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Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells
ACS Chemical Neuroscience ( IF 5 ) Pub Date : 2017-12-11 00:00:00 , DOI: 10.1021/acschemneuro.7b00391
Kallol Bera 1 , Anand Kant Das 1 , Ananya Rakshit 1 , Bidyut Sarkar 1 , Anoop Rawat 1 , Barun Kumar Maity 1 , Sudipta Maiti 1
Affiliation  

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.

中文翻译:

荧光检测活细胞中单胺类神经递质

单胺神经传递是神经调节的关键,但是在活神经元中成像单胺仍然是一个挑战。在这里,我们表明,外部添加的苯二甲醛(OPA)可以渗透活细胞,并与细胞内单胺(例如5-羟色胺,多巴胺和去甲肾上腺素)和L-DOPA形成明亮的荧光加合物,可以使用常规单光子灵敏地对其成像。在荧光显微镜下激发。峰值激发和发射波长(5-羟色胺的λex = 401 nm和λem = 490 nm;多巴胺的λex = 446 nm和λem = 557 nm;以及λex = 446 nm和λem对于大多数现代共聚焦成像仪器而言,去甲肾上腺素分别为544 nm和544 nm)。通过使用三光子激发显微镜对血清素进行准同时成像,可以确定血清素能RN46A细胞中含有单胺的结构(可能是神经递质囊泡)的身份。细胞提取物和体外溶液的质谱分析有助于我们确定加合物的化学性质并建立反应机理。我们的方法毒性低,选择性高,并且能够直接报告活细胞中单胺的位置和浓度。
更新日期:2017-12-11
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