Intracerebroventricular injection

Before drug injections, mice were anesthetized with 2% isoflurane and placed in a stereotaxic head frame on a heating pad. For each mouse, the bregma was located without exposure of the skull (60). A guarded 23-gauge needle was used to punch a hole 0.2 mm posterior to the bregma and 1.0 mm lateral to the midline. The Nanofil syringe (WPI) was used to inject NPS (5857, Tocris) (0.1 or 1 nmol in 2 μl of saline) or vehicle (saline) into the right cerebral ventricle at a depth of 2.5 mm from the skull at ZT11. Mice were allowed to recover for 5 min and then placed back in the home cage. Vehicle or SHA 68 (SML1459-25MG, Sigma-Aldrich) [50 mg/kg in phosphate-buffered saline (PBS), 10% cremophor EL] were injected (intraperitoneally) 10 min before NPS. Brain tissues were collected 1 hour after the intracerebroventricular injections.