Figure 3.

Converse effects of α-synuclein overexpression/absence on recycling pool kinetics. A, DIV 17–21 transgenic (or WT) boutons were loaded with FM4-64, a string of boutons were selected (#1–3, “pre-bleach”), and a single bouton (#2) was selectively photobleached (“post-bleach”). Note recovery of fluorescence over time in the grayscale (left) and pseudocolor (right) images. Scaled image below shows axon continuity (arrowheads). Variations in intensities of the three boutons over time are graphically represented below. Note partial recovery of FM4-64 in the bleached bouton, reflecting exchange between boutons (see Results). B, Top, Maximum FM4-64 recovery inversely correlated with α-syn:GFP intensities in the same boutons (r = 0.56; p < 0.0001). Bottom, Kinetics of FM4-64-recovery was also significantly diminished in the upper 50^th percentile of overexpressers (see Results). N ∼40 boutons/group, ***p < 0.0001. C, Conversely, the rate of FM4-64 FRAP recovery was significantly faster in DIV 17–21 α-synuclein^−/− neurons, compared with their WT/+/− littermates (containing α-synuclein). (N ∼ 60–90 boutons/group, ***p < 0.0001). D, Pooled FM4-64-FRAP data from all experiments show an incremental diminution of FM4-64 mobility upon increasing α-synuclein dosage. ***p < 0.0001, one-way ANOVA.