AAV-mediated MC4R knockdown in the PVN. (A) Representative images of in situ hybridization autoradiograms showing reduction of MC4R mRNA in the PVN by stereotaxic microinjection of AAV-MC4R-shRNA-EGFP vectors in comparison with AAV-control-shRNA vectors 35 days after injection. Quantitative data indicate that (B) AAV-MC4R-shRNA-EGFP significantly reduces the level of MC4R mRNA expression in the PVN, but exhibits (C) no significant effects on aVP mRNA expression in the PVN. Also, the levels of MC4R mRNA expression in the (D) dorsal medial hypothalamus (DMH), (E) amgydala, and (F) dorsal striatum were not significantly affected. AAV-control-shRNA-EGFP, n=5; AAV-MC4R-shRNA-EGFP, n=4. **P<0·01.