Stereotaxic Viral Injections in Mice

AAV injections were performed when Cre⁻ and Cre⁺ mice were 12–14 weeks old. Animals were anesthetized with isoflurane (3.0% for induction and 2.0–2.5% for maintenance, wt/vol) and placed in a stereotaxic apparatus (Narishige Scientific Instrument) with bregma and lambda skull landmark levels. According to the experimental protocols of Sherman and colleagues^{22 (link)}, two holes were drilled in the skull at predefined coordinates relative to bregma to allow the insertion of glass pipettes containing a mixture of two virus solutions (ChR2: 0.9–1.7 × 1013 genome copies per ml; eNpHR: 1.4 × 1013 genome copies per ml), which were held by a stereotaxic micromanipulator (SMM-100, Narishige) and connected to a Hamilton syringe with a pressure injection system (Motorized Stereotaxic Microinjector, IMS-20, Narishige). Each animal had bilateral injections targeted precisely to a location between the PreBötC and BötC (6.70 mm caudal to bregma, 1.25 mm lateral to the midline, and 4.65 mm ventral from the dorsal surface of the brain) according to the mouse brain atlas⁶⁰ . Once situated, 500 nl per side was slowly injected (100 nl/min) and the pipette was left in place for at least 5 min after injection to minimize backflow. The wound was closed with instant adhesives. AAV injections were performed by an investigator blinded to the animals.