Stereotaxic injection of hSyn-hM3D(Gq)-mCherry AAV

The hSyn-hM3D(Gq)-mCherry AAV virus (University of North Carolina Vectore Core, Chapel Hill, NC) was injected into the CEA of 24 Wistar rats. The surgeries were performed in a biosafety level 2 (BSL-2) virus injection facility. The rats were anesthetized and their head positioned in a stereotaxic apparatus with the Bregma and Lambda in the horizontal plane. Through a burr hole in the skull, a glass pipette (20-μm outer tip diameter) connected to a Nanoject II/Nanoliter 2000 microinjector (Drummond Scientific Co. or WPI Inc.) was lowered into the brain at stereotaxic coordinates corresponding to the medial part of CEA (CEAm) (anteroposterior: −2.4 mm, mediolateral: −3.9 mm, dorsoventral: −8.2 mm), or to the laterocapsular part of CEA (CEAlc) (anteroposterior: −2.4 mm, mediolateral: −4.45 mm, dorsoventral: −8.25 mm) based on the atlas of Paxinos and Watson (Paxinos and Watson 1998). The virus containing solution (80–100nl; 4.5×10¹² virus/ml) was injected unilaterally into the CEA with 5nl/sec speed. Five minutes after the injection, the pipette was removed slowly, the scalp was sutured and the rats were housed in BSL-2 quarantine for 2 weeks before experimentation.