Distal effects of BoNT/A.

<p>Long-range effects of BoNT/A were assessed by immunofluorescence (<b>A, B</b>) and western blot, either <i>in vitro</i> (<b>A</b>–<b>C</b>) or <i>in vivo</i> (<b>D</b>), using an antibody that specifically recognises the fragment of SNAP25 generated by BoNT/A. Motor neurons grown in MFC were incubated with 10 nM BoNT/A added to the axonal compartment only of MFC. Samples were processed as described in Experimental Procedures. (<b>A</b>) Tile scan of a representative MFC treated with BoNT/A (left) and the untreated control (right). Arrowheads indicate the microgrooves whilst the hatched lines indicate the boundaries between the cell body and the axonal side of the MFC. Scale bar, 50 µm. (<b>B</b>) High magnification images of MFCs treated as in <b>A</b>. Controls were run in parallel and the microscope settings used for image acquisition were kept constant. Scale bar, 10 µm. Similar results were obtained using two independent primary motor neuron cultures, and replicated twice per condition. (<b>C</b>) Western blot revealed the presence of BoNT/A-cleaved SNAP25 (24 kDa) in the somatic compartment of MFC treated only on the axonal side with full length BoNT/A. No signal was present in control MFCs. This result demonstrates that BoNT/A undergoes axonal retrograde transport in a fully active form and was confirmed in two independent experiments. A representative western blot is shown in (<b>C</b>). β-tubulin (50 kDa) was used as loading control. (<b>D</b>) Representative western blot for BoNT/A-cleaved SNAP25 on extracts from lumbar spinal cord segments of a naive (control) animal and a rat injected with BoNT/A into the hind limb muscles 10 d earlier. These results were confirmed in two independent experiments, replicated three times. A representative western blot is shown in (<b>D</b>). α-tubulin was used as loading control (51 kDa).