BoNT/A has no significant impact on synaptic degeneration.

<p>(A) Percentages of healthy (open circles) and degenerating synapses (filled circles) in the CA1 stratum radiatum during prion disease progression. Degenerating terminals increase while normal boutons decrease in frequency from 10 to 16–18 weeks after delivery of the ME7 agent (one way ANOVA followed by Holm-Sidak test, p<0.05). Numbers of terminals analyzed are as follows: 10 weeks, n = 263; 12 weeks, n = 371; 16 weeks, n = 367; 18 weeks, n = 398. (B) Percentages of healthy (open bars) and degenerating synapses (filled bars) in prion+vehicle (prion+Veh) and prion+BoNT/A mice at 12 weeks into the disease. Percentages are identical in the two groups (t-test, p = 0.97). (C) Density of normal synapses (open bars) and degenerating boutons (filled bars) in prion+vehicle (prion+Veh) and prion+BoNT/A mice at 12 weeks into the disease. There are no statistically significant differences between the two groups (t-test, p>0.19). Number of terminals analyzed are as follows: prion+vehicle, n = 327; prion+BoNT/A, n = 723.</p

Neuropathological changes in a subset of BoNT/A-treated terminals at 4

<p> <b>weeks.</b> (A) Representative electron micrograph showing a degenerating, dark synaptic terminal with curved PSD. Scale bar  = 250 nm. (B) Percentage of degenerating synapses in vehicle (VEH)- and BoNT/A-treated animals. Dark terminals with a curved PSD (<120 deg) were scored as degenerating synapses. *, p<0.05. A total of 100 terminals were scored for each of three vehicle- and BoNT/A-injected animals at 4 weeks.</p

Lack of significant microglial activation 8 weeks after BoNT/A.

<p>(A, B) Representative immunostaining for the microglial marker Iba-1 in the CA1 stratum radiatum of mice treated with vehicle (A, VEH) and BoNT/A (B). Scale bar  = 100 µm. (C) Cell countings reveal no significant differences in microglial density between vehicle and BoNT/A-infused animals (t-test, p>0.05). (D, E) Immunostaining for cleaved SNAP-25 (red) demonstrates strong labeling in the CA1 stratum radiatum of BoNT/A-treated animals (E). No labeling is evident in vehicle mice (D). Green, Yoyo-1 nuclear counterstaining. Pyr, stratum pyramidale; s.r., stratum radiatum. Scale bar  = 100 µm.</p

Ultrastructural hallmarks of prion disease are not impacted by BoNT/A treatment.

<p>Electron micrographs of CA1 stratum radiatum of the hippocampus illustrating control, vehicle-exposed synapses (A, VEH) and neuropathological changes in prion+vehicle (prion + VEH; B) and prion+BoNT/A animals (C) at 12 weeks following delivery of the ME7 agent. High magnification pictures are shown in the insets. Note degenerating boutons with curved PSDs in both prion+vehicle and prion+BoNT/A samples. Scale bar  = 500 nm (250 nm for insets).</p

Ultrastructure of BoNT/A-treated hippocampal synapses.

<p>Representative electron micrographs of CA1 stratum radiatum in animals treated with vehicle (VEH) and at different times (2, 4 and 8 weeks) after BoNT/A infusion. There was no significant difference between the control groups at 2, 4 and 8 weeks after vehicle injection and the data from these groups has been merged. High magnification pictures are shown in the insets. Note enlarged presynaptic terminals following BoNT/A. Scale bar = 500 nm (250 nm for insets).</p