For all parts of the quantitative analysis, the researcher was kept blind to the treatment of the animal. Digital images were captured at a magnification of 50,000×, by making systematic sweeps within grid squares to avoid resampling of synaptic profiles. Within the two-dimensional digitized images, synapses were identified as asymmetric and thus presumably excitatory, based on the presence of thick postsynaptic densities (PSDs) aligned within the intracellular surfaces of plasma membranes juxtaposed by vesicle-filled axon terminals. So as to maximize randomness, we evaluated all synapses encountered, strictly in the order that they were encountered. Usually, at the magnification of 50,000×, we could not resolve PEG particles until the digitized images were averaged across multiple frames. This feature of image capturing allowed us to ensure that we did not preselect synapses to analyze or not analyze, based on immunoreactivity.
