Neonatal Intracerebroventricular AAV Delivery

Intracerebroventricular (ICV) injections of AAV were performed as previously described (Chew et al., 2015 (link); Chew et al., 2019 (link)). Briefly, each AAV solution was diluted to a concentration of 1.5 × 10¹⁰ genomes/μl. C57BL/6 J pups at P0 were cryoanesthetized on ice for approximately 3 min or until they exhibited no movement. Two microliters of either AAV2/9-mycATXN3-Q28 or AAV2/9-mycATXN3-Q84 solution was manually injected into each cerebral ventricle using a 32-gauge needle (Hamilton Company) attached to a 10 μl syringe (Hamilton Company). The needle was inserted at a 30-degree angle from the surface of the head at a point approximately two-fifths the distance between the lambda suture and the eye and held at a depth of approximately 2 mm when injecting. After injections, the pups were placed on a heat pad until they completely recovered from anesthesia; they were then returned to their home cages.