2.8. High-Performance Liquid Chromatography (HPLC)

Neurochemical analysis was performed similarly to described in previous studies [59,60]. Briefly, frozen brain samples were sonicated in 0.5 mL of 0.4 N perchloric acid (HClO₄) on ice. Samples were then centrifuged at 16,400× g for 35 min at 4 °C. The supernatant was transferred to a 0.22 mm Spin-X tube with nylon filter (Corning, Corning, NY, USA) and centrifuged at 1000× g for 15 min at 4 °C. Samples were then stored at −80 °C until HPLC analysis.

The HPLC system consisted of a Dionex Ultimate 3000 Model ISO-31000BM pump, a model WPS-3000TBSL autosampler, Coulochem III electrochemical detector and an ESA Coulochem data station (ThermoScientific, Waltham, MA, USA) was used to measure neurotransmitter levels including GABA, glutamate, dopamine (DA), 3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) in the brain tissues of SVZ, STR, HP, and OB.

Neurochemicals were separated on a Waters XBridge reverse-phase G18 column (150 × 3.0 mm, 3.5 μm particle size) (Water Corp, Milford, MA, USA). For monoamine separation, the mobile phase was: 80 mM NaH₂PO₄, 10% methanol, 2 mM octanesulfonic acid, 0.025 mM ethylenediaminetetraacetic acid and 0.2 mM trimethylamine, at pH 2.4. Monoamines were detected by analytical cell set at E1 = −150 mV and E2 = +350 mV. For quantification of GABA and glutamate, the mobile phase was 0.1 M Na₂HPO₄, 22% methanol and 4% acetonitrile, at pH 6.75. Quantification of amino acid neurotransmitters was accomplished through derivatization. The samples were mixed with a derivatization agent containing 0.M o-phthaladehyde (OPA), 0.05% 2-mercaptoethanol, 10% methanol in OPA diluent, prior to separation. Neurotransmitters in the sample were detected by analytical cell set at E1 = −150 mV and E2 = +550 mV. Levels of neurotransmitters tested here were calculated using area under the curve by comparison with standard curve. Levels were normalized to total protein amount and expressed as ng neurotransmitter/mg protein. DA and 5-HT turnover rates were also calculated.