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      Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

      1 , 1 , 2 , 3 , 1 , 4 , 5 , 6 , 7 , 8 , 9 , 8 , 1 , 1 , 1 , 1 , 4 , 4 , 1 , 1 , 1 , 7 , 1 , 6 , 2 , 5 , 4 , 1 , 3 , 1

      Frontiers in Molecular Neuroscience

      Frontiers Media S.A.

      calcium imaging, genetically encoded calcium indicator, multi-color imaging, protein engineering, optogenetics

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          Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca 2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca 2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca 2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

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          Most cited references 84

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            The green fluorescent protein.

            In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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              Neocortical excitation/inhibition balance in information processing and social dysfunction.

              Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

                Author and article information

                Front Mol Neurosci
                Front Mol Neurosci
                Front. Mol. Neurosci.
                Frontiers in Molecular Neuroscience
                Frontiers Media S.A.
                04 March 2013
                : 6
                1Janelia Farm Research Campus, Howard Hughes Medical Institute Ashburn, VA, USA
                2Medical Research Council Laboratory of Molecular Biology Cambridge, UK
                3Department of Chemistry, University of Puerto Rico - Río Piedras San Juan, PR, USA
                4Institute of Biochemistry and Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University Frankfurt Frankfurt, Germany
                5Experimentelle Biophysik, Humboldt Universität zu Berlin Berlin, Germany
                6Department of Neurology, University Medicine Göttingen Göttingen, Germany
                7Laboratory of Neural Circuits and Behavior, Howard Hughes Medical Institute, The Rockefeller University New York, NY, USA
                8Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University Cambridge, MA, USA
                9Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown Lisboa, Portugal
                Author notes

                Edited by: Piotr Bregestovski, Institut national de la santé et de la recherche médicale, Université de la Méditerranée, France

                Reviewed by: Robert E. Campbell, University of Alberta, Canada; Thomas Grutter, University of Strasbourg - CNRS, France

                *Correspondence: Loren L. Looger, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, 19700 Helix Dr., VA 20147, USA. e-mail: loogerl@

                †Present address: Lin Tian, Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.

                Trevor J. Wardill, Marine Biology Laboratory, Program in Sensory Physiology and Behavior, Woods Hole, MA, USA.

                Copyright © 2013 Akerboom, Carreras Calderón, Tian, Wabnig, Prigge, Tolö, Gordus, Orger, Severi, Macklin, Patel, Pulver, Wardill, Fischer, Schüler, Chen, Sarkisyan, Marvin, Bargmann, Kim, Kügler, Lagnado, Hegemann, Gottschalk, Schreiter and Looger.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

                Page count
                Figures: 9, Tables: 3, Equations: 0, References: 112, Pages: 29, Words: 21113
                Original Research Article


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