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      An optimized fluorescent probe for visualizing glutamate neurotransmission

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          Abstract

          We describe an intensity-based glutamate-sensing fluorescent reporter (“iGluSnFR”) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted post-synaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

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          Author and article information

          Journal
          101215604
          32338
          Nat Methods
          Nat. Methods
          Nature methods
          1548-7091
          1548-7105
          24 November 2014
          13 January 2013
          February 2013
          17 June 2015
          : 10
          : 2
          : 162-170
          Affiliations
          [1 ] Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA
          [2 ]Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06511, USA
          [3 ]Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA
          [4 ]Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
          [5 ]Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Av. Brasília, Doca de Pedrouços, 1400-038 Lisboa, Portugal
          [6 ]Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT 06511, USA
          Author notes
          [7]

          current address: Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento CA 95817, USA

          []Correspondence should be addressed to L. L. L. ( loogerl@ 123456janelia.hhmi.org )
          Article
          NIHMS429924
          10.1038/nmeth.2333
          4469972
          23314171

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