Caudo-rostral spreading of hα-syn reaches midbrain and forebrain regions. (a) Rats received a single injection of hα-syn-AAV prep 1 (open bars) or prep 2 (filled bars) into the vagus nerve. The number of axonal projections immunoreactive for hα-syn was counted in the left (injected side) caudal midbrain (cMB), rostral midbrain (rMB) and forebrain (FB) at 6 (n = 7/group) and 12 (n = 5/group) weeks post treatment. Values are means ± SEM. **P <0.01 and *P <0.05. T test: F1,13 = 7.88 and F1,9 = 5.88 in the cMB at 6 and 12 weeks, respectively; F1,13 = 10.10 and F1,9 = 5.86 in the rMB at 6 and 12 weeks, respectively; F1,9 = 9.94 in the FB at 12 weeks. (b,e) Representative sections of the forebrain at (b) Bregma: −2.40 mm and (e) Bregma: +0.48 mm were obtained from rats injected with AAV prep 2 and sacrificed at 12 weeks post treatment. (c, d, f) Higher magnification images from the (c) hypothalamus, (d) amygdala and (f) bed nucleus of the stria terminalis were generated by z-stacking. They show axonal projections immunostained with an anti-hα-syn antibody. Scale bar (for c, d and f) in (f) = 30 μm.