title: Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals.
abstract: Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.
authors.affiliation: Brain Science Institute, Saitama University, Saitama, Japan. mohkura@mail.saitama-u.ac.jp
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ohkura
authors.forename: Takuya
authors.initials: T
authors.lastname: Sasaki
authors.forename: Junko
authors.initials: J
authors.lastname: Sadakari
authors.forename: Keiko
authors.initials: K
authors.lastname: Gengyo-Ando
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kagawa-Nagamura
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kobayashi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ikegaya
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics*; D002478:Cells, Cultured; D049229:Dendritic Spines / Q000378:metabolism; D018891:Dentate Gyrus / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis / Q000378:metabolism; D056347:Neuroanatomical Tract-Tracing Techniques; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism; D051381:Rats
chemical_list: D007202:Indicators and Reagents; D049452:Green Fluorescent Proteins; D002118:Calcium