Fig. 3

Areas harboring α-synucleinopathy send first-order projections to the site of infusion in the OB/AON. a The retrograde tracer FluoroGold (blue) was infused at the same stereotaxic coordinates as the α-synuclein fibrils. Animals were perfused after 7 days and sagittal sections were stained with the nuclear marker DRAQ5 (purple). Sections were imaged under UV and CY5 illumination. Dual-stained brain regions appear red (not purple or blue) in panel a a, such as the AON and the horizontal limb of the diagonal band (HDB). Dense FluoroGold label at multiple afferent sites is illustrated at higher magnification in panels c, e, g, i, k, and m. Nuclear DRAQ5 label on the same sections is shown in panels b, d, f, h, j, and l. Note the dense FluoroGold labeling in the horizontal limb of the diagonal band of Broca and the absence of FluoroGold label in the olfactory tubercle (Tu; f-g). Neither of these regions developed α-synucleinopathy. The olfactory tubercle is densely innervated by OB/AON projection neurons but does not project reciprocally back to the OB/AON. Abbreviations in Additional file 1: Table S1. b-c The transmission of α-synucleinopathy after infusions of waterbath-sonicated fibrils can be generalized to other neuroanatomical circuits. Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into CA2/CA3 of the hippocampus. Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, coronal brain sections were collected. Shown are stitched montages of pSer129 and Hoechst staining in the hippocampus and entorhinal cortex in PBS and fibril-infused animals—both groups were stained in parallel and captured at the same exposure and intensity scaling. The fibril injection in this animal extended from CA2/CA3 into CA1. Note the dense label in the entorhinal cortex, suggesting transmission of pathology through the perforant path, and the absence of label in the thalamus and most of the overlying cortex, demonstrating the lack of nonspecific uptake by neighboring sites from diffusion through the cerebrospinal fluid and interstitial space. For the antibody preadsorption control on coronal hippocampal tissue, see Additional file 1: Figure S6. To view the hippocampus at the same level after infusions of fibrils that were sonicated for 24 h, please examine Additional file 1: Figure S7. Abbreviations in Additional file 1: Table S1. d Electron micrographs of α-synuclein fibrils before and after sonication (1 min probe sonication and 1 h or 24 h waterbath sonication). Most sonicated fibrils were positively, not negatively stained with uranyl acetate. To view the pSer129 pathology in the entorhinal cortex and transentorhinal region in panel c, please zoom in and out of the high-resolution versions of these files at the link at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files