Development and application of the fluorescent analogue of dopamine – From a model dopaminergic cells in vitro to the retinal dopaminergic amacrine cells of zebrafish in vivo

• Main Authors: Lin, Hui-Jen, 林慧貞
• Other Authors: Li, Yaw-Kuen
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/wa4gck

博士 === 國立交通大學 === 應用化學系碩博士班 === 104 === Quantitative and sensitive assessment of the uptake, secretion and transport of neurotransmitters is important to improve our understanding of neurotransmission. We report a chemical strategy through direct conjugation of a fluorescent dye (FITC) to a dopamine molecule to develop a novel fluorescent analogue of dopamine (FITC-DA), and demonstrate its application on a model dopaminergic cell line (PC12) in vitro and the retinal dopaminergic amacrine cells of zebrafish in vivo. Our results showed that FITC-DA exhibits many essential features of natural dopamine. It accumulated abundantly in cells that express dopamine transporters (DAT) (such as PC12 and SK-N-SH) but not in cells with no DAT (such as HepG2, HeLa and NB41A3). The uptake of FITC-DA by PC12 cells was increased with the differentiation of cells while inhibited by a pretreatment of an inhibitor of DAT (GBR 12935) or by a cotreatment of natural dopamine. These results indicate that the uptake of FITC-DA was mediated mainly by DAT rather than by some unspecific pathways. Besides, FITC-DA acted as a substrate of the vesicular monoamine transporter expressed on the vesicular membrane of PC12 cells, and high potassium solution evoked the secretion of FITC-DA from PC12 cells. Moreover, FITC-DA labeled predominantly dopaminergic neurons in acute brain slices. Besides, FITC-DA possesses similar pharmacologic activity as dopamine by increasing the heart rate of rats. To extend the application in vivo, FITC-DA was tested on living zebrafish; immunostaine confirmed that FITC-DA selectively labeled the dopaminergic amacrine cells on the retina of larval zebrafish and potassium ion evoked the release of FITC-DA from the dopaminergic amacrine cells. As a demonstration, we show that it is feasible to ablate single dopaminergic amacrine cells on living zebrafish with femtosecond-laser pulses under the guidance of fluorescence imaging. Our strategy is simple and easy to be adapted, which should facilitate applications in varied fields such as stem cells research, neuropharmacology and neurotransmission. Finally, we anticipate that our work may shed light on the structure-function relation between the neurotransmitter and its transporter or receptor and may arouse new routes of the design of neuroactive drugs.