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