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1 Janelia Publications

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    02/15/22 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission
    Abhi Aggarwal , Rui Liu , Yang Chen , Amelia J Ralowicz , Samuel J Bergerson , Filip Tomaska , Timothy L Hanson , Jeremy P Hasseman , Daniel Reep , Getahun Tsegaye , Pantong Yao , Xiang Ji , Marinus Kloos , Deepika Walpita , Ronak Patel , Paul W Tilberg , Boaz Mohar , GENIE , Loren L Looger , Jonathan S Marvin , Michael B Hoppa , Arthur Konnerth , David Kleinfeld , Eric R Schreiter , Kaspar Podgorski
    bioRxiv PrePrint. 2022 Feb 15:. doi: 10.1101/2022.02.13.480251

    The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

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