Transgenic animals were anesthetized for surgery with subcutaneous injections of ketamine (75 mg/kg) and medetomidine (100 mg/kg). The right side of the face was carefully shaved and then depilated with Nair hair removal cream (Church and Dwight Co, Inc, Princeton, New Jersey) prior to skin preparation with alcohol swabs. Great care was taken to avoid caustic or exposure injury to the eye. The skin was incised 1 mm anterior and inferior to the tragus and carried in an arch posteriorly. Following the tragal pointer medially, the facial nerve trunk was identified at an original magnification of ×16 as it exits the stylomastoid foramen. The main nerve trunk was then crushed with a No. 5 Jeweler's forceps for 30 seconds,46 and the crush site was marked with a 10-0 nylon stitch. The wound was then irrigated, and skin was reapproximated with interrupted 6-0 nylon sutures. Animals recovered on a heated surface following anesthesia reversal with atipamezole hydrochloride (1 mg/kg). Animals were inspected and weighed weekly to ensure that nerve damage did not impair the ability to eat or cause ocular damage. At the time of imaging evaluation, animals were reanesthetized, and the surgical site was prepared. Following this, they were perfused transcardially with heparinized paraformaldehyde, 4%, in 0.1M phosphate-buffered saline (pH 7.4). The superficial subcutaneous tissue of the face was then dissected completely off of the underlying superficial musculoaponeurotic system, and the vibrissae were transected under the skin. Images were then taken under an Olympus MVX10 dissecting microscope equipped with fluorescent cube filters (Olympus Corporation, Center Valley, Pennsylvania), followed by removal of the superficial musculoaponeurotic system and imaging of the nerve trunk and its branches. The zygomaticus muscle was removed with careful dissection, placed on a Sylgard resin-coated dish (Dow Corning Corporation, Midland, Michigan) and rinsed with phosphate-buffered saline. It was then stained with α-bungarotoxin Alexa Fluor-594 (10 μg/mL; Invitrogen, Carlsbad, California) for 30 minutes at room temperature. The muscle was then rinsed again with phosphate-buffered saline and mounted under a coverslip in Vectashield (Vector Laboratories, Burlingame, California) for confocal microscopic imaging.