Figure 14.

Impairments in synaptic vesicle endocytosis and membrane trafficking in Ub^G76V-GFP-Syb2 mice. A–D, EM images of synaptosomes isolated from control (A, B) and Ub^G76V-GFP-Syb2 mice (C, D). A and C show examples of synaptosomes at rest; B and D show examples of synaptosomes containing HRP-labeled SVs (arrowheads) after incubation with 60 mm KCl for 15 min. E, Bar graphs showing the average number of HRP-positive SVs per synaptosome after depolarization with 60 mm KCl for 15 min. A total of 215 synaptosomes from Ub^G76V-GFP-Syb2 mice (N = 3) and 209 synaptosomes from control mice (N = 3) were analyzed. The average numbers of HRP-positive SVs per synaptosome from Ub^G76V-GFP-Syb2 mice are significantly (p = 0.0493) reduced in Ub^G76V-GFP-Syb2 mice (6.24 ± 1.66 vesicles, N = 3 mice) compared with control mice (11.42 ± 1.55 vesicles, N = 3 mice). F, Schematic diagram of GFP-Syb2-mOrange and Ub^G76V-GFP-Syb2-mOrange. G, Average traces of fluorescence changes in synaptic boutons expressing GFP-Syb2-mOrange (n = 8) and Ub^G76V-GFP-Syb2-mOrange (n = 9). H, Analysis from the same experiments as in G. The peak fluorescence generated during 20 Hz stimulation is normalized to the peak fluorescence generated after NH₄Cl treatment. The average peak values in synaptic boutons expressing Ub^G76V-GFP-Syb2-mOrange are significantly (p = 0.0067) lower than those expressing GFP-Syb2-mOrange. Scale bar: A–D (in C), 300 nm.