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J Vis Exp. 2016 May 12;(111). doi: 10.3791/53290.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo.

Author information

1
Department of Neuroscience, Icahn School of Medicine at Mount Sinai.
2
Department of Physics, University of California, San Diego.
3
Department of Physics, University of California, San Diego; Section of Neurobiology, University of California, San Diego; dk@physics.ucsd.edu.
4
Department of Neuroscience, Icahn School of Medicine at Mount Sinai; Paul.Slesinger@mssm.edu.

Abstract

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred ┬Ám, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

PMID:
27214050
PMCID:
PMC4939270
DOI:
10.3791/53290
[Indexed for MEDLINE]
Free PMC Article

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