Name: single drosophila ommatidium dissection and imaging
Uploaded: 2011-08-19T12:00:00
Description: The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

We thank Bernard Charroux for discussions. This work was funded by the KCL Biomedical School.

1. Dissection of the Drosophila retina
<ol>
<li>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.</li>
<li>Once the flies are anesthetized, pick up a single fly using forceps and drop it into the small dish of PBS.</li> 
<li>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.</li>
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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...

<table border="1">
	<tbody>
 	<tr>
	 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Portable CO2 Dispenser</td>
 <td>Flystuff</td>
 <td>59-150 </td>
 <td>Refills also available</td>
 </tr>
 <tr>
 	<td>DUMONT Tweezer No 5</td>
 <td>AGAR Scientific</td>
 <td>T5034</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>3-Well Glass Slide</td>
 <td>EMS</td>
 <td>71561-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Micro scissors</td>
 <td>VWR</td>
 <td>HAMMHSB516-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Schneider's Medium</td>
 <td>VWR</td>
 <td>733-1663</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>LysoTracker Red DND-99</td>
 <td>Invitrogen</td>
 <td>L7528</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Superfrost Slides</td>
 <td>VWR</td>
 <td>631-0108</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Vectashield with Dapi</td>
 <td>Vector Labs</td>
 <td>H-1200 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Coverslips</td>
 <td>VWR</td>
 <td>631-1336</td>
 <td>&nbsp;</td>
 </tr>
 
 
 </tbody>
</table>

single drosophila ommatidium dissection and imaging

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).

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.

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Research

JoVE Journal

Neuroscience

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Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...