Figure 4

AAV-BRI-Aβ42 and AAV-UBI-Aβ42 induce functional, morphological, and structural synaptic alterations. (a) Flow cytometry FASS-LTP identifies synaptosomes by size. Based on calibrated beads, we set a threshold in the forward scatter (FSC-H) channel, as well as a gate region. We excluded small particles having a size equivalent to 0.5 µm calibrated beads (red particles), while selecting particles of ~ 1.0 µm calibrated beads (blue particles). (b) Forward-Side (FSC-SSC) profile of particles in the synaptosomal P2 fraction isolated from the hippocampus. The inside rectangle (gate) selects putative synaptosomes according with to size (~1.0 µm = size-gated synaptosomes). (c) In size-gated synaptosomes, FASS-LTP identifies potentiated synapses by tracking GluA1 and Nrx1β surface staining. To induce cLTP in samples from each experimental group (EGFP, AAV-BRI-Aβ42 and AAV-UBI-Aβ42 samples were run in parallel), synaptosomal P2 fractions maintained in Mg2+ -free external solution were sequentially stimulated using 500 µM glycine (15 min) and 37 mM KCl (30 min). As controls, equivalent volumes of external solution were added to a parallel set of synaptosomal fractions maintained in external solution (basal). Representative two-color parameter plots show GluA1 (x-axis) and Nrx1β (y-axis) surface levels in basal and cLTP conditions. Thresholds for endogenous/non-specific fluorescence for each marker were set by staining with secondary antibodies only. (d) Values normalized to the basal state in each experimental group, mean ± SEM. Basal vs cLTP: EGFP, *p = 0.041 (n = 6); AAV- BRI-Aβ42, P = 0.244 (n = 7); AAV-UBI-Aβ42, P = 0.461 (n = 6).