類多巴胺螢光分子應用於專一性細胞成像及分析 - 從體外培養細胞到活體斑馬魚之視網膜多巴胺無軸突細胞

Abstract

定量與靈敏的分析神經傳導物質的回收 (uptake)、分泌 (secretion) 與運送 (transport) 對了解神經傳遞 (neurotransmission) 的機制極為重要。我們提出一個化學策略，將FITC螢光分子與多巴胺分子結合，設計出「類多巴胺螢光分子 (fluorescent analogue of dopamine)」，應用螢光成像在體外培養細胞及活體斑馬魚觀察及分析多巴胺神經傳導物質。此「類多巴胺螢光分子」 (以下稱為 FITC-DA) 仍保留許多天然多巴胺的重要生物特性。從體外培養細胞的實驗發現，FITC-DA 會被表現多巴胺轉運子 (dopamine transporter；DAT) 的細胞 (例如 PC12) 回收 (uptake)，而且此回收具細胞選擇性且回收率會隨著 PC12細胞的分化程度而提高，而天然多巴胺或 DAT的抑制劑均降低 PC12 細胞回收 FITC-DA。此結果顯示FITC-DA 可被PC12細胞膜上的多巴胺轉運子 (dopamine transporter；DAT) 選擇性辨識且回收。我們也觀察到FITC-DA 可被神經細胞之囊泡輸送，在鉀離子刺激下則會釋放至細胞外，顯示 FITC-DA 也是神經細胞之囊泡膜上的轉運子 (vesicular transporter) 的受質 (substrate)。與以上結果相符的是，FITC-DA可以在大鼠活性腦切片中選擇性標定多巴胺神經細胞 (dopaminergic neurons) 的分布區域。此外，FITC-DA 也具有與天然多巴胺類似的藥理活性，可以增加大鼠的心率。在此基礎下，我們應用斑馬魚 (zebrafish) 幼魚探討應用 FITC-DA 於活體動物多巴胺神經細胞研究之可行性。結果顯示 FITC-DA 對斑馬魚幼魚毒性極低，透過免疫染色法我們也證實 FITC-DA可選擇性標定斑馬魚幼魚視網膜上之無軸突多巴胺神經細胞 (retinal dopaminergic amacrine cell)，而且注入高鉀離子溶液則能刺激無軸突多巴胺神經細胞釋放FITC-DA。我們也展現在即時螢光成像的導引下，可應用飛秒脈衝雷射 (femtosecond-pulsed laser)，在活斑馬魚體，將單一多巴胺無軸突細胞燒蝕 (ablation)。由於合成方法簡易且可應用於顯微螢光成像，我們預期此「類多巴胺螢光分子」將在包括幹細胞分化、神經藥物作用、神經物質傳導及抑制的分析等領域有廣泛應用。此外，我們的工作也對「結構與功能相關性 (structure-function relation)」有興趣的研究者在探討神經傳導物質轉運子或受體如何選擇性辨識神經傳導物質以及藥物開發研究者在設計神經藥物，提供了全新的思考方向。

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.