Experiments on the 2-photon microscope were performed in the FAU Stiles-Nicholson Brain Institute Advanced Cell Imaging Core. We would like to thank the Jupiter Life Science Initiative for financial support.

All animals used for the protocol were of the species Drosophila melanogaster. There are no ethical issues surrounding the use of this species. Ethical clearance was not necessary to carry out this work. The details of the Drosophila species, reagents, and equipment used in the study are listed in the Table of Materials.
1. Breeding Drosophila and selecting the correct larval stage
<ol>
	<li>Choose a Gal4 driver line that drives expression in the cells that are to be ablated and recombine it with or cross it to a UAS-GFP reporter line. Raise flies on standard fly food at 25 &#176;C. 
	NOTE: For giant fiber (GF) ablation, R91H05-Gal4 or A307-Gal4 was recombined with UAS-GFP. ShakB(lethal)-GAL4 recombined with UAS-GFP was used for TTMn ablations (see Table of Materials).</li>
	<li>Select larvae that have started to emerge from the food and crawl up the side of the food vial. Those larvae are in the wandering stage, which is the preferred stage for performing GF cell ablation. 
	NOTE: Ablation can be performed at any larval stage. The last larval stage was important for the manipulation of the development of the GFS because&#160;the GF cell bodies can be easily identified in the brain, but the GF axons have not made connections to their targets at this stage.</li>
</ol>
2. Preparing the 3rd instar larva
NOTE: Larvae were anesthetized using a method similar to Burra et al.9. To keep procedure times short, only prepare one larva at a time. Short exposure to anesthetic will enhance the survival of experimental animals10.
<ol>
	<li>Prepare a glass dish with a tightly fitting lid. Place a cotton ball in the dish, and under a fume hood, add about 3-5 ml of ethyl ether (dangerous, flammable liquid, lab coat, gloves, goggles). Cover the dish with the lid.</li>
	<li>Collect individual 3rd instar larvae from fly vials. At the 3rd larval stage, larvae leave the food medium and crawl up along the side of the food vial11.
	<ol>
		<li>Ensure that larvae are still moving and haven&#39;t started puparium formation. Larvae that are stationary and show a shortening of the body are starting pupation and are unsuitable for ablation. Pick larvae up with a small paintbrush. 
		NOTE: The 3rd larval stage starts after the second molt at 72 h of development (after egg laying), and lasts until puparium formation at 120 h for flies raised at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Transfer one larva into a small, open container. Place the container in the glass dish with ethyl ether and close the dish tightly. When opening the glass dish, place it under a snorkel or in a fume hood to prevent fumes from escaping.
	<ol>
		<li>In intervals of 30 s, remove the lid with the larva from the dish and check the larva for mobility under a dissection microscope. The larva will generally be immobilized within 1 min. Once the mouth hooks stop twitching, the larva is ready for mounting. Discard any larvae that are not fully immobilized after 3 min. 
		NOTE: The top of an upside-down lid of a microcentrifuge tube (lid removed from the tube) can be used as a container.</li>
	</ol>
	</li>
</ol>
3. Mounting larvae on slides for ablation
<ol>
	<li>Place the anesthetized larva on a glass microscope slide. Submerge the larva in a drop of insect saline; this will wash away some of the food debris that might stick to the larva.</li>
	<li>Under the dissection microscope, remove most of the saline with a paper tissue. Position the larva dorsal side up for GF ablation or the ventral side up for TTMn ablation. Slowly lower a glass coverslip onto the larva. Add saline to the side of the coverslip to fill in the space between the glass slide and the cover slip.</li>
