This work was supported by a grant from the DFG to G.M.T.

We have used two slightly different variants of the single neuron labeling method in our laboratories. The differences relate to the embryo collection, dechorionisation, devitellinisation and embryo filleting steps. Figure 1 gives an overview of the common and diverging steps of the variants.
1. Preparation of Micro-needles for Embryo Dissection and Micropipettes for Dye Injection
<ol>
 <li>Glass needles for embryo dissection are drawn from glass capillaries (1 mm diameter a...

<ol>
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J., Sink, H., Kolodkin, A. L., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+II+can+function+as+a+selective+inhibitor+of+specific+synaptic+arborizations.">Semaphorin II can function as a selective inhibitor of specific synaptic arborizations.</a> Cell. 81, 631-639 (1995).</li><li>Murray, M. J., Whitington, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+roundabout+on+growth+cone+dynamics,+filopodial+length,+and+growth+cone+morphology+at+the+midline+and+throughout+the+neuropile.">Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile.</a> Journal of Neuroscience. 19, 7901-7912 (1999).</li><li>Murray, M. J., Merritt, D. J., Brand, A. H., Whitington, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+axon+pathfinding+in+the+Drosophilia+CNS:+a+time-lapse+study+of+an+identified+motorneuron.">In vivo dynamics of axon pathfinding in the Drosophilia CNS: a time-lapse study of an identified motorneuron.</a> J. Neurobiol. 37, 607-621 (1998).</li><li>Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+and+other+cell+types+in+whole-mount+Drosophila+embryos+and+larvae+using+antibody+probes.">Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes.</a> Methods Cell Biol. 44, 445-487 (1994).</li><li>Rickert, C., Kunz, T., Harris, K. -. L., Whitington, P. M., Technau, G. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+grasshopper+to+Drosophila:+a+common+plan+for+neuronal+development.">From grasshopper to Drosophila: a common plan for neuronal development.</a> Nature. 310, 203-207 (1984).</li><li>Whitington, P., Harris, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+axonogenesis+in+the+embryo+of+a+primitive+insect,+the+silverfish+Ctenolepisma+longicaudata.">Early axonogenesis in the embryo of a primitive insect, the silverfish Ctenolepisma longicaudata.</a> Development Genes and Evolution. 205 (5-6), 272-281 (1996).</li><li>Whitington, P., Meier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation,+neurogenesis+and+formation+of+early+axonal+pathways+in+the+centipede,+Ethmostigmus+rubripes+(Brandt).">Segmentation, neurogenesis and formation of early axonal pathways in the centipede, Ethmostigmus rubripes (Brandt).</a> Development Genes and Evolution. 199, 349-363 (1991).</li><li>Whitington, P. M., Seifert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+growth+from+limb+motorneurons+in+the+locust+embryo:+the+effect+of+target+limb+removal+on+the+pattern+of+axon+branching+in+the+periphery.">Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery.</a> Developmental Biology. 106, 438-449 (1984).</li><li>Whitington, P. M., Leach, D., Sandeman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+change+in+neural+development+within+the+arthropods:+axonogenesis+in+the+embryos+of+two+crustaceans.">Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans.</a> Development. 118, 449-461 (1993).</li><li>Williams, D. 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The authors declare that they have no competing financial interests.

One major advantage of Drosophila as a model system is that it allows analysis of development and function on the level of single cells. This is especially helpful regarding the nervous system, where the diversity of cell types is exceptionally high and the function and morphology of neighboring cells can be totally different.
The method we present here allows the labeling of individual neurons with a dye that can either be transformed into a permanent stain or examined directl...

