L dopa ethyl ester

The 95% ethanol extracts of the leaf and root of Patrinia villosa (Thunb.) Juss. (lPv-EE and rPV-EE, respectively) were obtained from the National Institute of Biological Resources (https://www.nibr.go.kr/), Incheon, Korea). (3-4-5-Dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), 5-hydroxy-2-hydroxymethyl-4H-pyranone (Kojic acid), monophenol monooxygenase (tyrosinase from mushroom), 4-hydroxyphenyl-β-D-glucopyranoside (arbutin), α-MSH, and L-DOPA ethyl ester were bought from Sigma Chemical Co. (St. Louis, MO, USA). The luciferase plasmids, which harbor promoter binding sites with CREB, were used as reported earlier [20 (link)]. TRIzol reagent was obtained from the Molecular Research Center, Inc. (Montgomery, OH, USA). Fetal bovine serum, Dulbecco’s modified Eagle’s media (DMEM), and phenol red-free DMEM were purchased from Gibco (Grand Island, NY, USA). B16F10 cells were received from ATCC (Rockville, MD, USA). All other chemicals were obtained from Sigma Chemical Co (St. Louis, MO, USA). Total and phospho-specific antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) as reported previously [33 (link)].

Various pharmacological agents were used to manipulate the zebrafish DA neurons. The neurotoxin 6-hydroxy-dopamine (6-OHDA, Sigma) was used at a concentration of 250–500 µM to illicit motor deficits by damaging DA neurons in the equivalent of the zebrafish nigrostriatal-like pathway, as established previously in the literature [24 (link)]. The l-DOPA DA precursor l-dopa ethyl ester (Sigma) was used at concentrations up to 10 mM, which was the limit of its solubility in the ERS control solution, and was used to elevate the neurotransmitter in deficient fish, starting at ranges prescribed previously for zebrafish embryos [32 (link),33 (link)].

The larvae were exposed to two different drugs during the behavioural recordings, pramipexole dihydrochloride and L-Dopa ethyl ester (both Sigma-Aldrich, St. Louis, USA, CAS: 104632-25-9 and 37178-37-3). Drug preparation was performed at the day of recording. Drugs were diluted using distilled water to reach their respective concentrations. For pramipexole and L-Dopa, larvae were submerged in 1 μM, 10 μM and 100 μM doses. Drugs were administered into the wells between 11:30 and 12:30 on the day of recording.