Rat intracerebroventricular injection.

Rats were anesthetized with 60 mg/kg pentobarbital and placed in a stereotaxic apparatus, with bregma and lambda kept in the same horizontal plane. A hole was drilled into their skulls, and a stainless steel guide cannula (Eicom) was inserted into the right lateral ventricle. Based on a rat brain atlas, the following stereotaxic coordinates were used: anterior-posterior (AP) = +0.6 mm, medial-lateral (ML) = +1.6 mm, dorsal-ventral (DV) = +4.5 mm from bregma. The guide cannula was implanted and fixed with dental acrylic, and a 30-gauge stainless steel stylet was placed into the guide cannula to prevent the entry of foreign materials. The experimental procedure was started 3–5 days after the surgery.

An injector (30 gauge) was fitted into the guide cannula, and intracerebroventricular infusions were made using 10 μL microsyringes (Hamilton Company) attached to the injector with a polyethylene tube. AM404 (10 or 50 nmol) or the control vehicle was injected with an automatic infusion pump at a rate of 1 μL/min and a total injection volume of 5 μL. After the experiments, Evans blue solution (5 μL) was injected through the cannula to confirm the injection site. If the injection site was incorrect, the animal was excluded from the analysis.