Fig 2: Quantitative variation of NMDA receptor subunit in PAG after opiate treatment Mice were treated with i.p. injection, twice daily, for 4 days with morphine (10 mg/kg) followed by one day of treatment with saline (Morph/Sal), morphine (Morph), methadone (2.5 mg/kg, Morph/Meth), or for 5 days with saline (Sal). The 6th day, NR1, NR2A and NR2B were quantified with western-blotting in PAG. Bars are the means ± SEM of subunit immunoreactivity expressed in percentage of saline-treated group. One-way ANOVA, F3,24 = 0.7198, p = 0.55 (A). One-way ANOVA, F3,17 = 8.995, p < 0.0001 (B). One-way ANOVA, F3,18 = 5.219, p < 0.01 (C). *p < 0.05, **p < 0.01, n.s. (not significant) vs Sal group ; N.S. (not significant) vs Morph group. Bonferroni post-hoc test, n = 5 to 8 determinants/group.

Fig 3: PKC?-A24E mouse shows highly PKC kinase activity. A, Western blot analysis of total PKC? protein and phospho-PKC substrates from organotypic slice cultures. Phosphorylation of PKC substrates was increased in Het and Homo PKC?-A24E mice. B, Western blot analysis normalized to actin shows PKC? protein reduction in PKC?-A24E mice, but phospho-PKC substrate is upregulated in PKC?-A24E mice. NMDAR-S890 is known to be phosphorylated by PKC?. Phospho-NMDAR-S890 protein expression is normalized to total NMDAR-N1 protein expression. This phosphorylation is increased in PKC?-A24E mice. The data were normalized to Wt as 100% from three independent experiments. C, Phospho-MARCKS is a known substrate of PKC. Phospho-MARCKS S152/S156 compared with total MARCK was decreased in PKC?-A24E mice and PMA-treated organotypic slice cultures from Wt mice (Wt = 100.4 ± 4.37%, n = 4; Het = 61.92 ± 15.25%, n = 4; Homo = 33.98 ± 5.83%, p = 0.0009, n = 4; Wt + PMA = 42.54 ± 15.4%, p = 0.0033, n = 4; Het + PMA = 34.58 ± 9.39%, p = 0.0010, n = 4; Homo + PMA = 16.89 ± 3.557%, p < 0.0001, n = 4). With added phosphatase inhibitor, the phospho-MARCKS reduction was partially rescued (Wt = 116.5 ± 11.8%, n = 3; Het = 64.68 ± 13.8%, n = 3; Homo = 97.15 ± 10.3%, n = 3). The two-tailed Mann–Whitney test was used to analyze the difference between each group, and Wt without any treatment is shown as 100%. Data are mean ± SEM.

Fig 4: Fenfluramine and norfenfluramine disrupt the association of s1Rs with NR1 subunits of NMDA receptors(A) In vitro assay to determine ligand activity at s1Rs. NHS-activated Sepharose beads covalently coupled to a sequence of NR1 subunits containing the transmembrane region plus C0–C1–C2 cytosolic segments were incubated with excess s1Rs (1:3). The unbound s1Rs were washed out, and the NR1-coupled s1Rs were exposed to serial concentrations of the ligands under study. The s1Rs that remained attached to the NR1 subunits were then evaluated by SDS-PAGE and immunoblotting. (B) Diminishing effect of ligands on the association of s1Rs with NR1 subunits. The assays were performed in the presence of 50 mM Tris–HCl (pH 7.5), 0.2% CHAPS and 2.5 mM calcium. Representative blots are shown. The ED50 values were computed using the software SigmaPlot V.13. (C) The s1R agonist PPCC diminishes the capacity of S1RA and fenfluramine to disrupt s1R–NR1 association. (D) S1RA, fenfluramine and norfenfluramine, but not PPCC, decreased the association of s1Rs with CaM binding site on NR1 subunits. Agarose-NR1 carrying the associated s1Rs was incubated with 2.5 mM CaCl2, in the absence or presence of 100 nM CaM. The assays were performed twice, and each point was duplicated. *Significant difference with respect to the control group, ? with respect to the group receiving only S1RA or fenfluramine; ANOVA, Dunnett multiple comparisons vs control group, p < 0.05.

Fig 5: Effects of chronic MK-801 treatment and pretreatment with olanzapine or haloperidol on N-methyl-D-aspartate (NMDA) receptor subunits in rat medial prefrontal cortex (mPFC) and hippocampus. Representative Western blots and relative bar graphs showing the expression levels of (A,B) GluN1 and its phosphorylated form, (C,D) GluN2A and its phosphorylated form, (E,F) GluN2B and its phosphorylated form. Values are expressed as means ± SEM (n = 5–6 per group). #p < 0.1, *p < 0.05, **p < 0.01. Veh, vehicle + vehicle; MK, vehicle + MK-801; HPD, haloperidol + MK-801; Ola, olanzapine + MK-801.