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

Abstract

Genetically encoded Ca 2+indicators (GECIs) based on fluorescent proteins (XFPs) and Ca 2+-binding proteins [like calmodulin (CaM)] have great potential for the study of subcellular Ca 2+signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca 2+concentration ([Ca 2+]) is complicated by the nonlinear interactions between Ca 2+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 [Ca 2+] indicators with similar affinities for Ca 2+. 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 [Ca 2+] revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca 2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively.