Figure 6: Pharmacological inhibition of TrkB activation abolishes the activity-dependent increase in CTB carrier flux undergoing retrograde axonal transport.

(a) Representative images of CTB retrograde flux inside the axon channels of hippocampal neurons pretreated with 0.5 μM ANA-12 or 100 nM K252a for 30 min before high K⁺ pulse-chase. Neurons pulse-chased with low or high K⁺ alone were used as controls. Time-lapse images were tracked with Imaris software, and the tracks shown are colour-coded based on the average speed. Scale bar, 10 μm. Both inhibitors significantly abolished thehigh K⁺ stimulation-induced CTB flux, as reflected by the decreased number of CTB carriers trafficking through the axon channels per second as quantified in b, (mean±s.e.m., n=78 (low K⁺), 81 (high K⁺), 83 (high K⁺+ANA-12) and 45 (high K⁺+K252a) tracks, data from 3 independent neuron preparations, *P<0.05, **P<0.01,***P<0.001, Student’s t-test). (c) Representative SIM images of axons in microfluidic chambers. Neurons were labelled with CTB (red) under either low K⁺ or high K⁺ conditions. In the bottom groups, neurons were pretreated with ANA-12 or K252a for 30 min before high K⁺ stimulation. TrkB antibody (green) was used to label endogenous TrkB receptors, Scale bar, 2 μm. Boxed regions are magnified in the right panels, Scale bar, 200 nm. (d,e) Both inhibitors significantly reduced the number of small CTB carriers with a diameter <150 nm (d), as well as the ratio of CTB overlapping with TrkB as quantified in (e). (Mander’s coefficient, mean±s.e.m., n=39 (low K⁺), 46 (high K⁺), 32 (high K⁺+ANA-12) and 25 (high K⁺+K252a) tracks, data from three independent neuron preparations, *P<0.05, **P<0.01,***P<0.001, Student’s t-test).