TgM83+/- mice were inoculated with 3 μL of a 5% mouse-passaged MSA brain homogenate. Inoculations were performed either by standard freehand injection (black) or by stereotactic inoculation into the hippocampus (Stereo. HC; red), thalamus (Stereo. Thal; green), or hypothalamus (Stereo. HTH; blue). (a) After the mice developed progressive neurological disease, the animals were euthanized and their brains were harvested. One half of each brain was fixed in formalin, cut, and immunostained for phosphorylated α-synuclein neuropathology (data in panel b). The other half was flash frozen, homogenized, and tested for α-synuclein prions in the α-syn140*A53T–YFP cell assay (× 103 A.U.; data in panel c). (b) Regardless of inoculation method or site, α-synuclein pathology measured in the HC, Thal, HTH, Mid, and pons was consistent across all four inoculation groups. (c) The α-synuclein prion concentration was consistent in terminal animals. * = P < 0.05; ** = P < 0.01.