1Junior Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen, 2Graduate School of Cellular and Molecular Neuroscience, University of Tübingen
Zhang, Y., Füger, P., Hannan, S. B., Kern, J. V., Lasky, B., Rasse, T. M. In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution. J. Vis. Exp. (43), e2249, doi:10.3791/2249 (2010).
Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.
A) Assembly of the imaging chamber
B) Anesthetization of the larva
D) Recovery from anesthetization
E) Time series
Figure 1 Assembly of the imaging chamber. (A) Place the larva and the plastic spacer onto the oil layer. (B) Place a 22 x 22 mm cover slip on the spacer and insert the plexiglas guide ring into the chamber, next (C) fix the position of the larva with the metal ring and (D) close the chamber. (E) Now the chamber is ready to be mounted on the microscope.
Figure 2 Body-wall muscles in Drosophila larvae. Muscles and NMJs were visualized by expression of a CD8-GFP-Sh fusion protein8. The muscles are shown as observed when focusing at the ventral side through the cuticle into the larva. In (A-C) the most superficial muscle, 27, is shown, in (K-L) the most interior muscles, 6 and 7, are shown. Scale Bar: 100 μm, ΔZ between slices is 2 μm.
Figure 3 Identity of body-wall muscles in Drosophila larvae. The identity of muscles in L3 Drosophila larvae, segment A3, is shown. Muscles and NMJs were visualized by expression of a CD8-GFP-Sh fusion protein8. The muscles are displayed as observed when focusing at the ventral side through the cuticle into the larvae. In A-C the most superficial muscle, 27, is shown, in K-L the most interior muscles, 6 and 7, are shown. Scale Bar: 100 μm, ΔZ between slices is 2 μm.
Figure 4 Identity of neuromuscular junctions of Drosophila larvae. (A-D) NMJs were visualized by expression of a DGluRIIA-mRFP fusion protein1. The NMJs are shown as observed when focusing at the ventral side through the cuticle into the larva. In (A-B) the most superficial NMJ, 27, is shown, in C-D the most interior NMJs, 6 and 7, are shown. Scale Bar: 100 μm, ΔZ between slices is 5 μm. (E) and (F) the identity of the superficial (E) and more interior (F) NMJs is given for reference.
The presented method was initially developed to study glutamatergic synapses on the body wall muscles of Drosophila melanogaster larvae. The Drosophila neuromuscular junction (NMJ) is characterized by a stereotypical cyto-architecture of muscles and neurons and is thus ideally suited for in vivo imaging. However, the described anesthetization protocol is not limited to imaging the NMJ; the transparency of Drosophila larvae facilitates the adaptation of the described protocol to study the development of organs, migration of cells, transport of axonal cargo and sub-cellular reorganization within cells.
No conflicts of interest declared.
We thank Andreas Schönle, Max-Plank-Institute for Biophysical Chemistry, Germany and David J. Sandstrom, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA for technical advice. We thank Frank Kötting, European Neuroscience Institute, Göttingen for constructing the imaging chamber and the anesthetization device. This work was supported by grants from the Alzheimer Forschung Initiative to T.M.R. Y.Z. was supported by a fellowship of the China Scholarship Council, S.B.H. by a fellowship of the Graduate School for Cellular and Molecular Neuroscience, University of Tübingen.