title: Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute/Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Pologruto
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yasuda
authors.forename: Karel
authors.initials: K
authors.lastname: Svoboda
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D008956:Models, Chemical; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D017966:Pyramidal Cells / Q000378:metabolism / Q000502:physiology; D051381:Rats; D046509:Tissue Culture Techniques; D014162:Transfection
chemical_list: D002147:Calmodulin; D008164:Luminescent Proteins; D002118:Calcium