Metabolite Extraction from Mouse Motor Cortex

Mice were deeply anesthetized with intraperitoneal injection of Ketamine (90 mg/kg, and Xylazine (10 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA, USA)^{16 (link)}. Intact brain was removed and motor cortex was dissected out, weighed and immediately frozen in liquid nitrogen. The tissue was homogenized in 1 ml chilled 80% chromatography grade methanol and vigorously vortexed three times. 200 µL tissue homogenate was mixed into a tube pre-added with 800 µL of ice-cold methanol/water 80% (vol/vol) followed by vortexing rigorously for 1 min, and then centrifuge at ~ 20,160g for 15 min in a refrigerated centrifuge. The metabolite-containing supernatant was separated into a fresh tube and stored at − 80 °C until metabolite profiling was performed^{77 (link)}.