Name: Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2013-03-04T12:00:00.000+00:00
Duration: 10 min 33 s
Description: Learn to label single neurons in the Drosophila melanogaster CNS using lipophilic dye and confocal microscopy. This technique enables detailed visualization of neuronal morphology in developmental neuroscience research.

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5 minutes

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

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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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>DAB</td><td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank"><img src="/img/new_window.png"> Sigma-Aldrich</a></td><td>&lt;a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank"&gt;D-5905&lt;/a&gt;</td><td>2-3 mg/ml in 100 mM TRIS-Hcl, pH 7.4</td></tr><tr><td>DiI</td><td><a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank"><img src="/img/new_window.png"> Invitrogen/Molecular Probes</a></td><td>&lt;a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank"&gt;D-282&lt;/a&gt;</td><td>1 mg/ml in ethanol</td></tr><tr><td>&lt;a href="http://www.merckmillipore.com/germany/chemicals/formaldehydl&amp;#8211;sung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWp&amp;#8211;fVhTl" target="_blank"&gt;Formaldehyde&lt;/a&gt;</td><td>Merck Millipore</td><td></td><td>7,4 % in PBS</td></tr><tr><td>Glycerol</td><td><a href="http://www.carlroth.com/catalogue/" target="_blank"><img src="/img/new_window.png"> &lt;a href="http://www.carlroth.com/catalogue/" target="_blank"&gt;Roth&lt;/a&gt;</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></td><td></td><td>1x</td></tr><tr><td>TRIS</td><td><a href="http://www.carlroth.com/catalogue/" target="_blank"><img src="/img/new_window.png"> &lt;a href="http://www.carlroth.com/catalogue/" target="_blank"&gt;Roth&lt;/a&gt;</a></td><td>4855</td><td></td></tr><tr><td>Vectashield</td><td><a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank"><img src="/img/new_window.png"> Vector Laboratories</a></td><td>&lt;a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank"&gt;H1000&lt;/a&gt;</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</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"><img src="/img/new_window.png"> &lt;a href="http://www.menzel.de/" target="_blank"&gt;Menzel&lt;/a&gt;</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"><img src="/img/new_window.png"> &lt;a href="http://www.menzel.de/" target="_blank"&gt;Menzel&lt;/a&gt;</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"><img src="/img/new_window.png"> &lt;a href="http://www.hilgenberg-gmbh.de/" target="_blank"&gt;Hilgenberg&lt;/a&gt;</a></td><td></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></td></tr><tr><td>Micromanipulator R</td><td>Leica</td><td></td><td>to perform the labelings</td></tr><tr><td>Model P-97</td><td>Sutter Instruments</td><td></td><td>to pull capillaries</td></tr><tr><td>Object Slides</td><td><a href="http://www.marienfeld-superior.com/" target="_blank"><img src="/img/new_window.png"> &lt;a href="http://www.marienfeld-superior.com/" target="_blank"&gt;Marienfeld Superior&lt;/a&gt;</a></td><td>1000000</td><td></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><tr><td colspan="4">&lt;p class="jove_content"&gt;&lt;strong&gt;Table 1.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

labeling of single cells in the central nervous system of drosophila melanogaster

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="...

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.

