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      Characterization and Subcellular Targeting of GCaMP-Type Genetically-Encoded Calcium Indicators

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          Abstract

          Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca 2+] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator—GCaMP2—in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17±10% ΔF/F [mean±SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302±50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca 2+ accumulations evoked by activation of synaptic NMDA receptors. We observed robust ΔF/F responses (range: 37%–264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.

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          Most cited references 49

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          Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

          Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
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            Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

            Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
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              A simple method for organotypic cultures of nervous tissue.

              Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2008
                19 March 2008
                : 3
                : 3
                Affiliations
                [1 ]Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
                [2 ]Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, United States of America
                [3 ]Laboratory for Memory and Learning, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan
                University of Southern California, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: svobodak@ 123456janelia.hhmi.org

                Conceived and designed the experiments: KS DO TM. Performed the experiments: DO TM VS. Analyzed the data: DO TM VS. Contributed reagents/materials/analysis tools: KS JN. Wrote the paper: KS DO TM.

                [¤]

                Current address: Department of Cellular and Systems Neurobiology, Max Planck Institute for Neurobiology, Martinsried, Germany

                Article
                07-PONE-RA-03091R1
                10.1371/journal.pone.0001796
                2262942
                18350138
                Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 10
                Categories
                Research Article
                Neuroscience

                Uncategorized

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