Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals

PLoS One. 2012;7(12):e51286. doi: 10.1371/journal.pone.0051286. Epub 2012 Dec 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Signaling / genetics*
  • Calcium* / chemistry
  • Calcium* / isolation & purification
  • Calcium* / metabolism
  • Cells, Cultured
  • Dendritic Spines / metabolism
  • Dentate Gyrus / cytology
  • Dentate Gyrus / metabolism
  • Green Fluorescent Proteins* / chemistry
  • Green Fluorescent Proteins* / genetics
  • HeLa Cells
  • Humans
  • Indicators and Reagents / analysis
  • Indicators and Reagents / metabolism
  • Neuroanatomical Tract-Tracing Techniques
  • Neurons / cytology
  • Neurons / metabolism
  • Pyramidal Cells* / cytology
  • Pyramidal Cells* / metabolism
  • Rats

Substances

  • Indicators and Reagents
  • Green Fluorescent Proteins
  • Calcium

Grants and funding

This work was partly supported by the Regional Innovation Cluster Program (City Area Type, Central Saitama Area) and by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to M.O. (nos. 22500285 and 24111509), T.S. (no. 10J05408), K.G.-A. (no. 22500353) and J.N. (no. 21500379). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.