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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Walsh, M.K. & Lichtman, J.W. In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron 37, 67–73 (2003).
Gray, N.W., Weimer, R.M., Bureau, I. & Svoboda, K. Rapid redistribution of synaptic PSD-95 in the neocortex in vivo. PLoS Biol. 4, e370 (2006).
Rasse, T.M. et al. Glutamate receptor dynamics organizing synapse formation in vivo. Nat. Neurosci. 8, 898–905 (2005).
Sigrist, S.J., Reiff, D.F., Thiel, P.R., Steinert, J.R. & Schuster, C.M. Experience-dependent strengthening of Drosophila neuromuscular junctions. J. Neurosci. 23, 6546–6556 (2003).
Zito, K., Parnas, D., Fetter, R.D., Isacoff, E.Y. & Goodman, C.S. Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22, 719–729 (1999).
Rasse, T.M. In Vivo Imaging of Long-term Changes in the Drosophila Neuromuscular Junction Dissertation. Göttingen, Germany: Ernst-August University (2004).
Qin, G. et al. Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila. J. Neurosci. 25, 3209–3218 (2005).
Kawasaki, F., Zou, B., Xu, X. & Ordway, R.W. Active zone localization of presynaptic calcium channels encoded by the cacophony locus of Drosophila. J. Neurosci. 24, 282–285 (2004).
Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).
Wagh, D.A. et al. Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49, 833–844 (2006).
Sandstrom, D.J. Isoflurane depresses glutamate release by reducing neuronal excitability at the Drosophila neuromuscular junction. J. Physiol. 558, 489–502 (2004).
Carlin, R.K. & Siekevitz, P. Plasticity in the central nervous system: do synapses divide? Proc. Natl. Acad. Sci. USA 80, 3517–3521 (1983).
Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656–4665 (2003).
Kittel, R.J. et al. Bruchpilot promotes active zone assembly, Ca2+-channel clustering, and vesicle release. Science 312, 1051–1054 (2006).
Klar, T.A., Engel, E. & Hell, S.W. Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes. Phys. Rev. E. Stat. Nonlin. Soft Matter Phys. 64, 066613 (2001).
Kasthuri, N. & Lichtman, J.W. Structural dynamics of synapses in living animals. Curr. Opin. Neurobiol. 14, 105–111 (2004).
Zhang, Y.Q., Rodesch, C.K. & Broadie, K. Living synaptic vesicle marker: synaptotagmin-GFP. Genesis 34, 142–145 (2002).
Wucherpfennig, T., Wilsch-Brauninger, M. & Gonzalez-Gaitan, M. Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J. Cell Biol. 161, 609–624 (2003).
Pilling, A.D., Horiuchi, D., Lively, C.M. & Saxton, W.M. Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol. Biol. Cell 17, 2057–2068 (2006).
Bachmann, A. et al. Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97. J. Cell Sci. 117, 1899–1909 (2004).
Bobinnec, Y., Morin, X. & Debec, A. Shaggy/GSK-3beta kinase localizes to the centrosome and to specialized cytoskeletal structures in Drosophila. Cell Motil. Cytoskeleton 63, 313–320 (2006).
Zhang, J. et al. Thirty-one flavors of Drosophila rab proteins. Genetics 176, 1307–1322 (2007).
Grieder, N.C., de Cuevas, M. & Spradling, A.C. The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127, 4253–4264 (2000).
Besse, F. et al. The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses. J. Cell Biol. 177, 843–855 (2007).
Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).
Yeh, E., Gustafson, K. & Boulianne, G.L. Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila. Proc. Natl. Acad. Sci. USA 92, 7036–7040 (1995).
Lin, D.M. & Goodman, C.S. Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13, 507–523 (1994).
Luo, L., Liao, Y.J., Jan, L.Y. & Jan, Y.N. Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes. Dev. 8, 1787–1802 (1994).
Aberle, H. et al. wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33, 545–558 (2002).
Osterwalder, T., Yoon, K.S., White, B.H. & Keshishian, H. A conditional tissue-specific transgene expression system using inducible GAL4. Proc. Natl. Acad. Sci. USA 98, 12596–12601 (2001).
Brand, A.H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).
Davis, G.W., Schuster, C.M. & Goodman, C.S. Genetic analysis of the mechanisms controlling target selection: target-derived Fasciclin II regulates the pattern of synapse formation. Neuron 19, 561–573 (1997).
Budnik, V. et al. Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron 17, 627–640 (1996).
Halfon, M.S. et al. New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes. Genesis 34, 135–138 (2002).
Schwille, P., Kummer, S., Heikal, A.A., Moerner, W.E. & Webb, W.W. Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 97, 151–156 (2000).
Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E. & Webb, W.W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069 (1976).
Rabut, G. & Ellenberg, J. Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP. In Live Cell Imaging—A Laboratory Manual (eds. Goldman, R.D. & Spector, D.) 101–127 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2005).
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
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Figure 1
Mechanical drawing of the modified petri dish, containing a hole in the base plate (PDF 82 kb)
Supplementary Figure 2
Mechanical drawing of the plastic spacer (PDF 77 kb)
Supplementary Figure 3
Mechanical drawing of the Plexiglas guide ring (PDF 111 kb)
Supplementary Figure 4
Mechanical drawing of the anodised metal ring (PDF 82 kb)
Supplementary Figure 5
Mechanical drawing of the main component of the Plexiglas lid (PDF 78 kb)
Supplementary Figure 6
Mechanical drawing of the hose connections to be attached to the Plexiglas lid (PDF 43 kb)
Supplementary Figure 7
Assembly of Plexiglas lid (PDF 38 kb)
Rights and permissions
About this article
Cite this article
Füger, P., Behrends, L., Mertel, S. et al. Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses. Nat Protoc 2, 3285–3298 (2007). https://doi.org/10.1038/nprot.2007.472
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2007.472
This article is cited by
-
Long-term in vivo imaging of Drosophila larvae
Nature Protocols (2020)
-
Rapid active zone remodeling consolidates presynaptic potentiation
Nature Communications (2019)
-
Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel–vesicle coupling
Nature Neuroscience (2016)
-
New Tools and New Biology: Recent Miniaturized Systems for Molecular and Cellular Biology
Molecules and Cells (2013)
-
Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
Nature Protocols (2008)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.