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<p>Page créée avec « They were allowed to habituate to a period of dimly lit environment for 10?min before contralateral and ipsilateral turns, regarding the side of the lesion, were recorded ... »</p> <p><b>Nouvelle page</b></p><div>They were allowed to habituate to a period of dimly lit environment for 10?min before contralateral and ipsilateral turns, regarding the side of the lesion, were recorded over 60?min. The results of the turning behavior test were calculated as the difference between the numbers of contralateral and ipsilateral turns and were expressed as turns/hour [22]. Animals previously treated with 6-OHDA [http://www.selleckchem.com/products/MS-275.html MS275] that failed to show ipsilateral rotations were considered as nonlesioned and therefore dismissed from the experiment. 2.7. Apomorphine-Induced Turning Behavior Eight weeks after 6-OHDA lesion, apomorphine was injected s.c. at a dose of 2?mg/kg [26] and rotations were monitored for 60?min using the same experimental setup as for amphetamine-induced turning behavior. Results were expressed as turns/hour. 2.8. Enzymatic Activity Assay of 3-HSOR The isolated striatum and SNs were homogenized in 2?mL of ice-cold 10?mM phosphate buffer (pH 6.5) containing 0.154?M KCI, 1?mM dithiothreitol, 0.5?mM EDTA, and 1?��M PMSF. The homogenate was centrifuged at 105000?��g for 60?min at 4��C in a Beckman Optima TL (Palo Alto, CA) ultracentrifuge equipped with a TLA-100.3 rotor. The supernatant fraction (cytosolic fraction) was stored at ?80��C until the day of enzyme assay. Enzyme activity assay of 3��-HSOR [27] was determined spectrophotometrically by measuring the oxidation rate of NADPH at 340?nm and 37��C in a 1.0?cm-path length cuvette with [http://www.selleckchem.com/products/GDC-0449.html www.selleckchem.com/products/GDC-0449.html] a Metrolab 1600 DR (USA) spectrophotometer. The reductase activity was measured in 100?mM phosphate buffer (pH 6.5) containing 0.1?mM NADPH, 0.08?mM 5��-DHP (substrate), and enzyme solution in a total volume of 1.0?mL. The reaction was initiated by addition of cofactor to the assay mixture. A blank sample without substrate was routinely included. Water-insoluble substrate was dissolved in ethanol, and the final concentration of ethanol in the assay mixture did not exceed 1%, a concentration that had no effect on the catalytic activity of the enzyme. Protein concentration was determined by the method of Lowry [28] using BSA as a standard. The enzymatic activity was expressed as nmol of substrate consumed by 1 milligram of total protein in 1 minute. 2.9. RNA Isolation and Multiplex RT-PCR Analysis of 3��-HSOR [https://en.wikipedia.org/wiki/Quinapyramine Quinapyramine] Total RNA was isolated using TRIZOL reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. Gel electrophoresis and ethidium bromide staining confirmed the integrity of the samples. Quantification of RNA was based on spectrophotometric analysis at 260?nm. Two micrograms of total RNA was reverse transcribed with 200 units of MMLV Reverse Transcriptase (Promega Inc.) using hexamer random primers in a 50?��L reaction mixture following manufacturer's instructions.</div>

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