(A) Representative images of NMJs on muscle six immunostained with antibodies that recognize GluRIIA, GluRIIC, and GluRIID receptor subunits in wild type (w1118), GluRIIA mutants (w1118;GluRIIASP16), and G14 > GluRIIARNAi (w1118;G14-Gal4/+;UAS-GluRIIARNAi/+). (B) Quantification of the mean fluorescence intensity of individual GluR puncta reveals GluRIIA subunits are virtually undetectable at NMJs of both GluRIIA mutants and G14 > GluRIIARNAi, while the essential subunits GluRIIC and GluRIID are moderately reduced, reflecting expression of the remaining GluRIIB-containing receptors. (C) Left: Schematic illustrating the composition of GluRIIA-containing and GluRIIB-containing postsynaptic receptor subtypes at the Drosophila NMJ. Right: Quantitative PCR analysis of GluR transcript levels for GluRIIA/B/C/D/E subunits in G14 >GluRIIARNAi normalized to G14/+. (D) Representative electrophysiological traces of EPSP and mEPSP recordings in the indicated genotypes. (E–G) Quantification of mEPSP amplitude (E), EPSP amplitude (F), and quantal content (G) in the indicated genotypes. Note that while mEPSP amplitudes are reduced to similar levels in GluRIIA mutants and G14 > GluRIIARNAi, EPSP amplitudes remain similar to wild type because of a homeostatic increase in presynaptic glutamate release (quantal content). (H) Quantification of mEPSP amplitude and quantal content values of the indicated genotypes normalized to wild-type values. Error bars indicate ± SEM. Asterisks indicate statistical significance using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test: (****) p<0.0001, (ns) not significant. Detailed statistical information for represented data (mean values, SEM, n, p) is shown in Supplementary file 1.