Name: Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes (Video) | JoVE Journal
Uploaded: 2020-01-12T16:00:00.000+00:00
Duration: 8 min 25 s
Description: Learn retrograde tracing of Drosophila embryonic motor neurons using lipophilic fluorescent dyes. This method enables detailed visualization of neuronal morphogenesis and connectivity in the motor neuron system.

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5 minutes

We thank members of the Kamiyama Lab for comments on the manuscript. This work was supported by an NIH R01 NS107558 (to M.I., K.B., and D.K.).

1. Equipment and Supplies
<ol>
	<li>Materials for collecting embryos and training adults to lay eggs
	<ol>
		<li>Prepare the filtration apparatus by severing a 50 mL tube and cutting open a hole in the cap to set a mesh filter with pores of 100 &#181;m (Table of Materials) in between the tube and the cap. 
		NOTE: Alternatively, cell strainers with pores of 100 &#181;m (Table of Materials) can be used for the filtration step of embryo collection.</li>
		<li>Make agar plates with grape agar premix (Table of Materials) according to the listed instructions. Briefly, gently stir 1 packet of the powder mix into 500 mL of room temperature (RT, 23 &#176;C) dH2O and microwave the dissolved mixture to vigorous boil. After cooling down to 70&#8722;75 &#176;C, pour the mixture into Petri dishes (60 mm). After the agar is solidified, store plates at 4 &#176;C.</li>
		<li>Prepare yeast paste by mixing active dry yeast (Table of Materials) and water to a paste consistency, and keep at 4 &#176;C.</li>
		<li>Use egg-collection cages (for 60 mm Petri dish, Table of Materials) that provide sufficient air flow.</li>
	</ol>
	</li>
	<li>Preparation of dissection needles and dye injection micropipettes
	<ol>
		<li>Prepare dye injection micropipette and dissection needle from the same capillary tubing with an inner diameter of 0.6 mm and an outer diameter of 1.2 mm (Table of Materials). Pull the capillary tubing by a micropipette puller at 7% from 170 V maximum output (Table of Materials) to create a sharp needle with a taper of ~0.4 cm in length.</li>
		<li>For dye injection, adjust the micropipette with a micropipette beveler (Table of Materials) by a bubble beveling technique described in instrument&#39;s manual.
		<ol>
			<li>In short, soak the grinder with a wetting agent (Table of Materials) to prevent the water from &#39;dragging&#39; the needle tip. Place the needle on the micropipette clamp at 25&#8722;30&#176; and lower the tip onto two-thirds of the radius out from the center of the beveling surface. Grind the needle while a syringe with tubing pushes air into the needle, to ensure that the micropipette will be clear of glass shavings.</li>
			<li>Mark the micropipette with a fine-tip permanent marker to indicate the position of the opening at the tip after beveling as it is challenging to locate the narrow opening of the micropipette that is formed at an angle.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation for Embryo Collection
<ol>
	<li>Ensure that the adult flies (20&#8722;40 wild-type Canton-S or white flies), males and females, are maintained in young (&#60;7 days) and healthy conditions for the ideal egg collection. 
	NOTE: To stimulate egg-laying, flies are trained in their egg collection cage a couple of days prior to egg collection on agar plates streaked with yeast paste at least once every day.</li>
</ol>
3. Embryo Staging
<ol>
	<li>Allow the flies to lay eggs overnight (or at least 15 h) at RT to collect the embryos at 15 h AEL, i.e., stage 1618, to view dendritogenesis of the aCC and RP3 motor neurons. In the morning, collect the plate with the eggs. 
	NOTE: The embryos at 15 h AEL will have a distinct 4-chamber gut18. For imaging different stages follow their specific morphological criteria and aging conditions.</li>
	<li>To collect the embryos, dechorionate the eggs laid on the plate with 50% bleach for 5 min.</li>
	<li>Once the chorions have cleared, pour the contents of the plate through the filtration apparatus or cell strainer to isolate the embryos. Using a squeeze bottle of water, dilute the bleach left on the plate and gather as many embryos as possible by decanting the mixture into the filter.</li>
	<li>Wash the embryos on the filter 3&#8722;4x with more water or until the bleach odor dissipates. Remove the filter from the apparatus and wash the embryos onto another clean plate with water. Decant the water from the new plate that the embryos are on.</li>
	<li>Prepare a glass slide by covering it with two layers of vinyl tape in the center, forming a rectangle. Cut a rectangular pool out of the tape using a razor blade. Place a thin strip of double-sided tape towards the upper end of the pool, this is where the embryos will be placed as shown in Figure 1.</li>
	<li>Using fine forceps, individually select 5&#8722;10 embryos at 15 h AEL and place them on the double-sided tape with the dorsal side facing up. Add insect Ringer&#39;s saline19 to the dissection pool to protect the embryos from desiccation (Figure 1).</li>
</ol>
4. Dissection and Staining
<ol>
