Figure 6. Accumulation of endosome‐like vacuoles (ELVs), reduction in clathrin‐coated membranes, and vesicle depletion of the membrane‐distal part of the ribbon indicate impaired synaptic vesicle regeneration in AP‐2μ‐deficient IHCs.

• A, B
  Electron micrographs of random ultrathin sections of ribbon synapses of resting and K⁺‐stimulated (50 mM for 15 min) AP‐2μ control (A) and AP‐2μ ^fl/fl :Cre:GFP (B) p14‐17 IHCs. Coated structures (white arrowhead) are present in resting and stimulated AP‐2μ control IHCs but clearly reduced in AP‐2μ ^fl/fl :Cre:GFP IHCs near synaptic ribbons. A strong accumulation of ELVs was observed near AZs of stimulated IHCs of both genotypes (black asterisks). A depletion of vesicles at the membrane‐distal part of the ribbon was found in stimulated AP‐2μ ^fl/fl :Cre:GFP IHCs. Analysis was performed on random sections of ribbon‐occupied AZs derived from several IHCs per genotype: AP‐2μ control rest n _{random section} = 27; AP‐2μ control stim n = 25; AP‐2μ ^fl/fl :Cre:GFP rest n = 25; and AP‐2μ ^fl/fl :Cre:GFP stim n = 56. 1–2 mice were used per condition. Scale bars, 100 nm.
• C–E
  Electron tomography of stimulated AP‐2μ control (C) and AP‐2μ ^fl/fl :Cre:GFP (D, E) IHC synapses corroborated the depletion of ribbon‐associated synaptic vesicles (black arrowhead in D; also see representative virtual sections (C, D, E, black arrowhead in D points to the membrane‐distal ribbon that lacks ribbon‐associated vesicles.) and models reconstructed from tomograms, C', D', E' (C'' and D'', top view on the AZ without ribbons); red: ribbon; magenta: presynaptic density; blue: AZ membrane; yellow: ribbon‐associated SVs; green: membrane‐proximal SVs; and light blue > 70 nm: ELVs). Budding of non‐coated vesicles (magenta arrowheads, E, E') from an ELV (yellow asterisk) was observed in AP‐2μ ^fl/fl :Cre:GFP IHCs near the membrane‐distal end of the ribbon. Scale bars, 100 nm.
• F
  Quantification of perisynaptic non‐SV membrane organelles in random sections: in addition to coated structures such as clathrin‐coated pits at the membrane and coated vesicles (largely absent in AP‐2μ ^fl/fl :Cre:GFP IHCs), we quantified ELVs in random sections, defined as non‐coated endocytic intermediates round or polymorphic in shape and > 70 nm in diameter. CCP/CCV and CCS are depicted in a different scale compared to ELVs.
• G, H
  (G) Fewer ribbon‐associated SVs but (H) normal number of membrane‐proximal SVs in random sections from stimulated AP‐2μ ^fl/fl :Cre:GFP IHCs.

Data information: Data are presented as mean ± SEM. Statistics in (F‐H) was done on random sections using one‐way ANOVA followed by Tukey's multiple comparisons in Igor Pro software. Statistical significance is indicated by *(P < 0.05), **(P < 0.01), or ***(P < 0.001).