Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics
By Jasper Akerboom, Nicole Carreras Calderón, Lin Tian, Sebastian Wabnig, Matthias Prigge, Johan Tolö, A. Gordus, M. Orger, Kristen E. Severi, J. Macklin and 16 more. Frontiers in Molecular Neuroscience, 2013
731 citations recorded by the source corpus.
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 Ca2+-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 [Ca2+]) 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 Ca2+ 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.
Subfields: Cellular and Molecular Neuroscience, Cognitive Neuroscience, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/67140edd71fcb26ab6259e41496b33d2b81fd907
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In Cellular and Molecular Neuroscience
In Molecular Biology
- Robert E. Campbell
- Michael Z. Lin
- Roger Tsien
- A. Palmer
- Sergey A. Lukyanov
- Konstantin A. Lukyanov
- Vasilis Ntziachristos
- Dmitriy M. Chudakov
Citations in the corpus
- The growing and glowing toolbox of fluorescent and photoactive proteins (2016, positive): “Parallel efforts from the GENIE project also led to a series of color variants that included blue, cyan, yellow, and red Ca2+ indicators [106].”
- Closed-loop and activity-guided optogenetic control. (2015, neutral): “…to optogenetic inhibition. from Mattis et al., 2012; Hochbaum et al., 2014; Inoue et al., 2015; Wu et al., 2013; Yizhar et al., 2011b; Klapoetke et al., 2014; Lin et al., 2013; Akerboom et al., 2012, 2013; Chen et al., 2013c; Berndt et al., 2014; Gradinaru et al., 2010; Chuong et al., 2014).”
- Physiology of Astroglia. (2018, neutral): “Several variants of GCaMP have been tested in astroglia, including GCaMP2 (722), GCaMP 3 (1609), GCaMP5 (16), and red RCaMPs (16).”