A microdialysis probe (1mm CMA-7, 6 kDa, CMA) connected to an infusion pump was inserted through the cannula placed in the striatum. Artificial cerebrospinal fluid (ACSF) was perfused at 1.2 μL/min. After the probe insertion we waited for 40 minutes to avoid artifacts evoked by mechanical manipulation. Each sample was collected for 5 minutes in awake animals moving freely on a custom designed jetball system. Immediately after collection, each sample was quantified by an HPLC system (Eicom). Chromatograms were analyzed with the software EPC-300 (Eicom). Dopamine concentration was determined using a dopamine solution of 0.5 pg/μL (Sigma-Aldrich). The temporal course of uncaged dopamine was normalized to the maximum peak evoked by LED irradiation. We used such normalization because the measurements of uncaged dopamine in the samples varied as a function of the distance between the fiber optic and the microdialysis probe.
Hplc system
Manufactured by Eicom
The HPLC system is a versatile analytical instrument used for the separation, identification, and quantification of various chemical compounds. It operates on the principle of high-performance liquid chromatography, which involves the use of a liquid mobile phase and a stationary phase to achieve efficient separation of analytes.
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3 protocols using hplc system
1
Quantifying Uncaged Dopamine Dynamics
2
Microdialysis for Extracellular Glutamate Measurement
3
HPLC Analysis of Midbrain 5-HT and 5-HIAA
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The concentrations of 5-HT and 5-HIAA in the midbrain were measured using a high-performance liquid chromatography (HPLC) system (Eicom) as previously described (36 (link)). The midbrain samples were dissected from 9- to 10-week-old mice and stored at −80°C until use. For analyses, the samples were homogenized in 0.2 M ice-cold perchloric acid, and the homogenates were cooled on ice for 30 min for deproteinization. The homogenates were centrifuged at 20,000g for 15 min at 0°C. The pH of the supernatants was adjusted to approximately 3 by adding 1 M sodium acetate, and the pellets were used for protein quantification. The supernatants were filtered through a 0.45-μm filter (Millipore), and 10 μl of the filtrate was applied to the HPLC system. The system had a 3 × 150 mm octadecylsilane column (SC-5ODS, Eicom) and an electrochemical detector (HTEC-500, Eicom) set to an applied potential of 750 mV versus an Ag/AgCl reference analytical electrode. The changes in electric current (in nanoamperes) were recorded using a computer interface. The mobile phase was composed of 0.1 M aceto–citric acid buffer (pH 3.5), methanol, 0.46 M sodium-1-octane sulfonate, and 0.015 mM disodium-EDTA at a ratio of 830:170:1.9:1. The flow rate was 0.5 ml/min. Protein quantification was performed using a Bio-Rad protein assay system (Bio-Rad) according to the manufacturer’s protocol.
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