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The microdialysis probes consist of 2.5 mm in length of semipermeable dialysis membrane from Spectra/Por in vivo Microdialysis Hollow Fibers, 2 μm OD, 13,000 MW cutoff. On the day of sampling, a microdialysis probe was inserted into the guide cannula directed to the IIC or the GIC. Artificial CSF (aCSF; 147 mM NaCl, 2.8 mM KCl, 1.2 mM CaCl₂, and 1.2 mM MgCl₂; pH 7.4) was perfused at a rate of 0.6 μl/min for a period of 300 min, after an acclimatization of 120 min, samples were collected and frozen on dry ice every 20 min for a total of 300 min.
The dialysate from the samples were analyzed for 5-HT using the Eicom HTEC-510 HPLC/ECD system (Eicom USA). Each sample was extracted from the vial and loaded on a C-18 reverse-phase column (PP-ODS II, 4.6 × 30 mm, Eicom USA) using a manual injection port (Rheodyne 9725i; 20-µl loop). The column was maintained at a temperature of 25°C with a mobile phase [0.1 M phosphate buffer pH 5.4, including 1.5% methanol, 500 mg/l decansulfonate sodium salt (DSS), and 50 mg/l 2Na-EDTA] set at a flow rate of 0.6 μl/min. Electrochemical detection of 5-HT was determined using a graphite working electrode (WE-3G, Eicom USA) maintained at a potential of +450 mV relative to an Ag/AgCl reference electrode (RE-500, Eicom USA).

High-performance liquid chromatography (HPLC) measurements of tissue serotonin (5-HT), dopamine (DA), norepinephrine (NE) and their metabolites 5-hydroxyin- doleacetic acid (5-HIAA), 3,4-Dihydroxyphenylacetic acid (DOPAC), and Homovanillic acid (HVA) were performed as described previously (Belov et al., 2019 (link)). Briefly, the striatum, the frontal cortex, the hypothalamus, and the hippocampus were dissected on ice, frozen in liquid nitrogen, and stored at −80°C. The samples for analysis were homogenized in 0.1 M HClO4, centrifuged (10 min, + 4°C; 14,000 × g), and filtered using centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size, 0.22 μm, Millipore, Burlington, MA, United States]. Measurements of monoamines in the tissue samples were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Japan) using +650 mV applied potential. The system was equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at 200 μl/min flow rate. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values for monoamines and metabolites levels were expressed as nanogram per milligram of wet tissue weight.

The striatum and the frontal cortex (FC) of animals were dissected on ice, frozen in liquid nitrogen, and stored at −80 °C. For analysis, the samples were homogenized in 0.1 M HClO₄ (20 μL per mg of tissue) containing 100 ng/mL 3,4-dihydroxybenzylamine (DHBA) as an internal standard. Homogenates were centrifuged for 10 min at 10,000× g (at +4 °C). Supernatants were filtered through centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size 0.22 μm, Millipore, Burlington, MA, USA] and then analyzed for levels of DA, using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Tokyo, Japan), using +650 mV applied potential. The separation was carried on a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at 200 μL/min flow rate. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 19.5% (vol/vol) methanol, pH 4.31. The volume of injection was 10 μL. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values were expressed as nanograms per milligram of wet tissue weight.

HPLC measurements of serotonin, and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Sapporo, Japan), with a carbon electrode WE-3G (Eicom, Sapporo, Japan), using +650 mV applied potential, and equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Sapporo, Japan), at a flow rate of 200 μL/min. Cells in logarithmic growth phase were trypsinized, washed 2 times with PBS, and immediately frozen and stored at −80 °C in a refrigerator. The cell samples were homogenized in 0.1 M HClO4, centrifuged (10 min, +4 °C; 14,000× g), and filtered using centrifuge filter units (PVDF membrane, pore size 0.22 μm, Millipore, Burlington, MA, USA). The mobile phase contained 100 mM PBS, 0.17 mM disodium ethylenediaminetetraacetate (EDTA), 1.8 mM octane sulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to the internal standard 3,4-dihydroxybenzylamine, and final values for serotonin and 5-HIAA were counted as ng/mL cell extract of equal number of cells.

Levels of catecholamines and their metabolites in the brainstem were determined using high-performance liquid chromatography with electrochemical detection (HPLC-ECD), as described previously [9 (link)]. Reversed-phase chromatographic analysis was performed using the HTEC-500 system (Eicom, Kyoto, Japan) with a reversed-phase column (Eicompack SC-5ODS, 3 mm φ  × 150 mm; Eicom) and a graphite working electrode (EICOM WE-3G; 12 mm φ) with an Ag/AgCl reference electrode. The potential of the working electrode was set at +750 mV. The acetate-citrate buffer (pH 3.5) used for separation (flow rate: 0.5 mL/min at 25°C) contained 0.053 M citric acid, 0.047 M sodium acetate, 5 mg/L EDTA, 193 mg/L sodium octyl sulfonate (Nacalai Tesque, Inc., Kyoto, Japan), and 17% methanol (v/v).

HPLC measurements of tissue 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were carried out as described before Belov et al. (2019 (link)). Briefly, the striatum, the frontal cortex, the hypothalamus, and the hippocampus were dissected on an ice, frozen in a liquid nitrogen, and stored at −80°C. The samples for analysis were homogenized in 0.1 M HClO₄, centrifuged (10 min, +4°C; 14,000× g) and filtered using centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size, 0.22 μm, Millipore, Burlington, MA, USA]. Measurements of 5-HT and 5-HIAA in the tissue samples were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Japan) using +650 mV applied potential. The system was equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at a flowrate of 200 μl/min. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values for 5-HT and 5-HIAA were expressed as nanogram per milligram wet tissue weight. The serotonin turnover rate was calculated as a ratio of 5-HIAA/5-HT.