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      Ultra-sensitive fluorescent proteins for imaging neuronal activity

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          Summary

          Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultra-sensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies, and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5 - 40 micrometers long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

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          Most cited references 68

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          The Psychophysics Toolbox.

          The Psychophysics Toolbox is a software package that supports visual psychophysics. Its routines provide an interface between a high-level interpreted language (MATLAB on the Macintosh) and the video display hardware. A set of example programs is included with the Toolbox distribution.
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            Enzymatic assembly of DNA molecules up to several hundred kilobases.

            We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
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              The VideoToolbox software for visual psychophysics: transforming numbers into movies.

              The VideoToolbox is a free collection of two hundred C subroutines for Macintosh computers that calibrates and controls the computer-display interface to create accurately specified visual stimuli. High-level platform-independent languages like MATLAB are best for creating the numbers that describe the desired images. Low-level, computer-specific VideoToolbox routines control the hardware that transforms those numbers into a movie. Transcending the particular computer and language, we discuss the nature of the computer-display interface, and how to calibrate and control it.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                24 July 2013
                18 July 2013
                18 January 2014
                : 499
                : 7458
                : 295-300
                Affiliations
                [1 ]Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA
                [2 ]Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida Brasília, Doca de Pedrouços, 1400-038, Lisbon, Portugal
                [3 ]Department of Neurobiology, University of California Los Angeles, Los Angeles, CA 90095, USA
                Author notes
                Correspondence should be addressed to M.B.O. ( michael.orger@ 123456neuro.fchampalimaud.org ) for zebrafish, V.J. ( jayaramanv@ 123456janelia.hhmi.org ) for flies, L.L.L. ( loogerl@ 123456janelia.hhmi.org ) for GCaMP protein structure information, K.S. ( svobodak@ 123456janelia.hhmi.org ) for mice, and D.S.K. ( kimd@ 123456janelia.hhmi.org ) for neuronal culture screen information and constructs
                [†]

                Present address: Marine Biological Laboratory, Program in Sensory Physiology and Behavior, 7 MBL Street, Woods Hole, Massachusetts 02543, USA

                Article
                HHMIMS489004
                10.1038/nature12354
                3777791
                23868258

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
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