(A) Representative image showing the heterogeneous distribution of GluRIIA-RFP (left panel) at a third instar muscle 4 NMJ. More uniform GluRIIB-GFP PSD puncta can also be observed over the much dimmer myrGCaMP6s (second panel). BRP distribution (third panel) and Pr heatmaps (right panel) for the same NMJ are shown. Several bright GluRIIA fields (intensity two standard deviations above average) are marked with white arrows. The correlation between AZ Pr and GluRIIA-RFP (B), GluRIIB (C) and BRP (D) fluorescence intensity is plotted. (E) Representative images showing distribution of GluRIIA, GluRIIB and BRP, without co-expression of myrGCaMP6s. Synapses containing bright GluRIIA puncta have GluRIIB predominantly localized to the periphery of the PSD (arrows), surrounding a GluRIIA core. These AZs have higher BRP intensities as well. (F) Average fluorescence line profiles showing GluRIIA, GluRIIB and BRP normalized to fluorescence range across average PSDs, separated into two groups according to their GluRIIA brightness, with ‘bright’ PSDs based on their GluRIIA intensity (two standard deviations above average). The peripheral distribution of GluRIIB around central GluRIIA cores was most obvious for bright GluRIIA-positive PSDs that were shown to be more active during stimulation. Correlation between GluRIIA-RFP (G) or GluRIIB-GFP (H) with BRP intensity at individual AZs.