title: In vivo performance of genetically encoded indicators of neural activity in flies.
abstract: 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.
authors.affiliation: Department of Systems and Computational Neuroscience, Max-Planck-Institute of Neurobiology, 82152 Martinsried, Germany. reiff@neuro.mpg.de
authors.forename: Dierk F
authors.initials: DF
authors.lastname: Reiff
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ihring
authors.forename: Giovanna
authors.initials: G
authors.lastname: Guerrero
authors.forename: Ehud Y
authors.initials: EY
authors.lastname: Isacoff
authors.forename: Maximilian
authors.initials: M
authors.lastname: Joesch
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
authors.forename: Alexander
authors.initials: A
authors.lastname: Borst
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004307:Dose-Response Relationship, Radiation; D004330:Drosophila; D004558:Electric Stimulation / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology / Q000528:radiation effects; D005818:Genetic Engineering / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D007814:Larva; D008164:Luminescent Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D015336:Molecular Probe Techniques; D009469:Neuromuscular Junction / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000528:radiation effects; D017729:Presynaptic Terminals / Q000378:metabolism / Q000528:radiation effects; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins