Preadsorption greatly reduced GluR1 immunolabeling at asymmetric synapses. At the dilution of 400 μg/ml of the control peptide, 93% of the synapses were devoid of even a single particle of PEG labeling (Fig. 5A). In contrast, ultrathin sections from paired animals’ LA that were incubated with the antibody but in the absence of the control peptide exhibited immunolabeling of approximately 30% of the synapses, and of these, 88% were labeled by just one PEG particle (Fig. 5C). Preadsorption reduced the proportion of asymmetric synapses labeled for GluR2/3 as well. In the presence of the control peptide, 95% of the synapses were devoid of PEG particles (Fig. 5B). In contrast, when the ultrathin section was incubated in the presence of the antibody but in the absence of the control peptide, only 30% of the synapses lacked PEG particles and approximately 70% of the synapses were immunolabeled with one, two, three, or more PEG particles. Of the GluR2/3-immunolabeled synapses, 48% exhibited one PEG particle (Fig. 5D). Based on these observations, we concluded that the association of one PEG particle with a spine or shaft synapse could be considered the threshold for GluR1 or GluR2/3 immunolabeling.
