Intracerebroventricular injection

Briefly, mice were anesthetized (using sodium pentobarbital 50 mg/kg, via i.p. injection) and positioned in a stereotaxic instrument with a mouse adapter. The stereotaxic coordinates for implantation of a guide cannula into the right lateral ventricle were as follows: AP, 0.5 mm relative to bregma; ML, 0.8 mm relative to bregma; and DV, −2.5 mm from the skull surface. The guide cannula was fixed with dental cement. All injections were administered over 1 min, and the syringe was kept in place for 2 min to prevent backflow after injection. To confirm successful entry into the right ventricle before our main experiments, 5 μL of trypan blue dye was injected into the cannula, and the mouse brain was cut into slices for observation. All animals were allowed to recover for 2 weeks after the operations before initiation of the subsequent experiments. Control animals received a vehicle solution. During drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannula and replaced by 27-gauge injection needles (1 mm below the tip of the guide cannula). Each injection was connected to a 10-μL Hamilton syringe using a polyethylene tube.