Dopamine h8502

Compounds UCM120 and UCM2550 were synthesized as described previously [38 (link), 51 (link)] and dissolved in DMSO before dilution to the needed concentration. Ergotamine and tryptamine were also dissolved in DMSO whereas Lysergic acid diethylamide (LSD), tyramine, octopamine, acetylcholine, histamine and dopamine were dissolved in distilled H₂O. 5-HT was dissolved in distilled H₂O at stock concentration of 5 mM. Stock solutions were either made up on the day of the experiment or taken from aliquots stored at -80°C for no longer than 1 week prior to use. After 0.22 μm filtration with Millex GV filter units (Millipore, Ireland), stock solutions were diluted to the corresponding final concentration (e.g. 0.1; 1; 10; 100; 500; 1000 and 2000 μM for 5-HT) in RPMI medium with high glucose (Gibco, USA). 5-HT (H9523), LSD (L7007), ergotamine (E1200000), tryptamine (193747), tyramine (T90344), octopamine (O0250), acetylcholine (A6625), histamine (H7125) and dopamine (H8502) were obtained from Sigma Chemical Company.

The dopamine quantification procedure was performed as described previously^{47 (link),63 (link)} with the following modifications. Thirty fly heads were homogenized in 600 μl homogenization buffer (0.1 M perchloric acid/3% trichloro acid) on ice, sonicated 5 times for 30 sec each, and then placed on ice for 30 min. Debris was removed by centrifugation at 15,000 × g at 4 °C for 15 min. Fifty microliters of the supernatant was utilized for the HPLC analysis using Nanospace SI-2 (Shiseido, Japan) with running buffer containing 180 mM chloroacetic acid, 50 μM EDTA, 160 mM sodium hydroxide, and 8.5% acetonitrile. Samples were separated on a CapCell Pak C18 UG120 column (Shiseido, Japan) at a 0.5 ml/min flow rate. Dopamine was electrochemically detected by Electrochemical Detector 3005 (Shiseido, Japan). Dopamine (H8502, Sigma-Aldrich) was used to build the standard curve at 0.0025, 0.005, 0.01, 0.02, and 0.04 μM. Differences in the dopamine levels of the examined samples were statistically analyzed using an ordinary two-way ANOVA with Tukey’s multiple comparisons test and graphed by GraphPad Prism 6.0 (GraphPad Software, USA).

β-alanine (A9920) and Dopamine (H8502) were purchased from Sigma. NBAD standards were provided by Professor Michael R. Kanost and Dr. Neal T. Dittmer (Department of Biochemistry, 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506-0116, USA). The β-alanine and aspartate standards used for quantitative analysis were provided by the Institute of Animal Nutrition, Sichuan Agricultural University (Chengdu, Sichuan, China).