Figure 11

GluN2 and GFP transgene expression in vivo mediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).