Single Drosophila Ommatidium Dissection and Imaging

Summary

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.

Abstract

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 techniques^6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Protocol

1. Dissection of the Drosophila retina

1. Fill a one well of a 3-Well Glass Slide with phosphate buffered saline (PBS 1X), and then anesthetize flies by CO₂. Both males and females can be used for this experiment.
2. Once the flies are anesthetized, pick up a single fly using forceps and drop it into the small dish of PBS.
3. Under a dissecting scope, pinch the proboscis with forceps, and then detach the head from the body by severing the neck using a second set of forceps.
4. Holding the fly head from the proboscis, cut in half along the left/right midline the fly head with microscissors to reveal the brain.
5. Use....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Coverslips	VWR international	631-1336