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.