Figure 6.

BoNT/C1 blocks Netrin-1-mediated chemoattraction of hippocampal axons. A–D, Examples of hippocampal explants cocultured in collagen gels with control or Netrin-1-expressing cell aggregates. Explants cocultured with control cells show a radial growth (A). Explants cocultured with Netrin-1-secreting cells exhibit chemoattraction toward the Netrin-1 source (B). Explants cocultured with Netrin-1-expressing cells and BoNT/A also display chemoattraction (C). Netrin-1-dependent chemoattraction is blocked in hippocampal explants cultured with BoNT/C1 (D). Low-power images are shown in the first row of panels. High-power magnifications of the proximal and distal quadrants are illustrated in the second and third rows. E, Western blots of protein samples from lysates of hippocampal explants cultured with BoNT/A, BoNT/C1, or left untreated (Ctrl), probed with α-Sytx1 and α-SNAP25 antibodies. Incubation with BoNT/A results in cleavage of SNAP25 and treatment with BoNT/C1 cleaves both SNAP25 and Sytx1. F, Western blots of conditioned media from control HEK293 cells or cells stably transfected with pCEP4-Netrin-1-myc cultured for 24–48 h in the presence or absence of BoNT/A or BoNT/C1, probed with an α-Myc antibody to detect Netrin-1 in the culture media. The Western blot did not show any differences in the secretion of Netrin-1 in the presence of botulinum toxins. G, Histograms illustrating proximal/distal ratios (attraction index) in hippocampal explants in different culture conditions. Significant differences are labeled by asterisks (**p ≤ 0.001). Scale bar: A, 300 μm. Error bars indicate SEM.