title: Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo.
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 µm, 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.
authors.affiliation: Department of Neuroscience, Icahn School of Medicine at Mount Sinai.
authors.forename: Emre
authors.initials: E
authors.lastname: Lacin
authors.affiliation: Department of Physics, University of California, San Diego.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Muller
authors.affiliation: Department of Neuroscience, Icahn School of Medicine at Mount Sinai.
authors.forename: Marian
authors.initials: M
authors.lastname: Fernando
authors.affiliation: Department of Physics, University of California, San Diego; Section of Neurobiology, University of California, San Diego; dk@physics.ucsd.edu.
authors.forename: David
authors.initials: D
authors.lastname: Kleinfeld
authors.affiliation: Department of Neuroscience, Icahn School of Medicine at Mount Sinai; Paul.Slesinger@mssm.edu.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Slesinger
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000502:physiology*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009638:Norepinephrine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D008164:Luminescent Proteins; D043643:Receptor, Muscarinic M1; D018340:Receptors, Adrenergic, alpha-1; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D000109:Acetylcholine; D002118:Calcium; D004298:Dopamine; D009638:Norepinephrine