Knowledge of neuronal morphology at the level of individual cells is a key prerequisite for understanding neuronal connectivity and CNS function. Thus, from the earliest days of neuroscience, researchers have sought to develop single cell labeling techniques (see 7 for a historical treatment of this issue). Classical methods such as Golgi staining provide excellent resolution of neuronal morphology but are not suitable if one seeks to label a particular type of neuron in a directed way, as staining occurs in a random fashion. The development of methods for staining single cells by intracellular or juxtacellular injection of dyes from a microelectrode addressed the requirement of specific labeling.
The application of single neuron dye injection to Drosophila presented a major challenge, because of the small size of both the organism and its neurons. Nonetheless, single neuron staining of embryonic Drosophila neurons was achieved in the mid-1980s in the laboratory of Corey Goodman 15. While the method has been eminently useful and has provided some key insights into mechanisms for neuronal development in Drosophila over the last 20-30 years (e.g. 2, 10), many workers have shied away from it, largely because of its technical demands.
The availability in more recent years of genetic techniques for neuronal labeling in Drosophila has also contributed to the unpopularity of single neuron dye injection. GAL4-directed expression of membrane-targeted GFP constructs can provide excellent resolution of neural morphology 1, 20. However, this method has certain limitations: the often-unavoidable expression of GFP in multiple cells can obscure the structure of individual neurons and a GAL4 driver line may not be available to drive expression in a particular neuron of interest. The MARCM (Mosaic Analysis with a Repressible Cell Marker) method 8 can provide labeling of essentially any neuron at the individual cell level, but cannot be successfully used in the embryo and early larva because of the slow turnover of the GAL80 protein.
Given these limitations of genetic labeling, we believe that single neuron dye injection in the Drosophila embryo remains a valuable technique and deserves broader application. To promote this goal, we provide here a detailed description of the method. An illustration of its power is provided by our recent account of the morphology of the complete set of interneurons in abdominal neuromeres of the late Drosophila embryo 14. This study, which revealed both the morphological variability of individual neuronal cell types and the principles of neuromere organization in the CNS of the embryo, would not have been possible with any other currently available labeling method.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>DAB</td>
 <td>Sigma-Aldrich</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a></td>
 <td>2-3 mg/ml in 100 mM TRIS-Hcl, pH 7.4</td>
 </tr>
 <tr>
 <td>DiI</td>
 <td>Invitrogen/Molecular Probes</td>
 <td><a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a></td>
 <td>1 mg/ml in ethanol</td>
 </tr>
 <tr>
 <td><a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">Formaldehyde</a></td>
 <td>Merck Millipore</td>
 <td>&nbsp;</td>
 <td>7,4 % in PBS</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td><a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a></td>
 <td>3783</td>
 <td>70% in PBS</td>
 </tr>
 <tr>
 <td>Heptane Glue</td>
 <td>Beiersdorf AG</td>
 <td>Cello 31-39-30 *</td>
 <td>dilute ca. 1 to 1 with n-Heptane</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>TRIS</td>
 <td><a href="http://www.carlroth.com/catalogue/" target="_blank">Roth</a></td>
 <td>4855</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vectashield</td>
 <td>Vector Laboratories</td>
 <td><a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Confocal Microscope</td>
 <td>Leica</td>
 <td>TCS SP2</td>
 <td>to view and document labelings in fluorescence</td>
 </tr>
 <tr>
 <td>DC - amplifier</td>
 <td>Dragan Corporation</td>
 <td>Cornerstone ION-100</td>
 <td>to perform the labelings</td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td><a href="http://www.menzel.de/" target="_blank">Menzel</a></td>
 <td>BB018018A1</td>
 <td>18 x 18 mm</td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td><a href="http://www.menzel.de/" target="_blank">Menzel</a></td>
 <td>BB024060A1</td>
 <td>24 x 60 mm</td>
 </tr>
 <tr>
 <td>Dissecting Microscope</td>
 <td>Leica</td>
 <td>MZ8</td>
 <td>to prepare and disect embryos</td>
 </tr>
 <tr>
 <td>Flat Capillaries</td>
 <td><a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a></td>
 <td>&nbsp;</td>
 <td>outer diameter 1 mm; glas thickness 0.1 mm</td>
 </tr>
 <tr>
 <td>Injection Capillaries</td>
 <td>Science Products</td>
 <td>GB 100 TF 8P</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micromanipulator R</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>to perform the labelings</td>
 </tr>
 <tr>
 <td>Model P-97</td>
 <td>Sutter Instruments</td>
 <td>&nbsp;</td>
 <td>to pull capillaries</td>
 </tr>
 <tr>
 <td>Object Slides</td>
 <td><a href="http://www.marienfeld-superior.com/" target="_blank">Marienfeld Superior</a></td>
 <td>1000000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Scientific Microscope</td>
 <td>Zeiss</td>
 <td>Axioskop 2 mot</td>
 <td>to view labelings after photoconversion</td>
 </tr>
 <tr>
 <td>Scientific Microscope</td>
 <td>Olympus</td>
 <td>BX50</td>
 <td>to perform the labelings</td>
 </tr>
 <tr>
 <td>Sony MC3255 Video Camera</td>
 <td>Sony/AVT Horn</td>
 <td>Sony MC3255</td>
 <td>to record labelings after photoconversion</td>
 </tr>
 </tbody>
</table>
Table 1.

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 lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Figure 4 illustrates typical results of the technique, we describe here. Figure 4A shows an example of a DiI filled single interneuron that was cleanly photoconverted. It nicely demonstrates the amount of detail these preparations offer. When viewed under DIC optics the spatial context of the labeled cell within the non-labeled surrounding tissue becomes visible, e.g. the position of the cell body within the cortex and of the fiber projection within the neuropile.
<p class="...

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

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

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

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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12.6K Görüntüleme.  University of Mainz. Biz, merkezi sinir sistemi içinde tek nöron etiketlenmesine yönelik bir yöntem (CNS) sunmak Drosophila Geçen ışık veya konfokal mikroskopi ya tarafından nöronal morfoloji analizi sağlar embriyolar,.