Fig. 1: Overexpressing of h-α-Syn in raphe 5-HT neurons in male mice.

Male mice received 1 μl of AAV2/5-CBh-WPRE3 construct to drive expression of h-α-Syn (AAV-h-α-Syn) or empty AAV2/5-CBh-WPRE3 vector containing non-coding (null) stuffer DNA (AAV-EV) into dorsal raphe nucleus (DR) and were euthanized at 4 and 8 weeks (W) post-injection. A Representative confocal image showing co-localization of h-α-Syn protein and TPH⁺ cells in the DR of mice after AAV2/5 injection. Scale bar: low: 200 μm, high: 50 μm. On the right, schematic representation of h-α-Syn⁺/TPH⁺ cell density. B Bar chart (top) showing progressive h-α-Syn accumulation in mouse DR. Bar chart (down) showing the proportion of TPH⁺ cells co-localized with h-α-Syn⁺ (n = 6 mice/group; *p < 0.05, ***p < 0.001, ****p < 0.0001 versus AAV-EV or AAV-h-α-Syn 4 W mice). C Representative confocal image showing co-localization of p-α-Syn protein and GAD67⁺ cells in the DR of mice at 8 W after AAV2/5 injection. Scale bar: low: 200 μm, high: 50 μm. D Bar chart (left) showing progressive accumulation of p-α-Syn protein in mouse DR. Bar chart (right) showing the number of double cells co-localizing GAD67⁺/p-α-Syn⁺ in mouse DR (n = 6 mice/group; ^*p < 0.05, ****p < 0.0001 versus AAV-EV or AAV-h-α-Syn 4 W mice). E Representative confocal image showing co-localization of h-α-Syn protein and TH⁺ cells in the DR of mice at 4 W and 8 W after AAV2/5 injection. Scale bar: low: 200 μm, high: 50 μm. F Bar chart showing the proportion of the total number of TH⁺ cells co-localized with h-α-Syn⁺ (n = 5 mice/group; ***p < 0.001 versus AAV-EV mice). G Representative confocal image showing co-localization of h-α-Syn⁺/SERT⁺ fibers in 5-HT projection brain regions, such as medial prefrontal cortex (mPFC), cingulate cortex (Cg), caudate putamen (CPu), and hippocampus (HPC) of mice at different time points after AAV2/5 injection. Scale bar: 25 μm. H Bar chart showing progressive accumulation of h-α-Syn protein in projection brain areas: prelimbic cortex (PrL), infralimbic cortex (IL), Cg, CPu, and different HPC subfields (n = 5 mice/group; *p < 0.05, **p < 0.01, ***p < 0.001 versus AAV-h-α-Syn 4 W mice). Values are presented as mean ± SEM. See Supplementary Figs. 1–3.