Esa coulochem 3 electrochemical detector

Mice striatal samples were homogenized with 0.1 M perchloric acid (1 mg tissues in 100 μL perchloric acid) and 0.1 mM EDTA, treated by ultrasonic and centrifuged at 20,000 rpm for 30 min as reported [27 ]. Then supernatant liquids were collected for measuring. The mobile phase is a mixed solution consisting of 90 mM sodium phosphate monobasic, 1.7 mM 1-octanesulfonic acid, 50 mM citrate, 50 μM EDTA, 10% acetonitrile with the flow rate of 0.2 ml/min. In parallel, for quantitative standard curve calculation, stock standards solution for dopamine and dihydroxy-phenyl aceticacid (DOPAC) (Sigma-Aldrich, USA) were prepared in 0.1 M HClO₄. Amount of 10 μl of prepared supernatant or standard solution was injected into the mobile phase and tested by ESA Coulochem III electrochemical detector (Coulochem III, Thermo Fisher Scientific). Samples were measured and peaks were quantified. The concentrations of monoamines were quantified by comparing with the standard using ClarityChrom software (Knauer, Germany) and then the contents in stratum were converted according to the dilution ratio. The standard curve of DA and DOPAC were shown in Supplementary Fig. 1a and b.

The levels of 5-HT and NE in the mouse hippocampus were determined by high-performance liquid chromatography (HPLC). The hippocampus was homogenized in extract solution, which consisted of 0.1 mM EDTA and 0.1 M HClO₄ buffer, and the mixture was centrifuged at a speed of 20,000 × g for 30 min at 4°C. Then, 50 µl of the resulting supernatant was injected into the liquid chromatography system equipped with a reversed phase C18 column (2.2 µm, 120 Å, 2.1×100 mm; Dionex; Thermo Fisher Scientific, Inc.). The mobile phase was composed of solutions A (0.1% formic acid, v/v) and B (acetonitrile), with a gradient elution as follows: 0–6 min, 90–65% A; 6–8 min, 65–0% A; 8–10 min, 0% A. The flow rate was maintained at 0.4 ml/min, and the column temperatures were maintained at 30°C, and was detected by ESA Coulochem^® III Electrochemical Detector (Dionex; Thermo Fisher Scientific, Inc.). The detector was set at 350 mV. The identification and purity were evaluated by the chromatographic peaks as well as their quantitative evaluation, which was measured by comparing their retention times and peak areas with those of standard solutions. 5-HT (cat. no. H9523) and NE (cat. no. N-069-1ML) standards were purchased from Sigma-Aldrich (Merck KGaA).