Cover glass

On the day of experimental imaging, ferrets aged P21-24 were anesthetized with 3 to 4% isoflurane and atropine (0.2 mg/kg) or glycopyrrolate (0.01 mg/kg) was administered. Animals were placed on a feedback-controlled heating pad to maintain an internal temperature of 37 °C. Animals were intubated and ventilated. Isoflurane was delivered between 1 and 2% throughout the surgical procedure to maintain a surgical plane of anesthesia. An intraperitoneal or intravenous catheter was placed to deliver fluids. Electrocardiogram, end-tidal CO2, and internal temperature were continuously monitored during the procedure and subsequent imaging session. The scalp was retracted and a custom titanium headmount adhered to the skull using C&B Metabond (Parkell). A 6- to 7-mm craniotomy was performed over areas of viral expression and the dura was retracted to reveal the cortex. Cover glass (round, #1.5 thickness, Electron Microscopy Sciences) adhered to the bottom of a custom titanium or 3-D printed plastic insert was placed onto the brain to gently compress the underlying cortex and dampen biological motion during imaging. Upon completion of the surgical procedure, isoflurane was gradually reduced (0.6 to 1.0%) and then vecuronium bromide (2 mg/kg/h) was delivered to reduce motion and prevent spontaneous respiration.