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The Journal of Neuroscience, May 11, 2005, 25(19):4766-4778; doi:10.1523/JNEUROSCI.4900-04.2005
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Cellular/Molecular
In Vivo Performance of Genetically Encoded Indicators of Neural Activity in Flies
Dierk F. Reiff,1 * Alexandra Ihring,1 * Giovanna Guerrero,2 Ehud Y. Isacoff,2 Maximilian Joesch,1 Junichi Nakai,3 andAlexander Borst1 1Department of Systems and Computational Neuroscience, Max-Planck-Institute of Neurobiology, 82152 Martinsried, Germany, 2Molecular and Cellular Biology, University of California Berkeley, Berkeley, California 94720, and 3Laboratory for Memory and Learning, RIKEN Brain Science Institute, Saitama 351-198, Japan
Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparative in vivo analysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of the Drosophila larval neuromuscular junction. Robust neural activity did not result in any or noteworthy fluorescence changes when Flash-Pericam, Camgaroo-1, and Camgaroo-2 were expressed. However, calculated on the raw data, fractional fluorescence changes up to 18% were reported by synapto-pHluorin, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, GCaMP1.3, GCaMP1.6, and the troponin C-based calcium sensor TN-L15. The response characteristics of all of these indicators differed considerably from each other, with GCaMP1.6 reporting high rates of neural activity with the largest and fastest fluorescence changes. However, GCaMP1.6 suffered from photobleaching, whereas the fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible, with TN-L15 showing the fastest rise of the signals at lower activity rates. We show for GCaMP1.3 and YC3.3 that an expanded range of neural activity evoked fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR). The expression level of the indicator biased the signal kinetics and SNR, whereas the signal amplitude was independent. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.
Key words: genetic indicators; GFP; neural activity; transgenic animals; optical imaging; calcium
Received Dec 1, 2004; revised March 24, 2005; accepted April 4, 2005.
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