Figure 4.

Representative confocal micrographs showing that the P2X7R immunoreactivity is located predominantly in nerve terminals, but not in glial cells, of all sub-regions of the hippocampus of MTLE-HS patients. Synaptic nerve terminals were identified with an antibody against the vesicle-associated membrane protein 1 (VAMP-1 or synaptobrevin 1), whereas astrocytes were stained with an antibody against the glial fibrillary acidic protein (GFAP). Note that VAMP-1-positive nerve terminals (red) are endowed with the P2X7R (green; panel A), but no significant co-localization was observed between P2X7R (green) and GFAP (red; panel B); scale bars = 50 μm. Data in panels (C) and (D) correspond to staining overlap and Pearson’s Coefficient (ρ) parameters calculated from three to four confocal micrographs per individual; at least three individuals from each group, control, and MTLE-HS, were analyzed. These parameters were automatically calculated per image with Olympus Fluoview 4.2 Software (Olympus FV1000, Tokyo, Japan) and were used to estimate the co-localization of P2X7R and type-specific cell markers (yellow staining). Overlap between two colors gives values between +1 (total overlap) and 0 (no overlap); the Pearson’s Coefficient (ρ) is a measure of the linear correlation between two variables (stainings), giving values between +1 and −1 inclusive, where 1 is total positive correlation, 0 is no correlation, and −1 is total negative correlation. Higher magnification images show that the P2X7R immunoreactivity also co-localizes with the synaptic vesicle glicoprotein synaptophysin (Synapt), which is one of the most commonly used neuronal cell markers in neuropathology (Panel E), but not with GFAP (Panel F). Nuclei are stained with DAPI; cross-reactivity for the secondary antibodies was tested in control experiments in which primary antibodies were omitted (negative controls).