Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.
Jasper Akerboom,Nicole Carreras Calderón,Nicole Carreras Calderón,Nicole Carreras Calderón,Lin Tian,Lin Tian,Sebastian Wabnig,Matthias Prigge,Johan Tolö,Andrew Gordus,Michael B. Orger,Michael B. Orger,Kristen E. Severi,John J. Macklin,Ronak Patel,Stefan R. Pulver,Trevor J. Wardill,Trevor J. Wardill,Elisabeth Fischer,Christina Schüler,Tsai Wen Chen,Karen S. Sarkisyan,Jonathan S. Marvin,Cornelia I. Bargmann,Douglas S. Kim,Sebastian Kügler,Leon Lagnado,Peter Hegemann,Alexander Gottschalk,Eric R. Schreiter,Eric R. Schreiter,Loren L. Looger +31 more
TLDR
Red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby are described and 2-color calcium imaging is demonstrated both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes.Abstract:
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 Ca(2+)-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 [Ca(2+)]) 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 Ca(2+) 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.read more
Citations
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Journal ArticleDOI
Ultrasensitive fluorescent proteins for imaging neuronal activity.
Tsai Wen Chen,Trevor J. Wardill,Trevor J. Wardill,Yi Sun,Stefan R. Pulver,Sabine L. Renninger,Amy Baohan,Amy Baohan,Eric R. Schreiter,Rex Kerr,Michael B. Orger,Vivek Jayaraman,Loren L. Looger,Karel Svoboda,Douglas S. Kim +14 more
TL;DR: A family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo are developed and provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
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Multimodal fast optical interrogation of neural circuitry
Feng Zhang,Liping Wang,Martin Brauner,Jana F. Liewald,Kenneth Kay,Natalie Watzke,Phillip G. Wood,Ernst Bamberg,Georg Nagel,Alexander Gottschalk,Karl Deisseroth +10 more
TL;DR: In this paper, an archaeal light-driven chloride pump (NpHR) was developed for temporally precise optical inhibition of neural activity, allowing either knockout of single action potentials, or sustained blockade of spiking.
Journal ArticleDOI
Physiology of astroglia
TL;DR: Astrocytes are tightly integrated into neural networks and act within the context of neural tissue; astrocytes control homeostasis of the CNS at all levels of organization from molecular to the whole organ.
Journal ArticleDOI
Sensitive red protein calcium indicators for imaging neural activity
Hod Dana,Boaz Mohar,Boaz Mohar,Yi Sun,Sujatha Narayan,Andrew Gordus,Jeremy P. Hasseman,Getahun Tsegaye,Graham T Holt,Amy Hu,Deepika Walpita,Ronak Patel,John J. Macklin,Cornelia I. Bargmann,Misha B. Ahrens,Eric R. Schreiter,Vivek Jayaraman,Loren L. Looger,Karel Svoboda,Douglas S. Kim +19 more
TL;DR: Improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a) are presented, with sensitivity comparable to GCaMP6, to facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.
Journal ArticleDOI
Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy
Robert Prevedel,Young-Gyu Yoon,Maximilian Hoffmann,Maximilian Hoffmann,Maximilian Hoffmann,Nikita Pak,Gordon Wetzstein,Saul Kato,Tina Schrödel,Ramesh Raskar,Manuel Zimmer,Edward S. Boyden,Alipasha Vaziri,Alipasha Vaziri,Alipasha Vaziri +14 more
TL;DR: This work demonstrates simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain with high-speed volumetric calcium imaging.
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