Figure 1: Analysis of CTB binding and endocytosis in primary hippocampal neurons.

(a) Cultured hippocampal neurons were washed once with low K⁺ buffer then incubated with CTB-Af555 for 5 min in either low or high K⁺ buffer before fixation. Neurons were processed for immunocytochemistry using an anti-VAMP2 antibody. Scale abr, 10 μm. (b,c) Fluorescence intensity of CTB in regions of interest (ROIs) alone (b) or in co-localization with VAMP2 (Pearson’s coefficient) (c) were determined for each condition, and no significant difference in either analysis was observed between low and high K⁺ treatments. (mean±s.e.m., n=3 independent experiments, 10 cells (2 ROIs each) per experiment, Student’s t-test). (d) Cultured hippocampal neurons incubated with CTB-HRP (10 μg ml⁻¹) for 5 min in either low or high K⁺ buffer before fixation. Cells were fixed, processed for DAB cytochemistry and imaged by electron microscopy. Compartments within the presynaptic terminal containing endocytosed CTB were identified using the DAB reaction product. Compartments were identified as vesicles (red arrowheads) or endosomes (blue arrows). Scale bar, 200 nm. (e–h) Cultured hippocampal neurons were treated as in a, then either fixed (5 min) or returned to growth medium for 4 h before fixation (5 min+4 h). Following processing, the number of small vesicles (<80 nm diameter) and endosomes (>80 nm diameter) per nerve terminal were quantified and the proportion of these compartments containing CTB was determined (mean±s.e.m., n=3–4 individual neuron preparations, >20 nerve terminals per preparation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant, Student’s t-test).