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2 Publications

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    03/24/16 | Sensitive red protein calcium indicators for imaging neural activity.
    Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS
    eLife. 2016 Mar 24;5:. doi: 10.7554/eLife.12727

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

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    10/03/12 | Optimization of a GCaMP calcium indicator for neural activity imaging.
    Akerboom J, Chen T, Wardill TJ, Marvin JS, Mutlu S, Carreras Caldero N, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr R, Takagi R, Kracun S, Shigetomi E, Khakh BS, Baier H, Lagnado L, Wang SS, Bargmann C, Kimmel B, Jayaraman V, Svoboda K, Kim DS, Schreiter ER, Looger LL
    The Journal of Neuroscience. 2012 Oct 3;32:13819-40. doi: 10.1523/​JNEUROSCI.2601-12.2012

    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.GCaMP5allows more sensitive detection of neural activity in vivo andmayfind widespread applications for cellular imaging in general.

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