluN2b, Gria1–GluA1, Gria2–GluA2. Each dot represents one animal; the bars indicate average values and error bars show standard deviations. One-way ANOVA, Grin1: F2,19 = 11.8, p < 0.001; Grin2a: F2,20 = 3.7, p < 0.05; F2,20 = 1.7, p = 0.2; Gria1: F2,20 = 1.8, p = 0.2; Gria2: F2,20 = 9.1, p < 0.01. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05; ** p < 0.01.
Fig 2: Western blotting data of proteins production in the dorsal hippocampus 7 days after pilocarpine-induced SE. For inserts, upper part in each line shows the chemiluminescent signal, lower part shows the Ponceau S. For bands c is calibrator sample, 1 is Ctrl, 2 is Pilo, and 3 is MTEP group. On charts each dot represents one animal; the columns indicate average values, and error bars show standard deviations. One-way ANOVA, GluN2A: F2,17 = 16, p < 0.001; GluN2B: F2,8.62 = 3.04, p = 0.10; GluA1: F2,16 = 25.4, p < 0.001, GluA2: F2,13 = 5.1, p < 0.05. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05; ** p < 0.01, *** p < 0.001.
Fig 3: Hepatolobectomy induced the selective loss of NMDA receptors in the hippocampus of LPS pretreated mice, but not in age-matched normal adult mice.Hippocampal synaptic changes analyzed by western blotting. (A-C) Hepatolobectomy reduced the levels of NR2A, NR2B, NR1 in the hippocampus of LPS pretreated mice on day 3. LPS pretreated mice vs. LPS pretreated mice + surgery, p = 0.004 for NR2A, p = 0.03 for NR2B, p = 0.02 for NR1 (D) No significant difference in SYP expression was observed across the groups, p>0.05. LPS pretreated mice (1d) correspond to 6 days survivors, LPS pretreated mice (3d) correspond to 8 days survivors. All data were presented as mean±SEM and analyzed by two-way ANOVA; n = 4 in control part, n = 3 in surgery part; *p<0.05, **p<0.01.
Fig 4: The effect of subchronic N-acetylcysteine (NAC) on glutamatergic genes. (A) Lever presses and infusions during nicotine self-administration and extinction training of rats tested for transcript expression following administration of 0 or 100 mg/kg NAC prior to the last four sessions of extinction and the reinstatement session. (B) No difference was found for the total number of infusions received throughout self-administration, prior to NAC or vehicle treatment. (C) High-dose NAC did not significantly reduce active lever pressing during a 2-h reinstatement test prior to sacrifice for gene expression analysis. (D) Glutamatergic transcripts of interest (Slc1a2, Gria1, Gria2, Grin2a, and Grin2b) relative to the endogenous control gene GAPDH revealed no differences in mRNA expression due to subchronic 100 mg/kg NAC treatment. Expression is shown as a percentage of control animals. N per group for each gene is listed with each bar. **P < 0.0001 main effect of session (extinction vs. reinstatement). #P < 0.05 main effect of drug (saline vs. nicotine). ‡P < 0.05 main effect of treatment (0 vs. 100 mg/kg NAC). §P < 0.05 interaction between drug and treatment. *P < 0.05 significant difference between 0 and 100 mg/kg NAC in saline animals. The bar in (C) indicates a significant main effect of session.
Fig 5: Increased PSD-95 and altered postsynaptic receptor expression in Panx1 KO cortical synaptosomes. A, Representative Western blots of cortical synaptosome preparations from WT and Panx1 KO (P14 and P29) probed for Panx1, PSD-95, and glutamate postsynaptic receptor subunits (GluA1, GluA2, GluN1, GluN2A, GluN2B). The Bio-Rad Stain-Free reagent (bottom) was used to quantify total protein for normalization. Molecular weight markers are indicated in kilodaltons. B, Quantification of protein expression levels of Panx1, PSD-95, and post-synaptic glutamate receptors. Expression levels for each protein were normalized to total protein and expressed as a percentage of WT P14 values; n = 5 animals per group analyzed in five independent experiments. Panx1 significantly decreased from P14 to P29 in WT cortical synaptosomes (P14 = 100 ± 9.4%; P29 = 13.4 ± 1.2%, p < 0.0001i3,4,7; simple effect ANOVA with Bonferroni’s multiple-comparison test; ****p < 0.0001). No Panx1 signal was detected in Panx1 KO cortical synaptosomes. PSD-95 significantly increased with age in both WT and Panx1 KO, and was also significantly higher in Panx1 KO relative to WT within age-matched controls (age: F(1,16) = 37.4, p < 0.0001j3; genotype: F(1,6 = 175.8, p < 0.0001j2; interaction: F(1,16) = 4.2, p = 0.0570j1; two-way ANOVA with Bonferroni’s multiple-comparison test; WT P14 = 100 ± 8.5%, KO P14 = 179.2 ± 9.1%, p < 0.0001j4; WT P29 = 248.5 ± 9.0%, KO P29 = 287.9 ± 11.8%, p = 0.0220j5; *p < 0.05, ****p < 0.0001). GluA1 and GluN2a also exhibited age-matched increases in expression in Panx1 KO cortical synaptosomes (GluA1: genotype, F(1,16) = 9.090, WT P14 = 100 ± 7.2%, KO P14 = 155.6 ± 24.4%; WT P29 = 93.42 ± 14.9%, KO P29 = 168.4 ± 31.7%, p = 0.0082k2; GluN2A: F(1,16) = 7.892, WT P14 = 100 ± 12.2%, KO P14 = 167.8 ± 31.20%; WT P29 = 121.4 ± 23.2%, KO P29 = 201.3 ± 33.3%, p = 0.0126n2); *p < 0.05, **p < 0.01, GluN1 developmental upregulation was more pronounced in the WT group (p = 0.0009m1-8); ***p < 0.001, whereas GluN2B immunoreactivity in Panx1 KO synaptosomes exhibited a steeper developmental decline at P29 compared to WT (age: F(1,16) = 4.547, p = 0.0488o3; WT P14 = 100 ± 6.5%, WT P29 = 97.1 ± 16.6%, p > 0.9999o4; KO P14 = 133.1 ± 11.9%, KO P29 = 88.6 ± 5.9%; p = 0.0240o5; two-way ANOVA with Bonferroni’s multiple-comparison test; *p < 0.05). Data are presented as mean ± SEM. For additional statistical information, see Table 1i1-o5.
Supplier Page from Abcam for Anti-NMDAR2A antibody