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The Journal of Neuroscience, July 16, 2008, 28(29):7399-7411; doi:10.1523/JNEUROSCI.1038-08.2008

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Cellular/Molecular
Fluorescence Changes of Genetic Calcium Indicators and OGB-1 Correlated with Neural Activity and Calcium In Vivo and In Vitro

Thomas Hendel,¹ Marco Mank,² Bettina Schnell,¹ Oliver Griesbeck,² Alexander Borst,¹ and Dierk F. Reiff¹

¹Department of Systems and Computational Neurobiology and ²Group of Cellular Dynamics, Max Planck Institute of Neurobiology, D-82152 Martinsried, Germany

Correspondence should be addressed to Dierk F. Reiff, Department of Systems and Computational Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, D-82152 Martinsried, Germany. Email: reiff{at}neuro.mpg.de

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential for direct measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (F) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ([Ca²⁺]_i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca²⁺ dyes and in vivo calibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca²⁺]_i at rest and [Ca²⁺]_i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated F of eight different GECIs in vivo to neural activity, [Ca²⁺]_i, and F of purified GECI protein at similar [Ca²⁺] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum F compared with YC3.3 and TN-L15 in vivo. Maximum F of GCaMP2 and GCaMP1.6 were almost identical. Small [Ca²⁺]_i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of [Ca²⁺]_i was massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIs were blind for [Ca²⁺]_i associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the K_D (dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum F was reduced. The latter could be attributed to resting [Ca²⁺]_i and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.

Key words: GFP; calcium, neural activity; fluorescence; genetic probes; in vivo imaging

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Received March 10, 2008; revised June 9, 2008; accepted June 11, 2008.

Correspondence should be addressed to Dierk F. Reiff, Department of Systems and Computational Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, D-82152 Martinsried, Germany. Email: reiff{at}neuro.mpg.de

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				V. Egger and O. Stroh Calcium buffering in rodent olfactory bulb granule cells and mitral cells J. Physiol., September 15, 2009; 587(18): 4467 - 4479. [Abstract] [Full Text] [PDF]

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