Figure 5: Activation of TrkB controls the activity-induced increase in CTB retrograde flux.
(a) Representative wide-field and SIM images of axons in microfluidic chambers that were labelled with CTB (magenta) under either low K+ (left) or high K+ (right) conditions. TrkB antibody (green) was used to label endogenous TrkB receptors. Boxed regions magnified in the lower panels, and aligned CTB vesicles are pointed out with arrowheads. Scale bar, 1 μm. (b,c) Quantification of the ratio of CTB overlapping with TrkB (b), and the ratio of TrkB overlapping with CTB (c) from SIM images. (Mander’s coefficient, mean±s.e.m., n=39 (low K+) or n=46 (high K+) channels from three independent neuron preparations. ***P<0.001, Student’s t-test). (d) Activation of th TrkB pathway was examined with an antibody against phosphorylated Tyr707/706 of TrkB receptors (top panels). Pretreatment with 0.5 μM ANA-12 or 100 nM K252a for 30 min significantly inhibited the high K+-induced p-TrkB increase; β-tubulin III and DAPI (bottom panels) were used to show the density of neuron layers. Scale bar, 20 μm. (e) Quantification of d. (mean±s.e.m., n=29 (low K+) or 30 (high K+) channels from three independent neuron preparations. ***P<0.001, Student’s t-test). (f) Western blot of DIV 14 hippocampal neurons pretreated with 0.5 μM ANA-12 or 100 nM K252a for 30 min showing the same abolition of the high K+ induced increase in p-TrkB and phosphorylated CREB protein (p-CREB), which represented the downstream response; total TrkB and CREB protein levels were used as controls. (g–h) Quantification of f. (mean±s.e.m., n=3, data from three independent cultures, *P<0.05, Student’s t-test).
