Fig. 2

BoNT/A enzymatically cleaves SNAP25 in wFMNs-projecting synaptic terminals. a General schematic view of BoNT/A-cleaved SNAP25₁₉₇ expression. b BoNT/A-cleaved SNAP25₁₉₇ was detected on the ipsilateral lFN to BoNT/A injection side in subgroups of three different dosages 10 days post-BoNT/A WIM injection. Right two lines, magnified view of the left two lines. Scale bar, 100 μm. c Quantificational analysis of BoNT/A-cleaved SNAP25₁₉₇ among subgroups of three different BoNT/A dosages. n = 4 cells from 3 mice per subgroup, respectively. One-way ANOVA followed by Bonferroni’s multiple comparisons test, F _{(2, 9)} = 99.68, P < 0.0001. Bonferroni’s multiple comparisons: 3 U/kg vs. 10 U/kg: P < 0.0001, 3 U/kg vs. 30 U/kg: P < 0.0001, 10 U/kg vs. 30 U/kg: P = 0.0169. Intensity of the immunostaining for each subgroup was normalized to the labelling level at the subgroup of 3 U/kg. d Representative images of cl.SNAP25₁₉₇ expression on lFN of three time points post-BoNT/A (10 U/kg) WIM pre-injection. The right image was the magnified view of the left one. Scale bar, 100 μm. e Quantificational analysis of the percentage of BoNT/A-cleaved SNAP25₁₉₇-positive signal areas among groups of three time points post-BoNT/A (10 U/kg) pre-injection, indicating that cl.SNAP25₁₉₇ was detected at 10 days, with a highest expression at 4 weeks, and existed persistently to at least 7 weeks post-BoNT/A WIM injection. n = 4 brain sections from 3 mice in the group of 10 days, n = 5 brain sections from 3 mice in the group of 4 weeks, and n = 6 brain sections from 3 mice in the group of 7 weeks, respectively. One-way ANOVA followed by Bonferroni’s multiple comparisons test, F _{(2, 12)} = 4.33, P = 0.0384. The P-value of Bonferroni’s multiple comparisons: 10 Days vs. 4 Weeks: P = 0.8069, 10 Days vs. 7 Weeks: P = 0.4491, 4 Weeks vs. 7 Weeks: P = 0.0382. Intensity of the immunostaining for each group was normalized to the labeling level at the group of 10 days post-BoNT/A pre-injection. f Co-immunostaining of SNAP25 with ChAT showed non-colocalization of the two proteins. Scale bar, 50 μm. g Co-immunostaining of SNAP25 and PSD95 indicated non-colocalization. Scale bar, 50 μm. *P < 0.05, ****P < 0.0001, n.s., non-significance; Ip., ipsilateral side of BoNT/A injection side; Col., contralateral side of BoNT/A injection location