NMDAR activation controls accumulation of active zone proteins at developing excitatory presynaptic terminals. (A) Fluorescence images of the AZ protein piccolo (red, left panel; white, right panel) in young axons (tau-1, green). Piccolo was at synapses since it co-localized with PSD95 (blue). Scale bars: 10 μm. (B) Treatment with APV decreased accumulation of piccolo at presynaptic terminals that co-localized with PSD95. (C) Piccolo (arrows; red, left panel; white, right panel) is recruited to presynaptic terminals induced by contact with neuroligin and actin-EGFP-expressing HEK-293 cells (green and outline). These piccolo puncta did not co-localize with PSD95 (blue). Axons were labeled with tau-1 antibodies (green) to identify contacts. (D) APV decreased accumulation of piccolo at presynaptic terminals that lack postsynaptic partners (left). APV also decreased the apparent sizes of piccolo puncta (right). Data are presented as normalized mean ± standard error of the mean, and the number of puncta quantified are indicated on each bar. *, significant difference (P-values are indicated in the figure).