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

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


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.

Publication types

  • Video-Audio Media

MeSH terms

  • Acetylcholine / metabolism
  • Animals
  • Biosensing Techniques / methods*
  • Calcium / metabolism
  • Cerebral Cortex / metabolism
  • Dopamine / metabolism
  • Flow Cytometry
  • Genes, Reporter / physiology*
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Mice
  • Norepinephrine / metabolism
  • Polymerase Chain Reaction
  • Receptor, Muscarinic M1 / metabolism*
  • Receptors, Adrenergic, alpha-1 / metabolism*
  • Receptors, Dopamine D2 / metabolism*
  • Receptors, G-Protein-Coupled / metabolism*
  • Transduction, Genetic


  • Luminescent Proteins
  • Receptor, Muscarinic M1
  • Receptors, Adrenergic, alpha-1
  • Receptors, Dopamine D2
  • Receptors, G-Protein-Coupled
  • Acetylcholine
  • Calcium
  • Dopamine
  • Norepinephrine