Fig. 7

Peripheral intramuscular (IM) injection data showing time-dependent and progressive spread of alpha-synuclein inclusions through known neuroanatomically connected pathways. a–c A53T SynGFP inclusions appear specifically in motor regions of interest (ROI) in a time-dependent manner. Fixed tissue images of representative brain sections along the rostral-caudal axis. a Representative cortex section that includes the primary motor cortex (motor ROI, pink square) and the somatosensory barrel cortex (control ROI, white square). b Representative midbrain section that includes cerebral peduncle and the zona incerta (motor ROI, blue square) and the primary somatosensory area (control ROI, white square). c Representative pons section that includes cerebral peduncle and the medial lemniscus (motor ROI, green square) and primary visual cortex (control ROI, white square). At the 4-month time point, A53T SynGFP inclusions are visible in motor ROI in the pons (c″) and to a lesser extent in the midbrain (b″), but are not detectablef in the cortex (a″) or in any of the corresponding control ROI (a′–c′). Scale bar 1 mm in a–c and 100 µm in a′–c′ and a″–c″. d–f A53T SynGFP inclusions appear specifically in motor regions of interest (ROI) in a time-dependent manner. Fixed tissue images of representative brain sections along the rostral-caudal axis. d Representative cortex section that includes the primary motor cortex (motor ROI, pink square) and the somatosensory barrel cortex (control ROI, white square). e Representative midbrain section that includes cerebral peduncle and the zona incerta (motor ROI, blue square) and the primary somatosensory area (control ROI, white square). f Representative pons section that includes cerebral peduncle and the medial lemniscus (motor ROI, green square) and primary visual cortex (control ROI, white square). At the 8-month time point, A53T SynGFP inclusions are visible in all motor ROI along the rostral caudal axis (d″–f″) but are not visible in any of the corresponding control ROI (d′–f′). Scale bar 1 mm in a–c and 100 µm in a′–c′ and a″–c″. g Schematic showing the rostral to caudal locations of serial brain sections analyzed for each animal. Specific sections were chosen based on the presence of a motor region of interest (ROI). Motor ROI were determined based on known descending motor pathways in the cortex (pink square), midbrain (blue square), and pons (green square). The specific ROI were chosen because they encompass white matter tracts and nuclei within the lateral descending motor pathways, specifically the lateral corticospinal tract and the rubrospinal tract. These tracts begin in the primary motor cortex or red nucleus, respectively, and terminate in the lower motor neurons of the anterior gray horn of the spinal cord. Both tracts are connected to the site of PFF injection in the gastrocnemius muscle through multiple synaptic connections, thereby representing sites of retrograde trans-synaptic spread. h Quantification of SynGFP inclusions in each region of interest (ROI) at each time point (months post-injection (mpi). Density of inclusions in each ROI (mm²) was calculated in ImageJ using particle analysis. ROI equaled the motor (m) or control (c) area in each brain region (cortex, midbrain, pons) at each timepoint (4 vs. 8 mpi). Group data shows that the density of inclusions was significantly different in the motor ROI in all brain areas at 4 and 8mpi, but control ROI did not differ at 4 and 8mpi (one-way ANOVA F(11,138) = 43.55), p < 0.0001; Sidak’s multiple comparisons test, 4 vs. 8 mpi motor ROI (m): cortex, midbrain, pons p < 0.0001 for all 3, 4 vs. 8 mpi control ROI: cortex, midbrain, pons p > 0.9999 for all 3; N = 6–20 ROIs/3–4 animals per group. Mean and SEM of the density of inclusions in each group are shown in Additional file 3: Table S3. Cortex = pink, midbrain = blue, and pons = green. i At 4mpi there was a significant difference between the motor and control ROI in the pons alone, indicating trans-synaptic spread only to the most caudal brain section. Sidak’s multiple comparisons test, motor vs. control in cortex, midbrain, and pons: p > 0.9999, p = 0.1287, p = 0.0126, respectively. Mean and SEM of the density of inclusions in each group are shown in Additional file 3: Table S3. Cortex = pink, midbrain = blue, and pons = green. j At 8mpi there was a significant difference between motor and control ROI in each section along the rostral-caudal axis indicating progressive trans-synaptic spread through connected brain areas along the descending motor pathway following IM PFF injection. Sidak’s multiple comparisons test, motor vs. control in cortex, midbrain, and pons: p < 0.0001 for all 3. Mean and SEM of the density of inclusions in each group are shown in Additional file 3: Table S3. Cortex = pink, midbrain = blue, and pons = green