Figure 2: The flux of retrograde CTB carriers is activity-dependent in cultured rat hippocampal neurons.

(a) CTB-Af488 labelling was performed in nerve terminals of hippocampal neurons cultured in microfluidic devices. CTB retrograde trafficking was captured in the observation window along the axon channels (boxed). Scale bar, 20 μm. (b) Hippocampal neurons cultured in microfluidic chambers were pulsed at 37 °C for 5 min with CTB in low- or high-K⁺ buffers, followed by wash-off of the pulse solutions before a chase in the original culture medium for 2 h. They were then live-imaged with confocal microscopy. (c) Time-lapse images of CTB carriers of axons as described in a; arrowheads delineate the retrograde carriers. Asterisks indicate stationary carriers. Scale bar, 10 μm. (d) Imaris tracing of CTB tracks in representative movies (colour-coded for average speeds of 0–1.5 μm s⁻¹). Scale bar, 10 μm. (e) Number of retrograde CTB carriers after high or low K⁺ treatment. (f) Representative kymographs of CTB carriers along a single axon. Tracing demonstrates track displacement and static periods (dwelling, red arrowheads). x— Scale bar, 5 μm; y— Scale bar, 20 s. (g,h) The average track speed (g) and dwelling time (h) were determined in low and high K⁺. (mean±s.e.m., n=15 and 20 channels for low and high K⁺ respectively, data from three independent experiments, Student’s t-test, **P<0.01, ***P<0.001).