	<li>Using a glass needle under a dissecting microscope (Table of Materials), cut through the midline of a single embryo at its surface from its posterior to its anterior end. Then drag the embryo out from the vitelline membrane from the tape onto the glass (boxed in Figure 1). Take care not to damage the interior tissues of the embryo.</li>
	<li>Flip the epithelial tissues from the center and attach the epidermal edge onto the surface of the glass slide (Figure 1, inset).</li>
	<li>Using a tube-connected needle with a tip opening of ~300 &#181;m (prepared by breaking the thin tip of a dissection needle), aspirate or blow air to detach and remove the dorsal longitudinal tracheal trunks as well as any remaining guts.</li>
	<li>Use 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) to fix the embryos for 5 min at RT. Wash the embryos 3x with PBS.</li>
	<li>Stain the embryos with 1 &#181;L of anti-horseradish peroxidase antibody conjugated with cyanine 3 dye (anti-HRP Cy3) (Table of Materials) in 200 &#181;L of PBS for 1 h. Wash the embryos with PBS 3x after staining. 
	NOTE: The dye of anti-HRP can be changed based on the lipophilic dyes of choice for injection.</li>
</ol>
5. Filling of the Injection Micro-pipette
<ol>
	<li>Heat lipophilic dyes (5 mg/mL of DiO or DiD, Table of Materials) to 60 &#176;C in a 1:10 mixture of ethanol:vegetable oil before use.</li>
	<li>Prepare an oil-dissolved dye slide for the injection micropipette. Place the micropipette into the capillary holder (Figure 2, #1). Using the micromanipulator (Table of Materials), adjust the micropipette to be over the dye slide. Then, adjust the stage to place the micropipette onto the dye (Figure 2, #2).</li>
	<li>To fill up the micropipette, use a microinjector (Table of Materials) (Figure 2, #3). Collect the dye in the micropipette by setting the Pi (injection pressure) between 200&#8722;500 hPa (hectopascal), the Ti (injection time) between 0.1&#8722;0.5 s and Pc (compensation pressure) to 0 hPa for 5 min (Figure 2, #4).</li>
	<li>Once the dye has been collected, remove the dye slide and place the sample onto the microscope stage. Next, increase the Pc to a range of 20&#8722;60 hPa before lowering the micropipette into the sample to prevent contamination of PBS by capillary action.</li>
</ol>
6. Dye Injection into Neurons
<ol>
	<li>Locate the embryo in the center using the microscope with 10x objective lens (Table of Materials) and align the micropipette with the embryo. 
	NOTE: The size of the dye droplet can be adjusted by changing the Pi or the size of the opening of the micropipette tip. The droplet should be 10&#8722;20 &#181;m, which is approximately the width of 1 muscle.</li>
	<li>Change the objective lens to a water-immersion 40x lens (Table of Materials) and submerge the lens into PBS to see the embryo.
	<ol>
		<li>Use fluorescence microscopy to check the neuronal morphology marked by anti-HRP Cy3 and determine the injection site.</li>
		<li>During injection, use brightfield microscopy to see the dye droplet. When the embryo is in focus, change the position of the micropipette to make gentle contact with the tip of the axon of interest (e.g., aCC, RP3).</li>
		<li>Drop the dye in a right abdominal (A2&#8722;A6) hemi-segment at the neuromuscular junction of aCC or RP3 (Figure 3) with either DiD or DiO, by using the neurons marked by anti-HRP Cy3. Using the hand control (mouse; Figure 2, 5) release the dye and remove the micropipette after dropping the dye with the micromanipulator and move onto the next injection site. 
		NOTE: Unlike other dyes (e.g., Lucifer yellow, calcein) which spread into neighboring cells through gap junctions, lipophilic dyes associate with cell membranes and do not transfer to neighbors. Due to the relatively large size of the dye droplet, however, this technique also results in labeling of the partnering muscles (Figure 3A).</li>
	</ol>
	</li>
	<li>Incubate the sample at RT for 1 h after dye-drop before imaging. 
	NOTE: The protocol can be paused here before mounting, and the sample can be kept at 4 &#176;C overnight. Lipophilic dyes can also be delivered using iontophoresis, if an intracellular direct-coupled (DC) amplifier is readily available20.</li>
</ol>
7. Imaging with a Confocal Microscope
<ol>
	<li>Remove the double-sided tape and vinyl tape from the glass slide with the help of forceps.</li>
	<li>Prepare a cover slip (22 x 22 mm2 No.1 cover glass) with a small amount of vacuum grease (Table of Materials) at the four corners and carefully place on the sample, avoiding air bubbles. Remove any excess PBS using task wipers.</li>
	<li>Push down the cover slip to adjust the working distance between the objective lens and the sample. Completely seal the edges of the cover slip with nail polish.</li>
	<li>Image at 10x and 100x magnification using a confocal microscope.</li>
	<li>Use ImageJ software for processing raw images from the microscope (Table of Materials). 
	NOTE: Observation must begin within 10 min after mounting for the best images. Otherwise, at RT, the dye will spread to sites adjacent to the injection site creating unwanted background for imaging. To slow down the diffusion of dye, the sample can be stored at 4 &#176;C for a couple of hours.</li>
</ol>

