Intracerebroventricular (ICV) Injections

ICV injections were performed following preciously published procedures (Navarro et al., 2015). Briefly, 2–3 days before the experiment, mice were anesthetized with isoflurane and a small hole was bored in the skull 1 mm lateral and 0.5 mm posterior to bregma with a Hamilton syringe attached to a 27-gauge needle fitted with polyethylene tubing, leaving 2.0 mm of the needle tip exposed. Once the initial hole was made, all subsequent injections were made at the same site. On the day of ICV injection experiments, mice were anesthetized with isoflurane for a total of 5–10 min, during which time 5 μl of solution were slowly and continuously injected into the lateral ventricle. The needle remained inserted for approximately 30 s after the injection to minimize backflow up the needle track. Mice typically recovered from the anesthesia within 3 min after the injection.