Figure 9: Activity-dependent CTB retrograde flux is dependent on synaptic activity but not endogenous BDNF secretion.

(a) Hippocampal neurons cultured in microfluidic chambers were pretreated with 100 pM BoNT/A for 24 h or 20 μg ml⁻¹ BDNF collators (BDNF blocking antibody; TrkB-Fc) for 30 min before 5 min CTB pulse in low or high K⁺ buffers as indicated. After wash-off, the chambers were chased in the original culture medium containing BoNT/A or BDNF collators for 2 h, then live-imaged with confocal microscopy. For western blotting, neurons were collected without the 2 h chase. (b) Imaris tracing of CTB tracks in representative live-imaging movies as treated in a, colour-coded average speed 0–3 μm s⁻¹. Scale bar, 5 μm. (c) Number of retrograde CTB carriers with indicated treatments (mean±s.e.m., n=14, 31, 26, 13, 17 and 17 for low K⁺, high K⁺, high K⁺+anti-BDNF, high K⁺+TrkB-Fc, low K⁺+BoNT/A and high K⁺+BoNT/A, respectively, data are from three independent cultures, student’s t-test, *P<0.05; ***P<0.001; NS, not significant). (d) Western blot of treated hippocampal neurons showing that the high K⁺-induced increases in p-TrkB and p-CREB were not affected by BDNF collators but were abolished by BoNT/A treatment; total TrkB and CREB protein levels were used as controls. (e) Quantification of (d). (mean±s.e.m., n=3, data from three independent cultures, *P<0.05; **P<0.01; ***P<0.001; NS, not significant; Student’s t-test).