<ol><li>Arzan Zarin, A., Labrador, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+axon+guidance+in+Drosophila%5BTitle%5D)+AND+%22Seminars+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">Motor axon guidance in Drosophila.</a> Seminars in Cell and Developmental Biology. 85, 36-47 (2019).</li>
<li>Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+neuromuscular+specificity+in+Drosophila%3A+novel+mechanisms+revealed+by+new+technologies%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Neuroscience%22%5BJournal%5D)">Generation of neuromuscular specificity in Drosophila: novel mechanisms revealed by new technologies.</a> Frontiers in Molecular Neuroscience. 5, 62(2012).</li>
<li>Kim, M. D., Wen, Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterning+and+organization+of+motor+neuron+dendrites+in+the+Drosophila+larva%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Patterning and organization of motor neuron dendrites in the Drosophila larva.</a> Developmental Biology. 336 (2), 213-221 (2009).</li>
<li>Manning, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+resource+for+manipulating+gene+expression+and+analyzing+cis-regulatory+modules+in+the+Drosophila+CNS%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">A resource for manipulating gene expression and analyzing cis-regulatory modules in the Drosophila CNS.</a> Cell Reports. 2 (4), 1002-1013 (2012).</li>
<li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> Journal of Visualized Experiments. (27), e1347(2009).</li>
<li>Chen, K., Featherstone, D. E., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+recording+in+the+Drosophila+embryo%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Electrophysiological recording in the Drosophila embryo.</a> Journal of Visualized Experiments. (27), e1348(2009).</li>
<li>Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+Patterning+in+the+Drosophila+CNS%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">Temporal Patterning in the Drosophila CNS.</a> Annual Review of Cell and Developmental Biology. 33, 219-240 (2017).</li>
<li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+neuroblasts%3A+a+model+for+stem+cell+biology%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Drosophila neuroblasts: a model for stem cell biology.</a> Development. 139 (23), 4297-4310 (2012).</li>
<li>Urbach, R., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroblast+formation+and+patterning+during+early+brain+development+in+Drosophila%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Neuroblast formation and patterning during early brain development in Drosophila.</a> Bioessays. 26 (7), 739-751 (2004).</li>
<li>Carrero-Martínez, F. A., Chiba, A. Cell Adhesion Molecules at the Drosophila Neuromuscular Junction. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCarrero-Mart%C3%ADnez%2C+FA+%22The+Sticky+Synapse%3A+Cell+Adhesion+Molecules+and+Their+Role+in+Synapse+Formation+and+Maintenance%22">The Sticky Synapse: Cell Adhesion Molecules and Their Role in Synapse Formation and Maintenance</a>. Umemori, H., Hortsch, M. , Springer. New York. 11-37 (2009).</li>
<li>Ritzenthaler, S., Suzuki, E., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postsynaptic+filopodia+in+muscle+cells+interact+with+innervating+motoneuron+axons%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Postsynaptic filopodia in muscle cells interact with innervating motoneuron axons.</a> Nature Neuroscience. 3 (10), 1012-1017 (2000).</li>
<li>Kohsaka, H., Takasu, E., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+induction+of+postsynaptic+molecular+assembly+by+the+cell+adhesion+molecule+Fasciclin2%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">In vivo induction of postsynaptic molecular assembly by the cell adhesion molecule Fasciclin2.</a> Journal of Cell Biology. 179 (6), 1289-1300 (2007).</li>
