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6 Janelia Publications

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    06/01/23 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.
    Aggarwal A, Liu R, Chen Y, Ralowicz AJ, Bergerson SJ, Tomaska F, Mohar B, Hanson TL, Hasseman JP, Reep D, Tsegaye G, Yao P, Ji X, Kloos M, Walpita D, Patel R, Mohr MA, Tillberg PW, GENIE Project Team , Looger LL, Marvin JS, Hoppa MB, Konnerth A, Kleinfeld D, Schreiter ER, Podgorski K
    Nature Methods. 2023 Jun 01;20(6):. doi: 10.1038/s41592-023-01863-6

    The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. 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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    05/25/20 | jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.
    Mohr MA, Bushey D, Abhi Aggarwal , Marvin JS, Kim JJ, Marquez EJ, Liang Y, Patel R, Macklin JJ, Lee C, Tsang A, Tsegaye G, Ahrens AM, Chen JL, Kim DS, Wong AM, Looger LL, Schreiter ER, Podgorski K
    Nature Methods. 2020 May 25;17(1):694-97. doi: 10.1038/s41592-020-0835-7

    Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

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    02/08/20 | A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies.
    Borden P, Zhang P, Shivange AV, Marvin JS, Cichon J, Dan C, Podgorski K, Figueiredo A, Novak O, Tanimoto M, Shigetomi E, Lobas MA, Kim H, Zhu P, Zhang Y, Zheng WS, Fan C, Wang G, Xiang B, Gan L, Zhang G, Guo K, Lin L, Cai Y, Yee AG, Aggarwal A, Ford CP, Rees DC, Dietrich D, Khakh BS, Dittman JS, Gan W, Koyama M, Jayaraman V, Cheer JF, Lester HA, Zhu JJ, Looger LL
    bioRxiv. 2020 Feb 8:. doi: https://doi.org/10.1101/2020.02.07.939504

    Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

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    07/29/19 | Kilohertz frame-rate two-photon tomography.
    Kazemipour A, Novak O, Flickinger D, Marvin JS, Abdelfattah AS, King J, Borden P, Kim J, Al-Abdullatif S, Deal P, Miller E, Schreiter E, Druckmann S, Svoboda K, Looger L, Podgorski K
    Nature Methods. 2019 Jul 29;16(8):778-86. doi: 10.1101/357269

    Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

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    07/15/19 | A genetically encoded fluorescent sensor for in vivo imaging of GABA.
    Marvin JS, Shimoda Y, Magloire V, Leite M, Kawashima T, Jensen TP, Kolb I, Knott EL, Novak O, Podgorski K, Leidenheimer NJ, Rusakov DA, Ahrens MB, Kullmann DM, Looger LL
    Nature Methods. 2019 Jul 15;16(8):763-770. doi: 10.1038/s41592-019-0471-2

    Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

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    11/01/18 | Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR.
    Marvin JS, Scholl B, Wilson DE, Podgorski K, Kazemipour A, Mueller JA, Schoch-McGovern S, Wang SS, Quiroz FJ, Rebola N, Bao H, Little JP, Tkachuk AN, Hantman AW, Chapman ER, Dietrich D, DiGregorio DA, Fitzpatrick D, Looger LL
    Nature Methods. 2018 Nov;15(11):9386-9. doi: 10.1038/s41592-018-0171-3

    Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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