	<li>For GF ablation, check the positioning of the brain under high magnification on the dissection microscope. Ensure that the brain is lying level and is visible through the cuticle. Often, the brain will be covered with fat tissue, making visualization and cell ablation impossible.
	<ol>
		<li>To displace any fat tissue covering the brain, apply slight pressure to the coverslip with forceps and move the coverslip from side to side. If the fat tissue can&#39;t be moved away from the brain this way, use a different larva instead.</li>
	</ol>
	</li>
</ol>
4. Locating the target cells
NOTE: The multi-photon system used for this study was mounted on an upright microscope. System-specific software was used to control acquisition and laser stimulation settings. The system was equipped with an epifluorescence light source to locate the samples. The objective lens was a water immersion lens with 25x magnification, a long 2 mm working distance, and an NA of 1.10.
<ol>
	<li>Place the sample on the multi-photon microscope&#39;s stage and use the GFP filter to locate the sample in epifluorescence mode. For GF ablation, the cell bodies can be identified in the brain, as seen in Figure 1A,C,D. For TTMn ablation, a cluster of four cells can be identified from the ventral side in the ventral nerve cord (VNC), as shown in Figure 1A,B. When done focusing, center the cells in the field of view and switch to 2-photon mode.</li>
</ol>
5. Setting up the ablation parameters
<ol>
	<li>Adjust laser settings and detector gain to view the GFP-expressing cells with the Galvano scanner. A wavelength of 870 nm works best for visualizing the GFP signal. Center the cell in the field of view.</li>
	<li>Use a circular region of interest (ROI) to define the area for ablation. Ensure that the ROI covers most of the surface of the cell (Figure 1E,G).</li>
	<li>Set up the ablation protocol in the software. Set one frame of acquisition, then stimulation, followed by another acquisition frame.</li>
	<li>Start the stimulation laser power setting at a lower value (10%-20 %) and run the stimulation protocol. A successful ablation can be recognized by a clear circle in the center of the cell soma at the location of the ROI and an increase in fluorescence surrounding the ROI (Figure 1F).
	<ol>
		<li>If the ablation was not successful and merely bleached the cell (Figure 1H), increase the laser power by increments of 5% or the number of loops one at a time and run the protocol again. The applied laser power was between 10% and 40% for the system used in the protocol. 
		&#8203;NOTE: Laser power settings for ablation depend greatly on how deep the cells are located in the tissue and other factors such as cuticle folding. If cells are bright and clearly visible, laser power will be on the lower end of the range. Laser power will be different for each system depending on the laser model and the age of the laser.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67053/67053fig01.jpg" /> 
Figure 1: Identification of neurons for laser ablation in intact Drosophila L3 larvae. (A) Schematic of the location of giant fiber (GF) soma in the brain and cluster of motor neurons containing the tergotrochanteral motorneuron (TTMn) in the ventral nerve cord (VNC). (B) Maximum intensity projection of shakB(lethal)-Gal4 driving expression of GFP in the larval VNC. The midline is indicated by the dotted line. The circle indicates the cluster of neurons containing the TTMn that are targeted for laser ablation. Scale bar: 50 &#181;m. (C) Partial projection view of the larval brain expressing GFP under the control of A307-Gal4. The circle indicates the GF soma targeted for laser ablation. Scale bar: 50 &#181;m. (D) Maximum intensity projection of the brain with R91H05-Gal4 driving UAS-GFP. Both GFs (circles) are identifiable by location, shape, and size. Scale bar: 50 &#181;m. (E) GF soma enlarged view before laser ablation. The magenta circle indicates the target region for laser ablation. Scale bar: 10 &#181;m. (F) GF soma enlarged view after delivery of localized laser power and successful ablation. Scale bar: 10 &#181;m. (G) GF soma enlarged view before laser ablation. The magenta circle indicates the target region for laser ablation. Scale bar: 10 &#181;m. (H) GF soma enlarged view after delivery of localized laser power and unsuccessful ablation (bleaching). Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67053/67053fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Recovering the larvae
<ol>
	<li>After successfully ablating the cell, remove the coverslip and gently pick up the larvae from the slide with a paintbrush. Place the larvae in a food vial. Larvae will start crawling around again within 30 min. 
	NOTE: Keeping procedure time short improves the survival rates of animals. With experience, the experiment can be performed in just under 10 min.</li>
	<li>Continue raising larvae at standard conditions until they eclose from the pupal casing.</li>
</ol>
7. Testing the functionality of the GFS in adult flies
NOTE: The following steps are explained in detail in Allan and Godenschwege12, and Augustin et al.13.
<ol>
	<li>Test adult flies at age 2-5 days after eclosion. Anesthetize flies with CO2 and mount them on dental wax by pushing the legs into the wax. Spread the wings at a 90&#176; angle and fix them in place with the wax. Secure the head by pushing the proboscis into the wax.</li>
	<li>Place stimulation wires into both eyes, a ground wire into the abdomen, and glass recording electrodes into the jump muscles (TTMs) on each side.</li>
	<li>Stimulate the GF circuit with single stimuli to measure response latency for both muscles. Stimulate the circuit with 100 Hz and 200 Hz trains of stimuli, respectively, to measure the following frequency for both muscles.</li>
</ol>
8. Dissection and labeling of the giant fiber system for confocal imaging
NOTE: Fly CNS dissection and dye injection are detailed in Boerner and Godenschwege14.
<ol>
	<li>Gently remove the fly from the dental wax with forceps and transfer it to a silicone elastomer-lined Petri dish under a dissection scope.</li>
	<li>Remove the legs, wings, and proboscis with scissors and use insect pins to secure the fly dorsal side up in the elastomer coating.</li>
	<li>Make a lateral incision along the midline from the middle abdomen, through the entire thorax, and up the neck, connecting the thorax to the head. Open the fly with insect pins. Remove organs and glands from the fly without damaging the nervous system. 
	NOTE: At this point, GF axons can be injected with dyes to label the GFs and electrically coupled neurons13.</li>
	<li>Remove the head capsule from the head to expose the brain.</li>
</ol>
9. Immunohistochemistry of the nervous system
<ol>
	<li>After CNS dissection, fix the fly in 4% PFA in PBS for 45 min at room temperature (RT). Wash for 20 min in PBS at RT (3 times).</li>
	<li>Block the sample for 1 h at RT in 3% BSA in PBS with 0.5% detergent solution.</li>
	<li>Incubate for 2-3 nights at 4 &#176;C in 3% BSA in PBS with 0.3% detergent solution and rabbit anti-GFP (1:500). Wash for 20 min in PBS at RT (6 times).</li>
	<li>Incubate overnight at 4 &#176;C in PBS with anti-rabbit Alexa 488. Wash for 20 min in PBS at RT (6 times).</li>
	<li>Dehydrate samples with an ascending ethanol series (50%, 70%, 90%, 100% ethanol) for 10 min each step. Remove the pins and trim off the abdomen and the flight muscles. Transfer the rest of the thorax containing the nervous system onto a slide and cover it with a mounting medium like methyl salicylate. Place a coverslip on the sample and seal it with nail polish.</li>
	<li>Image the nervous system on a confocal microscope to analyze the anatomy of the GF terminal.</li>
</ol>

