Fig 2: Functional expression of NMDAR in HEK293 cells using baculovirus. (A) NR1 and NR2A protein levels in HEK293 cells transduced with baculovirus. HEK293 cells were transduced in the presence of 1 mM Ketamine with different amounts of baculovirus encoding human NR1 and NR2A. Cells were harvested 16 h after transduction, and NR1 and NR2A protein levels were analyzed by immunoblotting. Representative images are shown. Molecular weight standards are indicated. NR2A shows an unspecific band at ~150 kDa. (B) Cell viability of HEK293 cells 16 h after transduction with different amounts of baculovirus encoding NR1 or NR2A. Cell viability was assessed using CellTiterGlo reagent. Data was normalized to non-transduced cells and represents the mean ± standard error of the mean (SEM) of three experiments. (C,D) Cell viability of HEK293 cells 16 h after transduction with baculovirus encoding NR1 or NR2A with concurrent treatment with MK801 (C) or Ketamine (D). Data was normalized to non-transduced cells in the presence of DMSO and represents the mean ± SEM of three experiments. (E) Model for lack of NMDAR activity in functional assay after protection with MK801 or Ketamine. NMDAR-ligands contained or secreted by the cells into the media activate NMDAR and lead to excitotoxicity and desensitization of the channel. Protection with MK801 or Ketamine prevent excitotoxicity, however, both compounds are difficult to wash out and do not prevent occupation of the ligand binding sites with endogenous ligand and prevent NMDAR activity in functional assays.

Fig 3: Characterization of the mode of action of positive allosteric modulators and activators of NMDAR (A,D) Effect of positive allosteric modulators of NMDAR on NR1/2A- or NR1/2B-mediated calcium flux. HEK293 cells were transduced with baculovirus encoding NR1 and NR2A or NR2B in the presence of MDL105,519 or CGP070667, and NMDAR-mediated calcium flux was measured as described in the presence of the indicated ligand concentrations. Cells were incubated with the indicated concentrations pregnenolone sulfate (PS) (A,B) or GNE-8324 (C,D) for 5 min before addition of ligand. The data was normalized to the activity in the presence of saturating amounts of both ligands in the presence of DMSO. Data represents the mean ± SEM of three experiments. (E) Sensitivity of NR1/NR2A-V783F to GNE-8324. HEK293 cells were transduced with baculovirus encoding NR1 and NR2A-V783F in the presence of MDL105,519 and NMDAR-mediated calcium flux was measured as previously described in the presence of the indicated concentrations of GNE-8324. The data was normalized to the activity in the presence of saturating amounts of both ligands in the presence of DMSO. Data represents the mean ± SEM of three experiments. (F) Sensitivity of NR1/NR2A-H44A/E226A to zinc and EDTA. HEK293 cells were transduced with baculovirus encoding for NR1 and either wildtype NR2A or NR2A-H44A/E226A in the presence of MDL105,519, and NMDAR-mediated calcium flux was measured at the EC50 of D-serine as previously described in the presence of the indicated concentrations of zinc or EDTA. The data was normalized to the activity of a DMSO-treated control. Data represents the mean ± SEM of three experiments.

Fig 4: Effects of cannabidiol (CBD) treatment on ketamine (KET)-induced alterations of NMDA receptor subunit protein expression levels in the dorsal hippocampus (top row) and the ventral hippocampus (bottom row). (A,F): GluN1; (B,G): GluN2A; (C,H): GluN2B; (D,I): GluN2A/GluN2B ratio; (E,J): A representative band of the protein of interest and the corresponding loading marker band. Data are presented as percentage of the SAL/VEH group. The optical density (OD) of each band was divided by the corresponding loading marker. Statistical analysis was performed by two-way ANOVA.

Fig 5: Effects of prenatal antipsychotics exposure on the expression levels of NMDA receptor subunits and apoptosis-related molecules in the adult hippocampus. (A) GluN1 subunit in the dorsal hippocampus (dHC) and (B) GluN1 subunit in the ventral hippocampus (vHC) were similar among groups. (C) GluN2A subunit in dHC, (D) GluN2A subunit in vHC, (E) GluN2B subunit in dHC, (F) GluN2B subunit in vHC, (G) the ratio of Bax and Bcl-2 in dHC, and (H) the ratio of Bax and Bcl-2 in vHC were similar among groups. *P<.05, Hal group vs Veh group. Hal, haloperidol; Ris, risperidone; Veh, vehicle.