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

The overall goal of this procedure is to label single neurons in the central nervous system of early stage 17 drosophila embryos. This is accomplished by collecting eggs and allowing them to develop to the right stage. The second step of the procedure is to manually chlorinate and align the eggs.Following this diva, colonization and fileting of the embryos makes the nerve cord accessible. The final step is to label single cells by Iono retic application of the lipophilic die die eye. Ultimately, results allow the visualization of single neuron morphology if desired in the context of known GFP expression patterns and or in mutant background.The main advantage of this technique over existing single cell labeling methods like markum is that it works in the embryo and allows to visualize identified cells Intentionally. Visual demonstration of this method is extremely helpful as many steps are delicate and therefore difficult to learn. Allow healthy flies to lay embryos on agar plates.Replace the plate every one and a half hours at 25 degrees Celsius and retain the embryos. Incubate the embryos on their plates at 25 or 18 degrees Celsius until they reach the required developmental stage. When ready, transfer the embryos to a slide covered with double-sided sticky tape.Avoid transferring any agar with the embryos as it will interfere with their adhesion to the tape. On the tape. Allow the embryos to dry for five to 10 minutes while waiting.Coat a 24 millimeter by 60 millimeter cover slip with heptane glue. Place a small drop of glue in the center of the slide and spread it into a very thin film using an 18 millimeter square cover slip. Once the embryos have dried, gently touch them with a needle.The corion will split open and the embryo will stick to the needle. Immediately transfer the dechlorinated embryos to an agar block to prevent them from drying. Align them there 10 in a row, ventral sides up.Cut away the excess agar from the block with a scalpel. Then gently touch the embryos with the heptane glue coated cover slip. They should adhere to the cover slip with their ventral sides down.Apply a frame of adhesive tape around the cover slip and attach it to a microscope slide. Keeping the embryos on top of the cover slip. Cover the embryos with generous amounts of PBS on the dorsal side of each embryo near its posterior end.Penetrate the vitel membrane with a glass needle tear open the membrane along the dorsal midline and drag the embryo out of the membrane. Now reorient the embryo ventral side down elsewhere on the heptane glue substrate. File the embryo with a glass needle by gently perforating the body wall and tearing along the dorsal midline.Using the needle, push down on the body wall so that it sticks to the slide. Tear through the gut at its anterior and posterior ends, and lift the gut away. Since several embryos will be file on the same slide, it is helpful to either be very accurate with their orientation or make a drawing of their orientation.Next, the embryos may be lightly fixed for 10 to 15 minutes in 7.4%formaldehyde in PBS followed by four washes in PBS use extreme care to avoid touching the embryos to the surface of the solution. The surface tension forces will destroy them under a 10 x objective. Center the field of view on a prepared embryo, increase the magnification and locate the cell of interest under DIC optics or under fluorescence.If the target cell expresses GFP, ensure that the field diaphragm of the microscope is opened just to the edge of the field of view, not beyond a narrow illumination. Beam will aid positioning of the micro pipette change back to the 10 x objective and place the bath electrode for the DC amplifier into the solution far from the embryo and the path of the micro pipette. Mount the injection micro pipet filled with lithium chloride solution to the holder and attach it to the micro manipulator.Using the course controls position the micropipet below the objective and well above the embryo. If the angle of the micro peppe is too steep, the tip will not get in focus. If it is too shallow, the shaft will foul against the bath wall center the tip in the field of view.Now lower the micro pipette and focus alternately until the pipette is in the PBS and not too far above the embryo. Now change to high power objective and center the pipette within the field of view. When the shaft is in focus, move the micro pipette in the X axis until its tip lies in the center of the field of view.The micro pipette tip should be well above the level of the embryo. Switch to fluorescence illumination and examine the tip. Check for leakage of dye eye.Adjust the DC current so that dye eye crystal does not build up at the tip. Now lower the micro pipet in the Z axis towards the embryo. Progressively adjusting the focus as the embryo comes into focus.Switch to using the fine control. Focus the cell to be injected at the center of the field and then refocus on the micro pipette tip. From this point onwards, it might be more convenient to use the stage control of the microscope to move the embryo relative to the micro pipette.Bring the micro pipet tip into contact with the cell and make a depression on its surface. Pass several nano amps of depolarizing current for a few seconds. A small crystal of dye eye will form on the cell to confirm that the cell has been labeled.Briefly open the shutter to illuminate the field with fluorescent light. If the body of the cell shows signs of labeling, apply current for several more seconds. Then turn off the current and quickly pull the embryo away from the micro pipette using the stage controls.If no D eye labeling was evident, the micro pipette may have been blocked and needs replacement. If the tip of the pipette snags other tissues on root to the cell and stains that tissue, try withdrawing the micro pipet slightly and reapproach the cell before resorting to replacing the micro pipette as needed. Continue labeling other cells in the same embryo or in other embryos on the same slide.Move the tip to the edge of the field of view and repeat the procedure. Remove most of the solution in the injection chamber. Then fix the embryos by adding 7.4%formaldehyde PBS for 10 minutes, followed by four washes with PBS.The preparation can now be photo converted or mounted for confocal microscopy using the outlined protocol. A dai filled inter neuron was flawlessly photo converted under DIC optics. The spatial context of the labeled cell within the non labeled surrounding tissue was well resolved.For example, the positions of the cell body within the cortex and of the fiber projection within the neuro pill could both be seen in this preparation. The dye drop was a little too big. Several neighboring cells became labeled simultaneously, which makes it difficult to relate individual projections to distinct cell bodies.Also notice that under the fluorescent microscope, the background resolution is much lower without photo conversion. Multiple cells in neighboring segments can also be injected in this preparation, an antibody against fast two stains of fales in the neuro pill. Despite the clear cell bodies, the preparation shows a possible side effect of prolonged photo conversion periods.The more intensely labeled cells tend to get overs stained and start to swell as compared to labeling a single cell with confocal optics. Dye eye labeling in embryos that carry GFP reporter constructs can provide spatial context and identity of the D filled cell within specific populations of GFP positive neuronal or glial cell types. After watching this video, you should have a good understanding of how to prepare and perform labelings of single neurons in the embryonic CNS after oph.But while attempting this procedure, please keep in mind that every single step is demanding, so expect a couple of repetitions until the whole procedure runs smoothly.

labeling-single-cells-central-nervous-system-drosophila

Research

JoVE Journal

Neuroscience

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection of Larval CNS in Drosophila Melanogaster

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

This video demonstrates the immunofluorescence staining of a transgenic Drosophila brain for single-cell imaging of glial cells. The brain expresses membrane-targeted proteins fused with different combinations of antigenic epitopes under the control of a glial-specific recombinase system. The isolated and fixed brains are stained with primary antibodies specific to the antigenic epitopes, followed by fluorophore-conjugated secondary antibodies. This labeling enables the simultaneous imaging of...

Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)This video demonstrates the method of retrograde labeling of Drosophila motor neurons using lipophilic dyes. The dye enters the axon, travels retrogradely, and labels the neurons green, allowing detailed visualization of their morphology and projections under a confocal microscope.

Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live Imaging of Drosophila Larval Neuroblasts

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

This article describes an efficient imaginal disc dissection and antibody staining protocol that is economical with reagents and facilitates handling of imaginal discs in small numbers.

Small Scale Dissection and Antibody Staining of Eye-Antennal and Wing Imaginal Discs from Drosophila melanogaster

Learn to label single neurons in the Drosophila melanogaster CNS using lipophilic dye and confocal microscopy. This technique enables detailed visualization of neuronal morphology in developmental neuroscience research.

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

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