Single-Neuron Ablation in Drosophila for Studying Nervous System Adaptation

<ol>
	<li>Chung, S. H., Mazur, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+ablation+of+neurons+in+C.+elegans+for+behavioral+studies.">Femtosecond laser ablation of neurons in C. elegans for behavioral studies.</a> Appl Phys A Mater Sci Process. 96 (2), 335-341 (2009).</li><li>Bower, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+branching+has+conserved+needs+for+local+parasympathetic+innervation+but+not+neurotransmission.">Airway branching has conserved needs for local parasympathetic innervation but not neurotransmission.</a> BMC Biol. 12, 92 (2014).</li><li>Angelo, J. R., Tremblay, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-mediated+cell+ablation+during+post-implantation+mouse+development.">Laser-mediated cell ablation during post-implantation mouse development.</a> Dev Dyn. 242 (10), 1202-1209 (2013).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+Development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: Development and function of the Drosophila giant fibre system.</a> Semin Cell Dev Biol. 17 (1), 31-41 (2006).</li><li>Hubel, D. H., Wiesel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binocular+interaction+in+striate+cortex+of+kittens+reared+with+artificial+squint.">Binocular interaction in striate cortex of kittens reared with artificial squint.</a> J Neurophysiol. 28 (6), 1041-1059 (1965).</li><li>Wiesel, T. N., Hubel, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effects+of+unilateral+and+bilateral+eye+closure+on+cortical+unit+responses+in+kittens.">Comparison of the effects of unilateral and bilateral eye closure on cortical unit responses in kittens.</a> J Neurophysiol. 28 (6), 1029-1040 (1965).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Allen, M. J., Drummond, J. A., Moffat, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+giant+fiber+neuron+of+Drosophila+melanogaster.">Development of the giant fiber neuron of Drosophila melanogaster.</a> J Comp Neurol. 397 (4), 519-531 (1998).</li><li>Burra, S., Wang, Y., Brock, A. R., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Drosophila+larvae+to+study+epidermal+wound+closure+and+inflammation.">Using Drosophila larvae to study epidermal wound closure and inflammation.</a> Methods Mol Biol. 1037, 449-461 (2013).</li><li>Kakanj, P., Eming, S. A., Partridge, L., Leptin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+Drosophila+larvae.">Long-term in vivo imaging of Drosophila larvae.</a> Nat Protoc. 15 (3), 1158-1187 (2020).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J Embryol Exp Morphol. 66, 57-80 (1981).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFs).">Electrophysiological recordings from the Drosophila giant fiber system (GFs).</a> Cold Spring Harb Protoc. 2010 (7), 5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+giant+fiber+pathway+of+d.+Melanogaster.">Electrophysiological recordings from the giant fiber pathway of d. Melanogaster.</a> J Vis Exp. (47), e2412 (2011).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+preparation+of+the+adult+Drosophila+ventral+nerve+cord+for+giant+fiber+dye+injection.">Whole mount preparation of the adult Drosophila ventral nerve cord for giant fiber dye injection.</a> J Vis Exp. (52), e3080 (2011).</li><li>Blagburn, J. M., Alexopoulos, H., Davies, J. A., Bacon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutation+in+shaking-b+eliminates+electrical,+but+not+chemical,+synapses+in+the+Drosophila+giant+fiber+system:+A+structural+study.">Null mutation in shaking-b eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: A structural study.</a> J Comp Neurol. 404 (4), 449-458 (1999).</li><li>Kennedy, T., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+electrically+coupled+neurons+support+development+of+the+Drosophila+giant+fiber+model+circuit.">Newly identified electrically coupled neurons support development of the Drosophila giant fiber model circuit.</a> eNeuro. 5 (6), 0346 (2018).</li><li>Mcfarland, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+arrival+times+and+physical+occupancy+establish+visual+projection+neuron+integration+on+developing+dendrites+in+the+Drosophila+optic+glomeruli.">Axon arrival times and physical occupancy establish visual projection neuron integration on developing dendrites in the Drosophila optic glomeruli.</a> bioRxiv. , (2024).</li></ol>

The authors have nothing to disclose.

Cell ablation with a 2-photon microscope proved to be a highly successful method to manipulate neuronal circuit development in Drosophila. Since this method is non-invasive, it causes minimal damage to the animal. The data support the usefulness of this cell-specific manipulation of known circuits.
Crucial for the success of the ablation was selecting the most appropriate Gal4 driver. Since the GFS is well studied, many specific Gal4 driver lines have been described7...

