Microdialysis probes (MD-2200, BASI) were stereotactically inserted into the NAc and perfused with artificial cerebrospinal fluid (aCSF) composed of either 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, and 0.8 mM MgCl2 in 10 mM phosphate buffer alone or, additionally, with either 10 nM naltrindole or a combination of 10 μM hexamethonium and 10 μM scopolamine (3.0 μl/min). Samples were collected every 20 min for 4 h with MStim occurring after the first 2 h. Samples were analyzed using a HPLC pump (Ultimate 3000, Dionex, Sunnyvale, CA, USA) and electrochemical detector (Coulochem III, ESA). The detector included a guard cell (5020, ESA) set at +270 mV, a screen electrode (5014B, ESA) set at −100 mV, and a detection electrode (5014B, ESA) set at +220 mV. Dopamine was separated using a C18 reverse phase column (HR-80, Thermo Fisher Scientific, Waltham, MA, USA). Mobile phase included 75 mM H2NaO4P, 1.7 mM sodium octane sulfonate, 25 μM EDTA, 0.714 mM triethylamine, and 10% acetonitrile at a flow rate of 0.5 ml/min.
Hr 80
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HR-80 is a high-resolution centrifuge designed for a wide range of laboratory applications. It features a high-speed motor and a durable, corrosion-resistant construction to provide consistent and reliable performance.
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6 protocols using hr 80
1
Microdialysis of Dopamine in Nucleus Accumbens
2
Simultaneous Quantification of TRP Metabolites
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Plasma aliquots (100 μL) were deproteinized with perchloric acid, and tyrosine added as internal standard. After centrifugation, supernatants were injected onto a reverse phase column (HR-80; 80 mm × 4.6 mm, 3 µm; Thermo Fisher Scientific). KYN was eluted using a mobile phase containing 0.1 M sodium acetate and 4% acetonitrile (adjusted to pH 4.6) and determined by UV detection (360 nm, Waters 2487) whereas TRP and KA were separated using a mobile phase containing 0.5 M sodium acetate (adjusted to pH 6.2), 0.25 M zinc acetate and 5% acetonitrile, and detected fluorometrically at excitation/emission wavelengths of 270/360 nm for TRP and 344/398 nm for KA (Waters 2475). 5-HT was separated using an ODS2-C18 column (150 mm × 4.6 mm, 5 µm, Waters) and a mobile phase consisting of 0.1 M sodium acetate (adjusted to pH 3.8) and 8% methanol, and detected fluorometrically at excitation/emission wavelengths of 290/337 nm.
QA was determined using a commercially available ELISA immunoassay (Cloud-Clone Corp., Houston, USA) according to instructions of the manufacturer.
The ratios of KYN or 5-HT to TRP and KA or QA to KYN were used as a measure of TRP degradation and of KA and QA formation, respectively.
QA was determined using a commercially available ELISA immunoassay (Cloud-Clone Corp., Houston, USA) according to instructions of the manufacturer.
The ratios of KYN or 5-HT to TRP and KA or QA to KYN were used as a measure of TRP degradation and of KA and QA formation, respectively.
3
Quantification of 3-Hydroxykunurenine by HPLC
4
In Vivo Dopamine Measurement via Microdialysis
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Microdialysis probes (MD-2200, BASI) were stereotactically inserted into the NAc (+1.6 AP, +1.9 ML, −8.0 DV) of 7 rats per group. Artificial cerebrospinal fluid (aCSF; pH ~7.4 and osmolarity of 300–310 mOSm) composed of 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, and 0.8 mM MgCl2 in 10 mM phosphate buffer was perfused through the probe at a rate of 3.0 µL/min. Samples were collected every 20 min for 4 h with reinstatement does of ethanol (2.5 mg/kg; IP) occurring after the first 2 h had elapsed. Determination of the DA concentration in microdialysis samples was performed using a HPLC pump (Ultimate 3000, Dionex, Sunnyvale, CA, USA) connected to an electrochemical detector (Coulochem III, ESA). The detector included a guard cell (5020, ESA) set at +270 mV, a screen electrode (5014B, ESA) set at −100 mV, and a detection electrode (5014B, ESA) set at +220 mV. Dopamine was separated using a C18 reverse phase column (HR-80, Thermo Fisher Scientific, Waltham, MA, USA). Mobile phase containing 75 mM H2NaO4P, 1.7 mM sodium octane sulfonate, 25 µM EDTA, 0.714 mM triethylamine, and 10% acetonitrile was pumped through the system at a flow rate of 0.5 mL/min.
5
Plasma and Tissue Kynurenine Quantification
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Plasma samples were deproteinized by adding 75 μl of 6% perchloric acid to the mixture of 50 μl plasma and 125 μl of water. The acidified plasma was vortexed and kept at room temperature for 10 min, and then centrifuged for 15 min at 16000 × g at 4°C. Limbic forebrain samples were homogenized in five volumes of deionized water by sonication (Labsonic 2000 U, B. Braun Melsungen AG, Germany) at 30% amplitude during 15 s. Samples were deproteinized by adding 25 μl of 6% perchloric acid per 100 μl of homogenate, vortexed and kept at room temperature for 10 min. Samples were then centrifuged for 15 min at 16000 × g at 4°C. After centrifugation, supernatants were collected and frozen at −80°C until analysis. Sixty (limbic forebrain) or twenty (plasma) microliters of supernatant were applied to a reversed phase column (80 mm × 4.6 mm, 3 mm; HR‐80; Thermo Fisher Scientific, USA), and KYN was isocratically eluted using a mobile phase containing 0.1‐M sodium acetate and 4% acetonitrile, pH 4.6, at a flow rate of 1 ml·min−1. KYN was measured by UV detection (360 nm, 2487 UV detector; Waters, USA).
6
Microdialysis of Dopamine in Nucleus Accumbens
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Microdialysis probes (MD-2200, BASI) were stereotactically inserted into the NAc and perfused with artificial cerebrospinal fluid (aCSF) composed of either 150 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, and 0.8 mM MgCl2 in 10 mM phosphate buffer alone or, additionally, with either 10 nM naltrindole or a combination of 10 μM hexamethonium and 10 μM scopolamine (3.0 μl/min). Samples were collected every 20 min for 4 h with MStim occurring after the first 2 h. Samples were analyzed using a HPLC pump (Ultimate 3000, Dionex, Sunnyvale, CA, USA) and electrochemical detector (Coulochem III, ESA). The detector included a guard cell (5020, ESA) set at +270 mV, a screen electrode (5014B, ESA) set at −100 mV, and a detection electrode (5014B, ESA) set at +220 mV. Dopamine was separated using a C18 reverse phase column (HR-80, Thermo Fisher Scientific, Waltham, MA, USA). Mobile phase included 75 mM H2NaO4P, 1.7 mM sodium octane sulfonate, 25 μM EDTA, 0.714 mM triethylamine, and 10% acetonitrile at a flow rate of 0.5 ml/min.
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