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Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses

Nature Protocols volume 2, pages 32853298 (2007) | Download Citation

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Abstract

Here we describe how to anesthetize and image Drosophila larvae as to follow 'the life history' of identified synapses and synaptic components. This protocol is sensitive, for example, the distribution of glutamate receptors expressed at physiological levels can be monitored. Typically, 2–20 time points can be recorded in the intact organism. Finally, we discuss how to extract the kinetic information on protein dynamics from two-color fluorescence recovery after photo-bleaching (FRAP) measurements and give advice how to keep the in vivo imager's five arch enemies—limited temporal and spatial resolution, injury of the animal, inactivation of proteins and movement artifacts—in check. While we focus on synapses, as model structure, the protocol can easily be adapted to study other developmental processes such as muscle growth, gut development or tracheal branching.

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Acknowledgements

We thank Yvonne Eisele and Wernher Fouquet for comments on the manuscript. We thank Andreas Schönle and David Sandstrom for technical advice. We thank Hubert Willmann for the mechanical drawings, and Frank Kötting for constructing the imaging chamber and the anesthetization device. We thank Michael Knopp, as well as all other laboratory and mechanics workshop members, for help and discussion. This work was supported by grants from the University of Tübingen (fortüne 1691-0-0 and fortüne 1626-0-0) and from the Landesstiftung Baden-Württemberg to T.M.R.

Author information

Affiliations

  1. Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller Strasse 27, Tübingen D-72076, Germany.

    • Petra Füger
    • , Laila B Behrends
    •  & Tobias M Rasse
  2. Institut für Klinische Neurobiologie und Rudolf-Virchow-Zentrum Universität Würzburg Zinklesweg 10, Würzburg D-97078, Germany.

    • Sara Mertel
    •  & Stephan J Sigrist

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Corresponding author

Correspondence to Tobias M Rasse.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Mechanical drawing of the modified petri dish, containing a hole in the base plate

  2. 2.

    Supplementary Figure 2

    Mechanical drawing of the plastic spacer

  3. 3.

    Supplementary Figure 3

    Mechanical drawing of the Plexiglas guide ring

  4. 4.

    Supplementary Figure 4

    Mechanical drawing of the anodised metal ring

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    Supplementary Figure 5

    Mechanical drawing of the main component of the Plexiglas lid

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    Supplementary Figure 6

    Mechanical drawing of the hose connections to be attached to the Plexiglas lid

  7. 7.

    Supplementary Figure 7

    Assembly of Plexiglas lid

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DOI

https://doi.org/10.1038/nprot.2007.472

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