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The Journal of Neuroscience, October 27, 2004, 24(43):9572-9579; doi:10.1523/JNEUROSCI.2854-04.2004

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

Thomas A. Pologruto,^1,2 Ryohei Yasuda,¹ and Karel Svoboda¹

¹Howard Hughes Medical Institute/Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, and ²Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts 02138

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.

Key words: two-photon; X-Rhod-5F; Fluo4-FF; GCaMP; Camgaroo2; Inverse Pericam

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Received July 15, 2004; revised September 10, 2004; accepted September 13, 2004.

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