Figure 2.

Knock-down of NR1 in the hippocampus. A, Knock-down constructs, including the design of the sh molecules. CAG, Cytomegalovirus immediate early enhancer–chicken β-actin hybrid promoter. B, Representative images of dYFP fluorescence at three different levels of the hippocampus, examined 6 weeks after unilaterally injecting shNR1-carrying vector. The arrowhead in the first panel points to an uninjected side for comparison. Panels 1 and 2 represent higher-magnification views of their corresponding boxes. Bodies of pyramidal cells (arrowhead) and their axonal projections (arrow) are shown in panel 1, and granule cells are shown in panel 2. Scale bars: Left, 500 μm; middle, 500 μm; right, 25 μm. C, Representative immunohistochemistry images from brains injected with AAV-shNR1 unilaterally. On the injected side, highlighted by the strong presence of dYFP, expression of NR1 and NR2A, but not of another glutamate receptor, mGluR1, are reduced (arrowheads) compared with the contralateral, uninjected side. Scale bar, 1 mm. D, E, Reduction in NR1 expression (D) and decreased binding of [³H]MK-801 (E) (arrowheads) in shNR1-injected compared with shdYFP-injected hippocampi. Scale bar, 500 μm. F, NR1 protein expression by Western blot in hippocampal extracts from uninjected, shdYFP-injected, and shNR1-injected brains. G, Quantitative immunoblot analysis of NR1 expression as in F. Results are the mean ± SEM of three independent experiments. *p < 0.05 (one-way ANOVA). H, Western blots showing a reduction in NR1, NR2A, and NR2B expression in shNR1-injected hippocampi compared with shdYFP controls. Differential presence of dYFP confirms expression from both constructs. F, H, β-Actin served as a loading control. Immunoblot experiments were repeated three times, yielding similar results.