Figure 6. NMDAR inhibition results in increased NR1 targeting to the PSD.

Neurons were exposed to 100 μM APV, 1 μM Pb²⁺, or both from DIV7 - DIV12

(A-D) Representative images of control (A), 100 μM APV (B), 1 μM Pb²⁺ (C), and Pb²⁺ + APV (D)-treated neurons stained for NR1 (green) and PSD-95 (red). Colocalization is shown as yellow or orange color. Scale bar= 20 μm.

(E) Quantification of colocalization of NR1 and PSD-95. Both Pb²⁺ and APV treatments increase colocalization but APV treatment does so to a higher magnitude. The colocalization calculation is: ${\text{Area}}_{(\text{colocalized}\text{pixels})}/{\text{Area}}_{(\text{Total}\text{dendritic}\text{NR}1)}$. APV significantly reduces total NR1 dendritic area, resulting in increased percent colocalization. Data are the result of 3 independent trials with 12–14 neurons per treatment.

(F) NR1 immunofluorescent parameters. APV exposure significantly reduces NR1 puncta parameters, but Pb²⁺ or Pb²⁺ + APV treatments do not. Data are the result of 3 independent trials with 14–15 neurons per treatment.

(G) PSD-95 immunfluorescent parameters. No changes in spine density, area, or intensity are observed after any treatment, but neurons treated with APV + Pb²⁺ exhibited a decrease in PSD-95 puncta intensity. Data are the result of 3 independent trials with 14–15 neurons per treatment.

(H-I) Representative immunoblot of whole cell protein probed for NR1 and Actin (H). Quantification in (I). APV significantly reduces NR1 levels, but Pb²⁺ and co-exposure treatments do not, although they both exhibit non-significant reductions in NR1 protein. Data are the result of 3 independent trials.

Data are represented as the mean ± SEM. * = significance from control, @ = significance from 1.0 μM Pb²⁺, and # = significance from all other conditions (Fisher’s Protected LSD).