Fig. 6: Axonal projections of the striatal astrocyte-converted neurons in the R6/2 mouse brain.

a A sagittal view of a R6/2 mouse brain section immunostained for vGAT (green) and tyrosine hydroxylase (TH, cyan). TH positive cell bodies were present in the substantia nigra (above the SNr) and dense TH innervation was observed in the striatum. Inset shows the mCherry channel only to illustrate the axonal projections from the striatum to the GP and SNr. Scale bar: 1 mm. b High-resolution images showing mCherry⁺ puncta co-stained with vGAT (arrowhead) in GP and SNr (38 dpi). Scale bar: 2 µm. c Quantified data showing vGAT intensity in the GP and SNr significantly enhanced in NeuroD1 plus Dlx2-treated R6/2 mouse brains. d Experimental design of CTB retrograde tracing of converted neurons in the R6/2 mouse brain. Mice were sacrificed for immunohistochemistry analysis at 7 days after CTB injection. e Retrograde tracing of striatal astrocyte-converted neurons by injecting CTB into the GP at 21 or 30 days after AAV2/5 NeuroD1 + Dlx2 injection. Few CTB (green)-labeled converted neurons (red) were detected in the striatum at 21 dpi group (arrowhead), but many more CTB-labeled converted neurons were observed at 30 dpi group (arrowheads). f CTB injection into the SNr to trace striatal astrocyte-converted neurons. Even fewer converted neurons were labeled by CTB at 21 dpi group, but CTB labeling was clearly identified among the converted neurons in the striatum at 30 dpi group (arrowheads). Note that, in both GP (e) and SNr (f), many non-converted preexisting neurons were retrograde labeled by CTB, as expected. Scale bar for (e) and (f): 20 µm. g Bar graphs showing the percentage of CTB-labeled converted neurons in the R6/2 mouse striatum, which showed a significant increase from 21 dpi (black bars, immature neurons) to 30 dpi (red bars, more mature neurons). *p < 0.05, **p < 0.01, unpaired Student’s t-test. Data are shown as box plot (boxes, 25-75%; whiskers, 10-90%; lines, median).