Figure 6. ST-projecting DA fibers in non-transduced SN regions are protected by hSNCA gene silencing.

Adult, male rats were stereotaxically injected into one SN with AAV vectors containing hSNCA and/or shRNA-SNCA plus hrGFP and DA neurons that maintained projections to the ST were bilaterally labeled with the retrograde tracer FG 5 days before euthanasia. Rats were euthanized 9 weeks after AAV injections and FG positive neurons in SN and VTA were counted. (a) AAV-CBA-hSNCA and/or AAV-H1-shRNA-SNCA reduced the number of neurons retrogradely labeled with FG in virus-transduced regions of the SN. Uninjected SN is shown in the left panel and AAV-injected SN is shown in the right two panels. FG is shown in yellow and GFP, representing shRNA-SNCA, is shown in green. Panels are representative of all treatment groups. Size bar, 100 μm. (b) FG positive DA neurons in the SN/VTA were counted. Total cell counts in SN/VTA, expressed as a ratio of injected SN/VTA / uninjected SN/VTA are shown in b. Data were analyzed using a Kruskal-Wallis one-way ANOVA, followed by Dunn’s post-hoc tests. In order to examine the effect of AAV injection on ST-projecting DA neurons more thoroughly, FG cell counts were divided into virus-transduced SN/VTA and adjacent un-transduced SN/VTA regions. c exemplifies the process by which cell counts were separated. The white outline represents the region of virus transduction in the injected SN. FG is shown in yellow and hrGFP, representing shRNA-SNCA is shown in green. Ratios of FG positive DA neurons in virus-transduced (gray bars) and adjacent un-transduced regions (striped bars) are shown in d. Mean±SEM; data were analyzed using two-way ANOVA followed by Bonferonni post-hoc tests. F_2,44=11.48, p<0.0001. Asterisks show significant differences between virus-transduced and adjacent regions: **=p≤0.01. Significant differences compared to the adjacent region in hSNCA-injected rats: ††= p≤0.01, †††= p≤0.001.