Monitoring Neural Activity and Ca2+ with Genetically Encoded Ca2+Indicators

Genetically encoded Ca2+ indicators (GECIs) based on fluorescent proteins (XFPs) and Ca2+-binding proteins like calmodulin (CaM) have great potential for the study of subcellular Ca2+ signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca2+ concentration (Ca2+) is complicated by the nonlinear interactions between Ca2+ binding and GECI fluorescence. We have characterized GECIs in pyramidal neurons in cultured hippocampal brain slices, focusing on indicators based on circularly permuted XFPs GCaMP (Nakai et al., 2001), Camgaroo2 (Griesbeck et al., 2001), and Inverse Pericam (Nagai et al., 2001). Measurements of fluorescence changes evoked by trains of action potentials revealed that GECIs have little sensitivity at low action potential frequencies compared with synthetic Ca2+ indicators with similar affinities for Ca2+. The sensitivity of GECIs improved for high-frequency trains of action potentials, indicating that GECIs are supralinear indicators of neural activity. Simultaneous measurement of GECI fluorescence and Ca2+ revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively.