Viral Manipulation of NMDAR Expression

We performed experiments with cell cultures to validate the viral-mediated manipulation of NMDAR expression. Primary neuronal cultures were prepared as previously described [29 (link)]. Briefly, cortical neurons at E18 from pregnant female rats were dissected, dissociated and plated in wells (~1 million cells/well) and cultured in standard media. Viruses were added at DIV5 stage. To validate GluN1 overexpression, we used the following viruses: AAV1-CMV-Cre-GFP (1 × 10¹³ GC/ml titer, 400 nL) and LV-Syn-DIO-GluN1–2A-tdTomato (2 × 10⁹ TU/ml titer, 2 μL). Using these viruses, we made the four well conditions: GluN1, no Cre; GluN1, Cre; no GluN1, Cre; no GluN1, no Cre (no viruses added). After at least 21 days of culture, we performed Western blot for GluN1 (#5704S, Cell Signaling, 1:500, rabbit monoclonal antibody) and beta-actin (#4967S, Cell Signaling, 1:1000, rabbit polyclonal antibody). To validate GluN2B overexpression, we used identical procedures except that we used LV-Syn-DIO-mTagBFP2–2A-GluN2B (1 × 10⁹ TU/ml titer, 2 μL) and GluN2B antibody (#4207S, Cell Signaling, 1:1000, rabbit polyclonal antibody). Bands were quantified using Bio-Rad Image Lab software and raw band intensity values were normalized to beta-actin levels.