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5 Publications

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    Keller LabLooger Lab
    06/22/21 | In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.
    Keller JP, Marvin JS, Lacin H, Lemon WC, Shea J, Kim S, Lee RT, Koyama M, Keller PJ, Looger LL
    Cell Reports. 2021 Jun 22;35(12):109284. doi: 10.1016/j.celrep.2021.109284

    Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 μM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

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    Looger LabDruckmann LabKeller Lab
    09/23/19 | Single-cell reconstruction of emerging population activity in an entire developing circuit.
    Wan Y, Wei Z, Looger LL, Koyama M, Druckmann S, Keller PJ
    Cell. 2019 Sep 23;179(2):. doi: 10.1016/j.cell.2019.08.039

    Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis.

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    Keller LabLooger Lab
    03/08/19 | In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.
    Keller JP, Marvin JS, Lacin H, Lemon WC, Shea J, Kim S, Lee RT, Koyama M, Keller PJ, Looger LL
    bioRxiv. 2019 Mar 8:. doi: 10.1101/571422

    Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 µM to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

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    Looger LabKeller Lab
    12/15/15 | Direct in vivo manipulation and imaging of calcium transients in neutrophils identify a critical role for leading-edge calcium flux.
    Beerman RW, Matty MA, Au GG, Looger LL, Choudhury KR, Keller PJ, Tobin DM
    Cell Reports. 2015 Dec 15;13(10):2107-17. doi: 10.1016/j.celrep.2015.11.010

    Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

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    Ahrens LabLooger LabKeller LabFreeman Lab
    07/27/14 | Light-sheet functional imaging in fictively behaving zebrafish.
    Vladimirov N, Mu Y, Kawashima T, Bennett DV, Yang C, Looger LL, Keller PJ, Freeman J, Ahrens MB
    Nature Methods. 2014 Jul 27;11(9):883-4. doi: 10.1038/nmeth.3040

    The processing of sensory input and the generation of behavior involves large networks of neurons, which necessitates new technology for recording from many neurons in behaving animals. In the larval zebrafish, light-sheet microscopy can be used to record the activity of almost all neurons in the brain simultaneously at single-cell resolution. Existing implementations, however, cannot be combined with visually driven behavior because the light sheet scans over the eye, interfering with presentation of controlled visual stimuli. Here we describe a system that overcomes the confounding eye stimulation through the use of two light sheets and combines whole-brain light-sheet imaging with virtual reality for fictively behaving larval zebrafish.

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