Lc 4c amperometric detector

For determination of NE, DA, and their metabolites (3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)), collected samples were loaded into an autosampler maintained at 4°C (Waters 717 Plus, Waters Chromatography Division, Milford, Massachusetts) and analyzed by isocratic liquid chromatography with electrochemical detection (LC-4C Amperometric Detector, BASi, West Lafayette, Indiana) at an oxidation potential of +700mV. Flow rate was 1.6 mL/min. The mobile phase consisted of 0.15 M monochloroacetate, pH 2.95, containing 1.3% acetonitrile (vol/vol), 1.7% tetrahydrofuran (vol/vol) (including 250 ppm butylated hydroxytoluene as an inhibitor), 0.86 mM sodium octylsulfate, and 0.18 mM EDTA, and was pumped through a 250 × 4.6mm, 5 μm Biophase ODS analytical column (PerkinElmer, Waltham, Massachusetts). Chromatographic peaks were quantified with EZChrom Elite software (Agilent Technologies, Pleasanton, California).

For measurement of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), dissected striata were homogenized in 0.1M perchloric acid and centrifuged (9,400 × g for 10 min at 4°C). The supernatant was filtered through a 0.22μm membrane (Millipore). Samples were analyzed using a Hypersil Gold C18 column (150 × 3mm; 5μm; Thermo Scientific) and a LC-4C Amperometric Detector (BASi) set at an oxidizing potential of +0.75V. The mobile phase contained 24mM Na₂HPO₄, 3.6mM 1-octanesulfonic acid, 30mM citric acid, 0.14mM EDTA in 19% methanol, adjusted to pH 4.7 using concentrated NaOH. 5-S-Cysteinyl-dopamine and 5-S-cysteinyl-DOPAC were measured as described previously (Hatcher et al., 2007 (link); Caudle et al., 2007 (link)). Briefly, striatal samples were sonicated in 0.1M perchloric acid containing 347μM sodium bisulfite and 134μM EDTA. Homogenates were centrifuged, filtered and separated on a C18 column. The electrochemical detector was set at an oxidizing potential of +0.65V. The mobile phase was MD-TM (ESA) containing 2mM NaCl and adjusted to pH 2.1 using concentrated HCl. Quantification of all neurochemicals was conducted by referring to calibration curves constructed from pure standards (purity >98%; dopamine, DOPAC and HVA from Sigma Aldrich; 5-S-cysteinyl-DA and 5-S-cysteinyl-DOPAC from NIMH Chemical Repository).

An oxygen microsensor (OX-25, Unisense, Denmark) equipped with three electrodes, was used to measure the oxygen partial pressure (pO₂) in the recording chamber (total volume 600 μl). The measurements were stored via digital logger. (NI USB-6008, National Instruments Co. TX, USA). The sensor has a rapid response to oxygen pressure changes and the current signal changes linearly with oxygen partial pressure (Yin et al., 2015 (link)), and was polarized at -800 mV (working & guard vs. reference) before use. Three-point calibration was performed with zero-oxygen solution (0.1 M NaOH + 0.1M sodium ascorbate), artificial cerebrospinal fluid (ACSF) saturated with carbogen mixture (5% CO₂ / 95% O₂) and OGD-based solution (see below) saturated with 95% N₂ / 5% CO₂. An LC-4C amperometric detector (BASi, IN, USA) was used as the potentiostat. Acute slices or organotypic hippocampal slices were treated with control solution for 10 min, oxygen-glucose deprivation (OGD) for 10 min, and reperfusion (RP), 30-60 min. ACSF gassed with 95% O₂ / 5% CO₂ is used for the control solution and RP, and MACSF gassed with 95% N₂ / 5% CO₂ is used in OGD. The individual values obtained were averaged and shown in Figure 2.