Rats were injected intrastriatally with equal amounts (8μg) of recombinant α-syn or PFF at 1-site or 2-sites. (a). Panel illustrates substantia nigra dopamine neurons visualized using tyrosine hydroxylase (TH) immunohistochemistry in both the recombinant α-syn or α-syn PFF treatment groups compared to naïve age-match control 6 months post injection. Rats in both α-syn groups developed substantial α-syn pathology over time in the substantia nigra; however, only rats in the α-syn PFF injected groups exhibited significant nigral dopamine neuron degeneration at the 6 month time-point. (b). Representative images of pSYN pathology in the more vulnerable ventral tier (red dashed line) of the substantia nigra pars compacta (SNpc) illustrating the accumulation of α-syn aggregates at two months and their subsequent reduction at 6 months (c) pi. (d). Stereology results show significant bilateral dopamine (TH+) neuron loss in both the 1- (d) and 2-site (f) PFF injected animals. * p<0.002 compared to either naïve or recombinant α-syn. (e) and (g). Both recombinant α-syn and PFF injections resulted in neuronal loss (cresyl counts) however, PFF-induced neuronal loss was significantly greater than recombinant α-syn injected rats. * p<0.001 compared to naïve. #p < 0.05 compared to recombinant α-syn. (h). Stereological assessment of TH+ neuron loss at 60d, 120d and 180d following unilateral 1-site (8μg) α-syn PFF or saline injection. Significant bilateral reductions in TH+ neurons were observed as early as 60d pi. * p<0.01, ** p<0.001, # p<0.05 compared to saline. (i) Stereological assessment of neuronal loss at 60, 120, and 180 days following unilateral 1-site (8μg) α-syn PFF or saline injection. Significant bilateral neuron loss was not observed until 180d following α-syn PFF injection. *p≤0.003 compared to saline. (j). Optical density analysis of TH+ immunoreactivity within the substantia nigra pars reticulata 180d after 1- or 2-site α-syn PFF injection. Within the SNpr, TH+ dendrites were significantly decreased on the side of the injection, but were unaffected on the contralateral side. Data represent mean +/−SEM. Abbreviations: pS129 = phosphorylated α-syn at the serine 129 site; PFF = α-syn pre-formed fibril; contra=contralateral hemisphere (relative to injection site); ipsi=ipsilateral hemisphere (relative to injection site); TH=tyrosine hydroxylase; α-syn=recombinant α-syn; SEM=standard error of the mean; pi=post injection.