A genetically encoded fluorescent sensor for in vivo imaging of GABA

Abstract

Protein engineering and experiment design: JSM, LLL Hippocampal slice imaging: TPJ, DAR Visual cortex volume imaging: KP, ON Mouse epilepsy model: YS, VM, ML, DMK Mitochondria experiments: ELK, NJL Zebrafish: TK, MBA Abstract (150 words)Current techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR (∆F/Fmax ~ 2.5, Kd ~ 9 µM, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

“…2g). Thus, there was a ~3-fold loss of ATP sensitivity and decrease in peak response in both sensors when they were extracellularly displayed compared to the soluble proteins (similar effects are seen with other membrane-displayed sensors [25][26][27] ). During these experiments, we noticed that cells expressing iATPSnFR 1.1 were dimmer than those expressing iATPSnFR 1.0 , and we found lower expression levels of iATPSnFR 1.1 in relation to iATPSnFR 1.0 ( Fig.…”

“…Hence, our experiences with iATPSnFR 1.0 in solution, in HEK293 cells, in astroglia and in hippocampal neurons demonstrate that the weakening of ATP binding and the decrease in dF/F resulting from tethering the sensor to the outside of the membrane bilayer is independent of the host cell. We have observed altered sensitivity with other sensors on the membrane [25][26][27] ; we speculate that it results from steric restriction from lipid head groups and membrane proteins.…”

Presynaptic MAST kinase controls opposing postsynaptic responses to convey stimulus valence in Caenorhabditis elegans

“…It remains to be determined whether KIN-4, MEC-2, and DGK-1 are involved in the regulation of such ion channels to impact on the release of neurotransmitter from AFD. Utilization of the recently developed optical sensors for neurotransmitters (57)(58)(59)(60) would unveil the dynamics of axonal computations and help dissect the mechanisms by which kin-4, mec-2, and dgk-1 regulate the release of either or both of glutamate and neuropeptide from the AFD neuron.…”

G‐protein‐coupled receptor‐based sensors for imaging neurochemicals with high sensitivity and specificity

“…These sensors were originally designed by combining a ligand‐binding protein as the ‘sensing scaffold’ with a circular‐permutated fluorescent protein (cpFP) (Baird et al ) as the ‘reporting module’. Previously, prokaryotic periplasmic‐binding proteins (PBPs) were used as the scaffold for detecting several neurochemicals, including glutamate (iGluSnFR), γ‐aminobutyric acid (iGABASnFR), and adenosine triphosphate (iATPSnFR), suggesting that this strategy may be applicable for sensor engineering (Marvin et al ; Marvin et al ; Lobas et al ). However, this design strategy is not feasible in cases which a suitable PBP is not available for the molecule of interest.…”