Stereotaxic Viral Targeting of Arc and PVH

Stereotaxic surgeries to deliver viral constructs were performed as previously described in ref. ^{43 (link)}. Briefly, mice were anesthetized with a ketamine/xylazine cocktail (100 and 10 mg/kg, respectively), and their heads were affixed to a stereotaxic apparatus. Viral vectors were delivered through a 0.5 µL syringe (Neuros Model 7000.5 KH, point style 3; Hamilton, Reno, NV, USA) mounted on a motorized stereotaxic injector (Quintessential Stereotaxic Injector; Stoelting, Wood Dale, IL, USA) at a rate of 40 nL/min. Viral preparations were made by the Baylor NeuroConnectivity Core with the sterotype DJ8 and titers more than 10¹² vg/mL. Viral delivery was targeted to the Arc or PVH through four local injections with two to each side (200 nL/side, anteroposterior (AP): −1.4 and −1.6 mm, mediolateral (ML):±0.2 mm, and dorsoventral (DV): −5.9 mm). AAV-EF1a-Flex-EGFP-P2A-mNachBac, AAV-EF1a-Flex-Kir2.1-P2A-dTomato, AAV-CAG-Flex-mPOMC-P2A-EGFP, AAV-CAG-Flex-Alpha-MSH, or AAV-CAG-Flex-Beta-endorphin, or AAV-CAG-Flex-MC4R-P2A-GFP were delivered bilaterally or unilaterally into the Arc of POMC-Cre mice, POMC-cre:ob/ob mice, or the PVH of MC4R-cre mice. Two mutations (E224G and Y242F) were made in the Kir2.1 construct so that the channel will be more effective in reducing neuron activity^{43 (link)}. AAV-Flex-GFP or AAV-Flex-mCherry injections were used as a control group.