2.3. Stereotaxic surgery

For stereotaxic viral injections, mice were placed in a stereotaxic frame (Kopf 1900, Cartesian Research Inc, CA) under isoflurane anesthesia. The skull was exposed with a small incision, and two small holes were drilled for unilateral microinjection (400 nl) of an AAV into the ARC of AgRP-IRES-Cre mice at stereotaxic coordinates based on the Mouse Brain Atlas [17] using an angled approach: Anterior/Posterior: 1.85 mm, Dorsal/Ventral: 6.2 mm, ∼12.5° angle from midline) as previously described [1,18]. Following viral injections, a fiber-optic ferrule (0.48 NA, Ø400 mm core; Doric Lenses, Quebec, Canada) was implanted using the same coordinates. All microinjections were performed using a Hamilton syringe with a 33-gauge needle at a flow rate of 100 nl/min (Micro4 controller, World Precision Instruments, FL), followed by a 5 min pause and slow withdrawal. Animals received a perioperative subcutaneous injection of buprenorphine hydrochloride (0.05 mg/kg; Reckitt Benckiser, VA). After surgery, mice were allowed 3 weeks to recover to maximize virally transduced gene expression and to acclimate animals to handling and experimental paradigms prior to study. Viral expression was verified post hoc in all animals, and any data from animals in which the virus was expressed outside the targeted area were excluded from the analysis.