Rodent intracerebroventricular AAV injections

Raise and house mice in compliance with your local Institutional Animal Care and Use Committee (IACUC) and ensure that all protocols and procedures have been approved by the appropriate ethical and regulatory committees.

Obtain mice from The Jackson Laboratory (male, 6-8 weeks).

Anesthetize mice using isoflurane (5% for induction, 1-3% for maintenance) with 95% O₂/5% CO₂ (1 L/min).

Head fix mice in a stereotaxic frame, shave head using clippers, and sterilize the area with chlorohexidine.

Administer 0.05 mL of 2.5 mg/mL bupivacaine subcutaneously above the skull.

Using a surgical blade, make an midline incision to reveal the skull. Clean the skull of blood and connective tissue and level the head in the stereotaxic frame.

Intraoperatively administer 1 mg/kg of buprenorphine SR and 5 mg/kg of ketoprofen via subcutaneous injection.

Drill 2 bilateral burr holes above the lateral ventricles using the following coordinates:

• Anterior-posterior: 0.6 mm posterior to bregma
• Medial-lateral: 1.15 mm from the midline

Using a 10 μL NanoFil syringe with a 33 guage beveled needle, aspirate viral vectors (5 x 10¹⁰ or 1.5 x 10¹¹ vector genomes in 3-5 μL) using a microsyringe pump.

Equipment

Value	Label
SUB-MICROLITER INJECTION SYSTEM	NAME
NanoFil Syringe	TYPE
World Precision Instruments	BRAND
NANOFIL	SKU

Equipment

Value	Label
33 guage beveled NanoFil needle	NAME
NANOFIL NEEDLES	TYPE
World Precision Instruments	BRAND
NF33BV-2	SKU

Equipment

Value	Label
UMP3 ULTRAMICROPUMP	NAME
Microsyringe pump and controller	TYPE
World Precision Instruments	BRAND
UMP3-3	SKU

Slowly lower the needle through the burr hole into the lateral ventricle (1.6 mm from the pial surface) and allow the needle to sit in place for approximately 5 minute. Inject viral vector at a rate of 300 nL/min.

Allow needle to stay in place for 5 minutes after injection has been complete. Slowly remove needle, and suture.

For 5-days post-surgery, mice were treated with 30 mg/kg of ibuprofen and 60 mg/kg of trimethoprim/sulfamethoxazole via water.