Figure 4: Local elevation of endocytic activity in proximal dendrites.

(a) Top, schematic diagram of the membrane topology of pHluorin-CD4-tdTomato in the endocytic pathway. Fluorescent and non-fluorescent pHluorin are indicated with filled and open pentagons, respectively. Bottom, dendritic regions observed by high-magnification imaging. (b) Representative images of the proximal and distal dendrites of C4da neurons expressing pHluorin-CD4-tdTomato (from 12 and 11 experiments, respectively). For each region, images are taken from the same branches at 0.5 and 4.5 h a.p.f. Green arrows, varicosities; blue arrows, thin regions; magenta arrowheads, tdTomato⁺ pHluorin⁻ endocytic vesicles; an open arrowhead, the primary dendrite; and a bracket, a dendritic region with no apparent structural changes during the imaging period. Scale bars, 20 μm. (c) High-magnification images of a boxed area in b. A ratiometric image by dividing the image of the green channel by that of the red channel is shown in the bottom. (d) Quantification of the number of tdTomato⁺ pHluorin⁻ endocytic vesicles in the proximal and distal dendrites at 0.5 and 4.5 h a.p.f. **P<0.001, paired t-test. NS, not significant. (e) Quantification of dendritic branch width in relation to the distance from varicosities. The thick line corresponds to the average of individual branches (grey). **P<0.05, Tukey’s test: compared with branch width at 54 μm from a varicosity (n=6 dendrites). (f) Histogram of the number of endocytic vesicles in relation to the distance from varicosities (n=71 vesicles).