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1MRC Centre for Developmental Neurobiology, King's College London
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The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.
Volpi, V., Mackay, D., Fanto, M. Single Drosophila Ommatidium Dissection and Imaging. J. Vis. Exp. (54), e2882, doi:10.3791/2882 (2011).
The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).
1. Dissection of the Drosophila retina
2. Dissection of the ommatidia
3. Treatment, staining and imaging
4. Representative Results:
The good execution of the protocol should leave a number of single ommatidia and of small groups of ommatidia kept together by fragments of lamina but nicely separated on the corneal side. These can be imaged as in this example to visualize Atg8:GFP and Lysotracker, showing autophagosomes, lysosomes and autophagolysosomes (Fig. 3). The method can be used for live imaging, however in this case it is to be noted that autofluorescence of the Schneider’s medium will make it difficult to distinguish low intensity fluorescent signals. An additional issue is that the pigment granules that remain attached to the photoreceptors are intensely fluorescent, especially in the red channel. A brightfield picture may be required to distinguish them from genuine organelles.
Figure 1. Flow chart of the dissection procedure to obtain single ommatidia or small groups of ommatidia from the fly retina.
Figure 2. Possible variations of the simple protocol here describe that involve fly genetics and ageing upstream of the dissection and staining or culturing for up to 24 hours and drug administration downstream of the dissection.
Figure 3. Confocal scan of autophagosomes and lysosomes in a single ommatidium dissected from a w; GMR-Gal4, UAS-Atro75QN; UAS-GFP::Atg8/+ fly, expressing a polyglutamine Atrophin mutant and aged 12 days at 29°C. The brightfield panel (top right) displays the almost intact structure of the ommatidium and the frame indicates the scanned area. Red marks Lysosomes, green the autophagosomes, auto-lysosomes, resulting by the fusion of the two organelles, appear yellow. The arrowheads indicate two ongoing fusion events between autophagosomes and lysosomes.
The single ommatidium dissection presented here enables collection of deeper cell biological information about processes like neurodegeneration, when modeled in the Drosophila eye. The photoreceptor neurons are more easily accessible than other neurons and they cytoplasm is particularly large, and thus suitable to study vesicle dynamics, in the very same cells used proficiently in many models to quantify neurodegeneration in vivo. The critical aspect of the dissection is mainly the ability to perform it on unfixed tissue that must never be exposed to air or dry up. The possibilities for expansion of this basic technique are such (Fig. 2) that it may as well revolutionise the information obtained from eye models of neurodegeneration, allowing all kinds of manipulations possible in a primary neuronal culture coupled to the amazing genetic modifications possible in flies.
In combination with mosaic techniques, extremely useful in neurodegeneration studies9 it may allow identification of cell biological alterations in a specific subset of mutant ommatidia, in presence of wt controls from the very same fly.
When dissected ommatidia are kept in culture, reponses to drugs like rapamycin10 or parquat11 may be better controlled and quantified than when administered to living flies. The main limitation of the system is constituted by the survival time of the dissected ommatidia in culture, which we have not been able to expand much beyond 24 hours.
No conflicts of interest declared.
We thank Bernard Charroux for discussions. This work was funded by the KCL Biomedical School.
|Portable CO2 Dispenser||Flystuff||59-150||Refills also available|
|DUMONT Tweezer No 5||Agar Scientific||T5034|
|3-Well Glass Slide||EMS||71561-01|
|Micro scissors||VWR international||HAMMHSB516-09|
|Schneider’s Medium||VWR international||733-1663|
|LysoTracker Red DND-99||Invitrogen||L7528|
|Superfrost Slides||VWR international||631-0108|
|Vectashield with Dapi||Vector Laboratories||H-1200|
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