FIGURE 9.

Knockdown of Egr-1 blocks NMDA-induced AMPAR endocytosis in mouse hippocampal neurons in culture. Neurons infected with Ln-shRNA-ctl or Ln-shRNA-Egr-1 were treated with NMDA or vehicle and subjected to antibody feeding protocol to analyze GluA2 endocytosis by confocal microscopy. Images were captured, and GluA2 puncta were quantified. Based on puncta numbers, relative distributions and relative endocytosis values were determined. A, representative micrographs of neurons infected and treated as indicated showing GFP, surface GluA2, and internal GluA2. In Ln-shRNA-ctl-infected neurons, GluA2 is found on both surface and internal pool. When these neurons are treated with NMDA, the number of surface GluA2 is significantly reduced with a concomitant increase in the internal pool. In shRNA-Egr-1-infected and vehicle-treated neurons, more GluA2s are on the surface than in the internal pool; and they remain on the surface when neurons are treated with NMDA. Thus, knockdown of Egr-1 blocks NMDA-induced GluA2 endocytosis. B, representative micrographs of dendrites of infected neurons in higher magnifications. C, relative distribution. Fifteen GFP-positive (hence infected) neurons were randomly chosen from three different cultures from each group. Number of puncta along 30 μm of dendrite in each neuron was scored using Volocity software. The average number of puncta within 10 μm of the dendrite from three independent experiments were 3.9 ± 0.3 (surface) and 2.7 ± 0.5 (internal) in Ln-shRNA-ctl-infected and vehicle-treated neurons, 1.4 ± 0.25 (surface) and 5.8 ± 1.5 (internal) in Ln-shRNA-ctl- and NMDA-treated neurons, 4.5 ± 0.4 (surface) and 1.3 ± 0.2 (internal) in Ln-shRNA-Egr-1 infected and vehicle-treated neurons, and 3.8 ± 0.4 (surface) and 1.6 ± 0.3 (internal) in NMDA-treated neurons. Based on these numbers, relative distribution values were determined. The relative distribution value is the number of puncta in a fraction (surface or internal) of a group divided by the total (sum of the puncta in the two fractions (surface and internal)) of that group. Values are expresses as the % of the total. D, relative GluA2 endocytosis. To determine the relative amount of NMDA-induced GluA2 endocytosis, the % value of surface GluA2 from NMDA-treated neurons from panel C of a group was subtracted from the corresponding % value of surface GluA2 from vehicle-treated neurons of that group. Resulting value is then expressed as the % of the total GluA2 (% value of surface GluA2 in corresponding vehicle-treated neurons of that group). **, p < 0.005 (n = 3) with respect to Ln-shRNA-ctl-infected cells.