5-HT and 5-HIAA content determination by HPLC in the olfactory bulb
All OB tissue homogenization was performed according to Chi et al., 1999. Briefly, the tissue was collected in 400 μl of 0.2 N perchloric acid and then homogenized in a glass-glass homogenizer. The homogenate was centrifuged at 12000×g for 15min at 4°C (Hermle LaborTechnik GmbH, model Z233MK-2) and the supernatant was injected into a high-performance liquid chromatography (HPLC) instrument coupled to electrochemical detection, to measure 5-HT, 5-HIAA. The pellet was resuspended in 1N NaOH for protein quantification by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Richmond, CA, USA) using bovine serum albumin as standard. The contents of 5-HT and 5-HIAA were expressed as picograms per milligram of total protein. 10 μl of each supernatant were injected into the HPLC system with the following setting: A isocratic pump, (model PU-2080 Plus, Jasco Co. Ltd., Tokyo, Japan), a UniJet microbore column (MF-8912, BAS, West Lafayette, IN, USA), and an amperometric detector (set at 650 mV, 0.5 nA; model LC-4C, BAS, West Lafayette, IN, USA). The mobile phase, containing 0.05 M NaH2PO4, 1.0 mM 1-octane sulfonic acid, 0.27 mM EDTA, 1.0% (v/v) tetrahydrofuran, and 4.0% (v/v) acetonitrile (CH3CN) (pH adjusted to 2.6) was pumped at a flow rate of 100μl/min. The level of neurotransmitters and metabolites was assessed by comparing the respective peak area and elution time of the sample with a reference standard. The quantification was performed using a calibration curve for each neurotransmitter (Program ChromPass, Jasco Co. Ltd., Tokyo, Japan). Under these experimental conditions, retention times were 33.3 for 5-HT and 25.6 for 5-HIAA. Standards, EDTA, and 1-octane sulfonic acid were purchased from Sigma-Aldrich, Inc. (St Louis, MO, USA), and all other reagents were of analytical grade.