title: Rational design of a high-affinity, fast, red calcium indicator R-CaMP2.
abstract: Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.
authors.affiliation: 1] Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. [2] Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Inoue
authors.affiliation: Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Atsuya
authors.initials: A
authors.lastname: Takeuchi
authors.affiliation: Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Horigane
authors.affiliation: Brain Science Institute, Saitama University, Saitama, Japan.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ohkura
authors.affiliation: Brain Science Institute, Saitama University, Saitama, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Gengyo-Ando
authors.affiliation: Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Fujii
authors.affiliation: 1] Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. [2] Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kamijo
authors.affiliation: 1] Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. [2] Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Takemoto-Kimura
authors.affiliation: Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kano
authors.affiliation: Brain Science Institute, Saitama University, Saitama, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
authors.affiliation: 1] Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. [2] Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kitamura
authors.affiliation: 1] Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. [2] Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Bito
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D017173:Caenorhabditis elegans / Q000528:radiation effects; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007202:Indicators and Reagents / Q000138:chemical synthesis*; D008027:Light; D051379:Mice; D009474:Neurons / Q000502:physiology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism
chemical_list: D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010446:Peptide Fragments; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002118:Calcium