An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials.
Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.
Brain Science Institute, Saitama University, Saitama, Japan. mohkura@mail.saitama-u.ac.jp
Masamichi
M
Ohkura
Takuya
T
Sasaki
Chiaki
C
Kobayashi
Yuji
Y
Ikegaya
Junichi
J
Nakai
D000200:Action Potentials / Q000502:physiology* / Q000528:radiation effects; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D000075402:Channelrhodopsins; D004926:Escherichia coli; D005456:Fluorescent Dyes; D015870:Gene Expression; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008027:Light; D015335:Molecular Probes; D009474:Neurons / Q000502:physiology* / Q000528:radiation effects; D017966:Pyramidal Cells / Q000502:physiology* / Q000528:radiation effects; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D059010:Single-Cell Analysis; D019076:Transgenes
D000075402:Channelrhodopsins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D011994:Recombinant Proteins; D002118:Calcium