Stereotactic injections

Before animals reached the age of 10 days (day of injection), a DNA sample was obtained for genotyping (SOD1^G93A-positive versus wild-type mice). For each litter, the investigator made sure that for each experimental condition (MCT1, GFP, or empty vector) at least one mouse was represented to always have littermate controls. Postnatal day 10 mice (C57BL/6 mice for transduction efficiency study and SOD1^G93A for the treatment study) were anesthetized with isoflurane for both induction (4%, 1l/min oxygen) and maintenance (2%, 1l/min oxygen) of anesthesia. Both male and female mice were used for experiments. Before surgery, mice received a subcutaneous injection of Vetergesic (0.05 mg/kg) and topical administration of Xylocaine (6 mg/kg). Mice were then placed onto a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA) using an adaptor for neonatal mice. 4 μL of the vector solution was injected into the lateral ventricles (x = 0.8 mm, y = −0.2 mm, z = 2 mm), equivalent to a dose of 5 × 10¹⁰ vg of the MBP-MCT1^Myc/FLAG-AAV9 and MBP-GFP-AAV9 vector stock, or 4 × 10¹⁰ vg of the MBP-Empty-AAV9 vector stock. Delivery was performed at a rate of 500 nL/min, using a microprocessor-controlled mini-pump (World Precision Instruments, Sarasota, FL, USA) connected to a 33G Hamilton syringe. The needle was left in place for 5 min prior to slowly retracting it from the ventricles. Afterward the pups were left underneath an infrared light to recover their mobility before returning as a group to their mother. Mice were observed until postnatal day 31 or death.