Figure 6.

2D Ultrastructural Analysis of Putative Endocytic Profiles in Dock10-Cre;Ai32 Slices after High-Frequency Stimulation (100 × 5 ms LPs at 20 Hz, 2 mM Ca²⁺)

(A and B) Transmission electron micrographs of stimulated MFBs. Membrane invaginations are captured at peri-active zonal (AZ) sites (white arrowheads).

(C–F) Peri-AZ endocytic pits (white arrowheads). Electron dense material occasionally observed at the invagination lacked the density or periodicity of clathrin coats.

(G–K) Bulk-like larger endocytic profiles (white arrowheads).

(L–O) Clathrin-coated vesicles (open black arrowheads) in the cytoplasm (L), in the process of forming at the plasma membrane (M), or from elongated organelles (N). Clathrin-coated protrusions emerging from invaginations were only extremely rarely observed (O). NS, 131 MFBs; ST, 120 MFBs; S, 118 MFBs.

(P) Relative incidence of coated and non-coated invaginations per MFB profile. NS, no stimulation (black); ST, stimulation in the presence of TTX (gray); S, stimulation (blue).

(Q) Relationship between pore diameter (P) and invagination depth (I) for coated intermediates across all experiments analyzed (Pooled data from Dock10-Cre;Ai32 and Nex-Cre;Ai32 slices). Dashed lines indicate respective mean values.

(R) Relationship between pore diameter (P) and invagination depth (I) for non-coated (R) endocytic intermediates. Dashed lines indicate respective mean values.

(S) Relationship between pore diameter (P) and invagination depth (I) for non-coated (R) endocytic intermediates detected in stimulated MFBs at the peri-AZ (within 0 to 200 nm from AZ; red) and more distal (>200 nm from AZ; green) sites. Dashed lines indicate respective mean values.

d, dendrite; mfb, mossy fiber bouton; PSDs, black arrowheads; sp, spine. Scale bars: (A and B), 1 μm; (C–K and O), 200 nm; (L–N), 100 nm. For (P, R, and S): NS, three cultures, three slices, 47 MFBs; ST, three cultures, three slices, 39 MFBs; S, three cultures, three slices, 38 MFBs.

See also Table S1E and Figures S6 and S7.