Laser ablation is a preferred tool for dissecting neural circuits in a wide variety of organisms. Developed in model genetic systems like worms and flies, it has been applied across the animal kingdom to study the structure, function, and development of the nervous system1,2,3. Here, single-neuron ablation was employed to investigate how neurons interact during circuit assembly in Drosophila. The escape system of the fly is a favorite circuit for analysis because it contains the largest neurons and the largest synapses in the adult fly, and the circuit has been well-characterized in the past decades4. The role neuron-neuron interactions play in the assembly of the Giant Fiber circuit is a focal point of this research.
One type of interaction that has been a focal point in neuroscience since the work of Hubel and Wiesel in the 1960s is &#34;synaptic competition&#34;5,6. In this protocol, laser ablation was used to revisit the role of competition through single-cell ablation in the giant fiber system (GFS) of Drosophila, where the molecular underpinnings of the phenomena might be discovered.
Ablation of neurons in the developing fly has been difficult for a variety of reasons, including visualizing the target neurons, the precision of the ablation method, and the survival of the specimen. To overcome these problems in the GFS, the UAS/Gal4 system7 was used to label neurons of interest, and a two-photon microscope was used to remove the presynaptic giant fiber or the postsynaptic jump motor neuron (TTMn).
In this study, to determine the role that neighboring bilateral neurons play in adjusting synaptic connectivity and synaptic strength in the GFS, one of the bilateral pairs of neurons (either presynaptic GF or postsynaptic motor neuron) was deleted just before pupal development. At this developmental stage, GF axonogenesis has not been completed8. The GF structure and function of the synaptic circuit in the adult were then examined, with particular attention given to the output of the remaining GF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jaxkson ImmunoResearch</td>
			<td>111-545-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-green fluorescent protein, rabbit</td>
			<td>Fisher Scientific</td>
			<td>A11122</td>
			<td>1:500 concentration</td>
		</tr>
		<tr>
			<td>Apo LWD 25x/1.10W Objective</td>
			<td>Nikon</td>
			<td>MRD77220</td>
			<td>water immersion long working distance</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B4287-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ti:Sapphire Vision II Laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Ball</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextra, Tetramethylrhodamine, 10,000 MW, Lysine Fixable (fluoro-Ruby)</td>
			<td>Fisher Scientific</td>
			<td>D1817</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila saline</td>
			<td></td>
			<td></td>
			<td>recipe from Gu and O&#39;Dowd, 2006</td>
		</tr>
		<tr>
			<td>Ethyl Ether</td>
			<td>Fisher Scientific</td>
			<td>E134-1</td>
			<td>Danger, Flammable liquid</td>
		</tr>
		<tr>
			<td>Fly food B (Bloomington recipe)</td>
			<td>LabExpress</td>
			<td>7001-NV</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl salicylate</td>
			<td>Fisher Scientific</td>
			<td>O3695-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover-slip 18x18 #1.5</td>
			<td>Fisher Scientific</td>
			<td>12-541A</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobiotin Tracer</td>
			<td>Vector Laboratories</td>
			<td>SP-1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R multi-photon microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>on an upright FN1 microsope stand</td>
		</tr>
		<tr>
			<td>NIS Elements Advanced Research</td>
			<td>Nikon</td>
			<td></td>
			<td>Acquisition and data analysis software</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Fisher Scientific</td>
			<td>T353-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Salin)</td>
			<td>Fisher BioReagents</td>
			<td>BP2944-100</td>
			<td>Tablets</td>
		</tr>
		<tr>
			<td>R91H05-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>40594</td>
			<td></td>
		</tr>
		<tr>
			<td>shakB(lethal)-GAl4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>51633</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost microscope glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>422355000</td>
			<td>detergent solution</td>
		</tr>
		<tr>
			<td>UAS-10xGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>32185</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser cell ablation in intact drosophila larvae reveals synaptic competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

This method can be used to manipulate the development of specific neuronal networks in the nervous system of Drosophila. The primary research question here was the formation of synaptic connections. Removing either the presynaptic GF or the postsynaptic TTMn enabled the investigation of reactive synaptogenesis at this central synapse and the molecular mechanisms crucial for synaptic function and development. As described in the protocol, laser cell ablation of one of the GFs or one of the TTMns was performed, an...

Author Spotlight: Investigating the Mechanisms of Neural Circuit Assembly and Synapse Formation in Drosophila

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

laser-cell-ablation-intact-drosophila-larvae-reveals-synaptic

Research

JoVE Journal

Neuroscience

354 Views.  Florida Atlantic University. This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

Video: Author Spotlight: Investigating the Mechanisms of Neural Circuit Assembly and Synapse Formation in Drosophila 

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Methods Article

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition