Mobilization of AMPA receptors into silent synapses via the activation of NMDA receptors by spontaneous synaptic activity. A, Double staining of high-density cultured cortical neurons by antibodies against AMPA receptor subunits (left, GluR1/2/3) and an NMDA receptor subunit (middle, NR1) after the neurons were incubated in aCSF in the presence (top panels) and absence (bottom panels) of d,l-APV. Right panels are overlays of the left and middle panels(GluR1/2/3, red; NR1, green).B, Double staining of high-density cultured cortical neurons by antibodies against AMPA receptor subunits (GluR1/2/3,red) and synaptophysin (green) after the withdrawal of d,l-APV. C, Double staining of NR1 (red) and synaptophysin (green) after the withdrawal ofd,l-APV. Scale bar: A–C, 10 μm.D, The number of AMPA receptor clusters (GluR1 and GluR2/3) is increased significantly after the withdrawal ofd,l-APV (filled bars) compared with control (open bars; neurons incubated in aCSF withd,l-APV). TTX and 0 mm Ca²⁺blocked this increase. E, Withdrawal ofd,l-APV had a negligible effect on the number of NR1 clusters. F, The effect of withdrawal ofd,l-APV on the apparent size of AMPA receptor clusters and NR1 clusters. G, The proportion of silent synapses was decreased significantly after the withdrawal of d,l-APV (filled bars) compared with control (open bars). Silent synapses refer to synapses that contain only clustered NR1 receptors but no clustered AMPA receptors (GluR1 and GluR2/3). *p < 0.05; ***p < 0.001.