<li>Kohsaka, H., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+recognition+at+the+tips+of+postsynaptic+filopodia%3A+accumulation+and+function+of+Capricious%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Target recognition at the tips of postsynaptic filopodia: accumulation and function of Capricious.</a> Development. 136 (7), 1127-1135 (2009).</li>
<li>Landgraf, M., Bossing, T., Technau, G. M., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origin%2C+location%2C+and+projections+of+the+embryonic+abdominal+motorneurons+of+Drosophila%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">The origin, location, and projections of the embryonic abdominal motorneurons of Drosophila.</a> Journal of Neuroscience. 17 (24), 9642-9655 (1997).</li>
<li>Kamiyama, D., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endogenous+activation+patterns+of+Cdc42+GTPase+within+Drosophila+embryos%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Endogenous activation patterns of Cdc42 GTPase within Drosophila embryos.</a> Science. 324 (5932), 1338-1340 (2009).</li>
<li>Furrer, M. P., Vasenkova, I., Kamiyama, D., Rosado, Y., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slit+and+Robo+control+the+development+of+dendrites+in+Drosophila+CNS%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Slit and Robo control the development of dendrites in Drosophila CNS.</a> Development. 134 (21), 3795-3804 (2007).</li>
<li>Kamiyama, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specification+of+Dendritogenesis+Site+in+Drosophila+aCC+Motoneuron+by+Membrane+Enrichment+of+Pak1+through+Dscam1%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1.</a> Developmental Cell. 35 (1), 93-106 (2015).</li>
<li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCampos-Ortega%2C+JA+%22The+embryonic+development+of+Drosophila+melanogaster%22">The embryonic development of Drosophila melanogaster</a>. , Springer-Verlag. Berlin, Germany. (1985).</li>
<li>Drosophila Ringer's solution. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Cold+Spring+Harbor+Protocols%22">Cold Spring Harbor Protocols</a>. 2007 (4), (2007).</li>
<li>Rickert, C., Kunz, T., Harris, K. -L., Whitington, P., Technau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Labeling+of+single+cells+in+the+central+nervous+system+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Labeling of single cells in the central nervous system of Drosophila melanogaster.</a> Journal of Visualized Experiments. (73), e50150(2013).</li>
<li>Fujioka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Even-skipped%2C+acting+as+a+repressor%2C+regulates+axonal+projections+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Even-skipped, acting as a repressor, regulates axonal projections in Drosophila.</a> Development. 130 (22), 5385-5400 (2003).</li>
<li>Sink, H., Rehm, E. J., Richstone, L., Bulls, Y. M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((sidestep+encodes+a+target-derived+attractant+essential+for+motor+axon+guidance+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila.</a> Cell. 105 (1), 57-67 (2001).</li>
<li>Furrer, M. P., Kim, S., Wolf, B., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robo+and+Frazzled%2FDCC+mediate+dendritic+guidance+at+the+CNS+midline%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Robo and Frazzled/DCC mediate dendritic guidance at the CNS midline.</a> Nature Neuroscience. 6 (3), 223-230 (2003).</li>
<li>Landgraf, M., Jeffrey, V., Fujioka, M., Jaynes, J. B., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+origins+of+a+motor+system%3A+motor+dendrites+form+a+myotopic+map+in+Drosophila%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila.</a> PLoS Biology. 1 (2), 41(2003).</li>
</ol>

