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      Optimization of a GCaMP calcium indicator for neural activity imaging.

      The Journal of neuroscience : the official journal of the Society for Neuroscience

      growth & development, Animals, Astrocytes, chemistry, ultrastructure, Caenorhabditis elegans, Calcium Signaling, Crystallography, X-Ray, Drosophila melanogaster, Female, Fluorescent Dyes, analysis, Fluorometry, methods, Genes, Synthetic, Genetic Vectors, Green Fluorescent Proteins, genetics, isolation & purification, HEK293 Cells, Hippocampus, cytology, Humans, Larva, Lasers, Mice, Models, Molecular, Mutagenesis, Site-Directed, Neuroimaging, Neuromuscular Junction, Neurons, physiology, Neuropil, Olfactory Receptor Neurons, Peptides, Photic Stimulation, Protein Conformation, Rats, Recombinant Fusion Proteins, Retinal Bipolar Cells, Synaptic Transmission, Zebrafish

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          Abstract

          Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

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          Author and article information

          Journal
          23035093
          3482105
          10.1523/JNEUROSCI.2601-12.2012

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