Monoamine Quantification in Mouse Brain

In WT and KOR null mice, the hypothalamus, prefrontal cortex and striatum were dissected on an ice-cold plate, and immediately frozen on dry ice and stored at −80 °C until analysis. Then tissue was homogenized and then centrifuged (14,000×g; 10 min at 4 °C). The remaining supernatant fraction was filtered and injected (20 μl/injection) into the High-Performance Liquid Chromatography (HPLC) system (Shimadzu LC Prominence; Shimadzu Corporation; Kyoto, Japan)^{56 (link),57 (link)}. Dopamine and its metabolite, DOPAC, 5-HT and NE were separated using a reverse phase analytical column (Waters Symmetry 300C18; Waters, Milford, MA, US). The mobile phase consisted of a 10% MeOH solution (pH = 4) containing 70 mM KH2PO4, 1 mM octanesulfonic acid and 1 mM EDTA, and was delivered at a rate of 1 ml min⁻¹. Detection was performed with a coulometric electrochemical detector (ESA Coulochem III; ESA, Chelmsford, MA). Data were processed with the Shimadzu LC Solution Software (Shimadzu Corporation; Kyoto, Japan) and were expressed as ng mg⁻¹ of wet tissue.