The authors have nothing to disclose.

The use of dye labeling for studying neuronal morphology has several advantages over genetic cell-labeling techniques. The dye labeling technique can minimize the amount of time needed for labeling and imaging the morphologies of motor neurons. The dye labeling process is quite fast as it takes less than 2 h and enables us to define the outline of neuronal projections. As an alternative, one can visualize the aCC motor neuron by choosing a GAL4 line that expresses the yeast GAL4 transcription factor in aCC, and crossing ...

The embryonic motor neuron system of Drosophila offers a powerful experimental model to analyze the mechanisms underlying the development of the central nervous system (CNS)1,2,3. The motor neuron system is amenable to biochemical, genetic, imaging, and electrophysiological techniques. Using the techniques, genetic manipulations and functional analyses can be carried out at the level of single motor neurons2,4,5,6.
During early development of the nervous system, neuroblasts divide and generate a large number of glia and neurons. The spatiotemporal relationship between the delamination and the gene expression profile of neuroblasts has been previously investigated in detail7,8,9. In the case of the motor neuron system, the formation of embryonic neuromuscular junction (NMJ) has been extensively studied using the aCC (anterior corner cell), RP2 (raw prawn 2), and RP5 motor neurons2,10. For instance, when the RP5 motor neuron forms a nascent synaptic junction, the pre-synaptic and post-synaptic filopodia are intermingled11,12,13. Such direct cellular communication is vital to initiate the NMJ formation. Contrary to what we know about the peripheral nerve branches, our knowledge of how motor dendrites initiate synaptic connectivity within the CNS is still primitive.
In this report, we present a technique that allows retrograde labeling of motor neurons in embryos by means of micropipette-mediated delivery of lipophilic dyes. This technique enables us to trace the 38 motor neurons innervating each of the 30 body wall muscles in a hemi-segment at 15 h after egg laying (AEL)14. By using this technique, our group has thoroughly investigated numerous gain-of-function/loss-of-function alleles15,16,17. We have recently unraveled the molecular mechanisms that drive initiation of motor dendrite connectivity and demonstrated that a Dscam1-Dock-Pak interaction defines the site of dendrite outgrowth in the aCC motor neuron17. In general, this technique is adaptable for the phenotypic analysis of any embryonic motor neurons in wild type or mutant strains, enhancing our ability to provide new insights into the functional design of the Drosophila nervous system.

