Clozapine-N-oxide Preparation

CNO powder (NIMH C-929, RITI International) was stored at −20 °C, protected from light. CNO solutions were freshly prepared on each experimental day, by first dissolving the drug at 66 mg/mL in 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis MO, USA) and then diluting with calcium- and magnesium-free phosphate-buffered saline (PBS, Corning, Corning, NY, USA) to a final concentration of 10 mg/mL in 15% DMSO. To prevent degradation due to light exposure, vials and syringes that contained CNO solution were wrapped in aluminum foil. In some experiments, immediately following administration, an aliquot of the CNO was placed in a sterile Eppendorf tube, frozen on dry ice, and kept at −80 °C until assayed.