Stereotaxic Viral Injections in Mice

Stereotaxic injections were performed using previously described procedures^{23 (link)}. Mice were anesthetized with xylazine (5 mg per kg) and ketamine (75 mg per kg) diluted in saline (0.9%) and placed into a stereotaxic apparatus (KOPF). For postoperative care, mice were injected intraperitoneally with meloxicam (0.5 mg per kg). A micromanipulator (Grass Technologies, model S48 stimulator) was used to deliver the viruses at 2–5 nl per min and the pipette was withdrawn 5 min after injection. AAV1-FLEX-ChR2(H134R)-mCherry, AAV9-FLEX-ChR2(H134R)-EYFP, AAV9-CAG-ChR2(H134R)-mCherry (University of Pennsylvania School of Medicine), AAV8-DIO-hM3Dq-mCherry, AAV8-DIO-hM4Di-mCherry, AAV8-hSyn-mCherry-Cre, AAV5-hSyn-mCherry (University of North Carolina Vector Core), AAV8-DIO-synaptophysin-mCherry (Virovek, Inc) were injected into the ARC (coordinates, bregma: AP: −1.35 mm, DV: −6.00 mm, ML: ±0.2 mm). Mice with ‘missed’ injections, incomplete ‘hits’ or expression outside the ARC were excluded from analysis after post hoc examination of mCherry/GFP expression. AAV8-DIO-GFP (University of North Carolina Vector Core) was injected into the PVH (coordinates, bregma: AP: −0.75 mm, DV: −4.85 mm, ML: −0.20 mm).