mTORC1 activation and GDNF upregulation in the Rheb(S16H)-transduced hippocampus. (A) Immunohistochemical staining results of p-4E-BP1, indicating mTORC1 activation, showed increases in p-4E-BP1 expression in neurons (arrow heads) and non-neuronal cells (arrows), as demonstrated in the magnified photomicrographs of the rectangular insets in the CA1 layer in the Rheb(S16H)-transduced hippocampus. Scale bars: upper images, 100 μm; center images (insets), 20 μm. Astrocytic mTORC1 activation in the Rheb(S16H)-expressed hippocampus was confirmed by double immunofluorescence staining for GFAP and p-4E-BP1 (green arrows). Scale bar: lower images, 20 μm. (B) Double immunofluorescence staining to visualize co-expression patterns of GDNF and NeuN, or GDNF and GFAP in the Rheb(S16H)-transduced hippocampus. Scale bar, 50 μm. (C) Western blot analysis for p-4E-BP1, 4E-BP1, and GDNF expression in the controls, AAV1-GFP- and AAV1-Rheb(S16H)-injected hippocampus of rat brains. Differences among groups were evaluated by one-way analysis of variance and Tukey’s post-hoc test (*p<0.05 vs. CON; n=3 per group). (D) Western blot analysis for mTORC1 activity (p-4E-BP1 and 4E-BP1) and GDNF expression in the hippocampus of rats treated with rapamycin (5 mg/kg) and AAV1-Rheb(S16H) compared to AAV1-Rheb(S16H) alone. Differences among groups were evaluated by one-way analysis of variance and Tukey’s post-hoc analysis [*p<0.05 vs. CON, #p<0.01 vs. Rheb(S16H) alone; n=3 per group].