Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections

Presynaptic proteins are readily identified by light microscopic immunocytochemistry, but immunodetection of postsynaptic proteins in brain sections proves difficult. We performed immunofluorescent double labeling for the NR1 subunit of the N-methyl-D-aspartate receptor (NMDAR) and the vesicular glutamate transporter 1 (VGLUT1). In material fixed with 4% paraformaldehyde, NMDAR staining in somatosensory cortex was restricted to the section surface, whereas presynaptic staining extended deeper into the tissue. Staining for postsynaptic proteins was enhanced in weakly fixed material and in tissue treated with pepsin, as previously reported, but tissue quality was impaired. Staining was also markedly enhanced, and without impairment of tissue quality, by treatment during perfusion with a mixture of inhibitors of proteases and the ubiquitin/proteosome system. We performed quantitative analysis of confocal images to study how immunostaining varies with depth into the tissue. Virtually all puncta immunopositive for VGLUT1 colocalized with synaptophysin puncta; these presynaptic puncta were most numerous 1-2 microm beneath the section surface. In contrast, puncta immunopositive for the NR1 subunit were most numerous at the surface, as were puncta immunopositive for the NR2 subunit, SynGAP, and CaMKII. Punctate staining for all postsynaptic proteins, but not presynaptic markers, was substantially enhanced in material pretreated with antiproteolytic agents. The large majority of NR1-positive puncta at the surface associated with VGLUT1 in this material are likely to represent synaptic contacts. Approximately eighty-five percent of VGLUT1-positive puncta in layers II-III of SI are associated with NR1-positive puncta, and approximately 80% are associated with NR2, SynGAP, and CaMKII. This approach may permit systematic analysis of the chemistry of glutamatergic synapses with light microscopic immunocytochemistry.