<table><tbody><tr><td>10x objective lens</td><td>Nikon</td><td></td><td>Plan</td></tr><tr><td>40x water-immersion lens</td><td>Nikon</td><td></td><td>NIR Apo</td></tr><tr><td>Capillary tubing</td><td>Frederick Haer&amp;amp;Co</td><td>27-31-1</td><td></td></tr><tr><td>Confocal microscope</td><td>Andor</td><td>N/A</td><td>Dragonfly Spinning disk confocal unit</td></tr><tr><td>Cover glass</td><td>Corning</td><td></td><td>22x22 mm Square #1</td></tr><tr><td>DiD</td><td>ThermoFisher</td><td>V22886</td><td></td></tr><tr><td>DiI</td><td>ThermoFisher</td><td>V22888</td><td></td></tr><tr><td>DiO</td><td>ThermoFisher</td><td>V22887</td><td></td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>N/A</td><td>SMZ-U</td></tr><tr><td>Double Sided Tape</td><td>Scotch</td><td>665</td><td></td></tr><tr><td>Dow Corning High-Vacuum Grease</td><td>Fisher Sci.</td><td>14-635-5D</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Egg collection cage</td><td>FlyStuff</td><td>59-100</td><td></td></tr><tr><td>FemtoJet 5247</td><td>Eppendorf</td><td>discontinued</td><td>FemtoJet 4i (Cat No. 5252000021)</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Image processing software</td></tr><tr><td>Micromanipulator</td><td>Sutter</td><td>MP-225</td><td></td></tr><tr><td>Micropipette beveler</td><td>Sutter</td><td>BV-10-B</td><td></td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-100</td><td></td></tr><tr><td>Nutri-Fly Grape Agar Powder Premix Packets</td><td>FlyStuff</td><td>47-102</td><td></td></tr><tr><td>Nylon Net Filter</td><td>Millipore</td><td></td><td></td></tr><tr><td>Paraformaldehyde 16% Solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Any EM grades</td></tr><tr><td>PBS</td><td>Roche</td><td>11666789001</td><td>Sold on sigmaaldrich, boxed 10x solution</td></tr><tr><td>Photo-Flo 200</td><td>Kodak</td><td>146 4510</td><td>Wetting agent</td></tr><tr><td>Upright fluorescence microscope</td><td>Nikon</td><td>N/A</td><td>Eclipse Ci with a LED light source</td></tr><tr><td>Vinyl Electrical Tape</td><td>Scotch</td><td>6143</td><td></td></tr><tr><td>VWR Cell Strainers</td><td>VWR</td><td>10199-659</td><td></td></tr><tr><td>Yeast</td><td>FlyStuff</td><td>62-103</td><td>Active dry yeast (RED STAR)</td></tr></tbody></table>

retrograde tracing of drosophila embryonic motor neurons using lipophilic fluorescent dyes

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

A representative image of the aCC and RP3 motor neurons is shown in Figure 3C to demonstrate the multicolor labeling of motor neurons at 15 h AEL. Their dendritic morphologies are largely invariant between embryos. The staining pattern obtained with anti-HRP antibody is shown in gray. A small droplet of DiO or DiD was deposited on the NMJ of muscle 1 or 6/7, respectively. Figure 4 demonstrates the capability to quantitatively measure the phenoty...

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

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JoVE Journal

Developmental Biology

We described structural features of the Glia-neuromuscular synapses in a novel  Inside-out  tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Electrophysiological Recording in the Drosophila Embryo

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This video demonstrates a protocol to study synaptic vesicle recycling at neuromuscular junctions in channelrhodopsin-expressing Drosophila larvae using a fluorescent dye. Blue light activates the channelrhodopsins, causing calcium influx and synaptic vesicle exocytosis. The dye integrates into the neuronal membranes and is internalized during endocytosis, resulting in enhanced fluorescence. Further blue light stimulation induces exocytosis and dye release, which is shown by reduced...

Analyzing Synaptic Vesicle Recycling and Fluorescent Dye Uptake in Drosophila Larvae

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

Source: Guan, W., et. al. Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle. J. Vis. Exp. (2018) This video demonstrates imaging of Drosophila legs with green fluorescent protein-expressing motor neurons using confocal microscopy. A laser and two detectors capture GFP and cuticle autofluorescence. Images are processed by splitting channels, subtracting autofluorescence, and merging into a colored composite image to visualize axons within the leg segment.

Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

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

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Learn retrograde tracing of Drosophila embryonic motor neurons using lipophilic fluorescent dyes. This method enables detailed visualization of neuronal morphogenesis and connectivity in the motor neuron system.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

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