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ACS Chem Neurosci. 2018 Mar 21;9(3):469-474. doi: 10.1021/acschemneuro.7b00391. Epub 2017 Dec 26.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells.

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1
Department of Chemical Sciences , Tata Institute of Fundamental Research , Homi Bhabha Road , Colaba, Mumbai 400005 , India.

Abstract

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.

KEYWORDS:

Falck Hillarp method; dopamine imaging; monoamine microscopy; norepinephrine imaging; serotonin imaging

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