Fig 2: Pharmacological inhibition of TrkB activation has no effect on LC3-positive autophagosomes.(a) Representative images of axons in microfluidic chambers labelled with CTB under high K+ or pretreated with ANA-12, K252a for 30 min. TrkB antibody was used to label endogenous TrkB receptors, and co-localization is indicated with arrowheads. Scale bar, 10 μm. (b–d) Both inhibitors significantly reduced the number of CTB carriers (b) but had no significant effect on the level of the autophagosome marker LC3 in the same region of interest (c). The ratio of CTB overlapping with LC3 was also not affected (d). (Mander’s coefficient, mean±s.e.m., n=39 (high K+), 39 (high K++ANA-12) and 38 (high K++K252a) channels, data from two independent neuron preparations, ***P<0.001, NS, no significant difference; Student’s t-test).

Fig 3: 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).

Fig 4: CTB retrograde carrier flux is composed of small aligned vesicular compartments.(a) Dual-colour SIM on axon channels of microfluidic chambers labelled with CTB under either low K+ (left) or high K+ (right) conditions, β-tubulin III was used to label the axon fibers. Scale bar, 5 μm. Boxes are magnified in the lower panels; original wide-field images are compared with the SIM images of the same region (Scale bar, 1 μm), and individual CTB carriers are further magnified in yellow boxes (Scale bar, 100 nm). The resolution is ∼100 nm. Quantification of total (b) and small CTB carriers (diameter<150 nm) (c) from SIM images (Mander’s coefficient, mean±s.e.m., n=27 for low K+, n=31 for high K+, ***P<0.001, data from four independent preparations, Student’s t-test). (d) Electron microscopy of axons from neurons pulse-chased with CTB-HRP as described in a. Small vesicular carriers—red arrows, multivesicular body—blue arrow. Scale bar, 200 nm. (e) Quantification of the number of carriers per μm neurite length per axon channel. (mean±s.e.m., n=7 (low K+) or 16 (high K+) channels from two independent neuron preparations. **P<0.01) (f) Size distribution of CTB carriers in low K+ and high K+ stimulated cells (n=3 independent neuron preparations). (g) Confocal microscopy of axons from neurons pulse-chased with CTB as described in (a). Endogenous TrkB immunostaining is shown in green, CTB vesicles that co-localized with TrkB are indicated with arrowheads. Scale bar, 5 μm. (h) Quantification of CTB-positive (CTB+) and TrkB-positive (TrkB+) carriers after low K+ or high K+ treatments (n=10 and 11 for high K+ and low K+, *P<0.05, **P<0.01, data from two independent preparations; NS, not significant, Student’s t-test).

Fig 5: PNN breakdown restores ipsilateral hemidiaphragm function 12 weeks after cervical SCI. a Experimental protocol. b, c Average amplitude of b ipsilateral and c contralateral diaEMG. Filled circles = responders; unfilled circles = animals in d–g; triangles = non-responders; stars = tonic activity shown. d–g Representative EMG recordings at baseline and following the 4 weeks of treatment for all groups. Data panels presented from the same animal. For b–g n = 6 per group, baseline n = 24. h–aa Immunohistochemistry and intensity readings at the ipsilateral C4 PMP utilizing Ctb (red) and either (green) h–l 5HT, m–q TrkB, r–v 2B6, or w–aa WFA (n = 4 per group). For all EMG or graph panels, treatment groups = baseline (grey); saline+air (yellow); saline+IH (red); ChABC+air (light blue); ChABC+IH (blue). *p < 0.05, **p < 0.01 and ***p < 0.001. If no post-hoc result is shown, comparison was not-significant. Scale bar = 50 μm. For all panels: values represent mean ± SEM