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

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 Ca2+-detector, TN-XXL – activation of the receptor leads to an increase in the FRET signal. Because a CNiFER clone utilizes the native receptor for a particular neurotransmitter (e.g. D2R for dopamine), it has nanomolar sensitivity and a temporal response of seconds. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred micrometers, 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.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

“…To circumvent elaborate and invasive transfection procedures HEK cell clone that stably expresses green fluorescent G-geNOp was used to quantify exogenously generated single-cell NO• profiles. As HEK cells normally do not produce NO• endogenously 29 , this cell type is suitable for the generation of a geNOp-based sensor cell line, which might be useful for many other applications in co-culture conditions with NO• producing primary cells or even in living animals 30 . However, in this study we demonstrate the capacity of various NO•-liberating compounds, of different concentrations and stabilities, to evoke intracellular NO• signals using the G-geNOp expressing HEK cell model.…”

A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors

“…Peptides tagged with large fluorescent proteins or reporters such as EGFP, pHluorin, and GCaMP provide a relatively fast readout of peptide release and describe knockout phenotypes related to peptide release in vitro , but this approach is limited to modified peptides, not the endogenous peptides, and is hardly possible to apply in vivo (Burke et al, 1997; de Wit et al, 2009; Ding et al, 2019; Lang et al, 1997; van den Pol, 2012; Xia et al, 2009). Finally cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) have been used to detect neuropeptides; however, the need to implant exogenous cells in specific brain regions limits the utility of this approach (Jones et al, 2018; Lacin et al, 2016; Xiong et al, 2021). Thus, the precise spatiotemporal dynamics and release patterns of endogenous peptides remain poorly understood.…”

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

“…To circumvent elaborate and invasive transfection procedures HEK cell clone that stably expresses green fluorescent G-geNOp was used to quantify exogenously generated single-cell NO• profiles. As HEK cells normally do not produce NO• endogenously 29 , this cell type is suitable for the generation of a geNOp-based sensor cell line, which might be useful for many other applications in co-culture conditions with NO• producing primary cells or even in living animals 30 . However, in this study we demonstrate the capacity of various NO•-liberating compounds, of different concentrations and stabilities, to evoke intracellular NO• signals using the G-geNOp expressing HEK cell model.…”