Name: Autor destacado: Investigación de los mecanismos de ensamblaje de circuitos neuronales y formación de sinapsis en Drosophila 
Uploaded: 2024-07-26T14:45:00
Description: The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

Los experimentos con el microscopio de 2 fotones se realizaron en el Núcleo de Imágenes Celulares Avanzadas del Instituto del Cerebro Stiles-Nicholson de la FAU. Nos gustaría agradecer a la Iniciativa de Ciencias de la Vida de Júpiter por su apoyo financiero.

Todos los animales utilizados para el protocolo fueron de la especie Drosophila melanogaster. No hay problemas éticos en torno al uso de esta especie. No era necesaria la autorización ética para llevar a cabo este trabajo. Los detalles de las especies de Drosophila , los reactivos y el equipo utilizado en el estudio se enumeran en la Tabla de Materiales.
1. Cría de Drosophila y selección del estadio larvario correcto
<ol><li>E...

Ablación de una sola neurona en Drosophila para estudiar la adaptación del sistema nervioso

<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>

La ablación celular con un microscopio de 2 fotones demostró ser un método muy exitoso para manipular el desarrollo de circuitos neuronales en Drosophila. Dado que este método no es invasivo, causa un daño mínimo al animal. Los datos respaldan la utilidad de esta manipulación específica de la célula de circuitos conocidos.
Crucial para el éxito de la ablación fue la selección del controlador Gal4 más adecuado. Dado que el GFS está bien estudiado, se han descrito muchas l...

La ablación láser es una herramienta preferida para diseccionar circuitos neuronales en una amplia variedad de organismos. Desarrollado en sistemas genéticos modelo como gusanos y moscas, se ha aplicado en todo el reino animal para estudiar la estructura, la función y el desarrollo del sistema nervioso 1,2,3. Aquí, la ablación de una sola neurona se empleó para investigar cómo interactúan las neuronas durante el ensamblaje del circuito en Drosophila. El sistema de escape de la mosca es uno de los circuitos favoritos para el análisis porque contiene las neuronas más grandes y las sinapsis más grandes de la mosca adulta, y el circuito ha sido bien caracterizado en lasúltimas décadas. El papel que juegan las interacciones neurona-neurona en el ensamblaje del circuito de fibra gigante es un punto focal de esta investigación.
Un tipo de interacción que ha sido un punto focal en la neurociencia desde el trabajo de Hubel y Wiesel en la década de 1960 es la "competencia sináptica"5,6. En este protocolo, la ablación con láser se utilizó para revisar el papel de la competencia a través de la ablación de una sola célula en el sistema de fibras gigantes (GFS) de Drosophila, donde se podrían descubrir los fundamentos moleculares de los fenómenos.
La ablación de las neuronas en la mosca en desarrollo ha sido difícil por una variedad de razones, incluida la visualización de las neuronas objetivo, la precisión del método de ablación y la supervivencia del espécimen. Para superar estos problemas en el GFS, se utilizó el sistema UAS/Gal47 para etiquetar las neuronas de interés, y se utilizó un microscopio de dos fotones para eliminar la fibra gigante presináptica o la neurona motora de salto postsináptica (TTMn).
En este estudio, para determinar el papel que juegan las neuronas bilaterales vecinas en el ajuste de la conectividad sináptica y la fuerza sináptica en el GFS, se eliminó uno de los pares bilaterales de neuronas (ya sea GF presináptico o neurona motora postsináptica) justo antes del desarrollo de la pupa. En esta etapa del desarrollo, la axonogénesis de GF no se ha completado8. A continuación, se examinó la estructura y la función del circuito sináptico del FG en el adulto, prestando especial atención a la salida del FG restante.

<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

El protocolo describe la ablación de una sola neurona con un sistema láser de 2 fotones en el sistema nervioso central (SNC) de larvas intactas de Drosophila melanogaster. Con este método no invasivo, el sistema nervioso en desarrollo puede manipularse de una manera específica para cada célula. La interrupción del desarrollo de las neuronas individuales en una red se puede utilizar para estudiar cómo el sistema nervioso puede compensar la pérdida de información sináptica. Las neuronas individuales fueron ablacionadas específicamente en el sistema de fibras gigantes de Drosophila, con un enfoque en dos neuronas: la fibra gigante presináptica (GF) y la neurona motora tergotrochanteral postsináptica (TTMn). El GF hace sinapsis con el TTMn ipsilateral, que es crucial para la respuesta de escape. La ablación de uno de los GF en el tercer estadio del cerebro, justo después de que el GF comienza el crecimiento axonal, elimina permanentemente la célula durante el desarrollo del SNC. El GF restante reacciona al vecino ausente y forma un terminal sináptico ectópico con el TTMn contralateral. Este terminal atípico, bilateralmente simétrico, inerva ambos TTMns, como se demuestra mediante el acoplamiento de colorantes, e impulsa ambas neuronas motoras, como se demuestra mediante ensayos electrofisiológicos. En resumen, la ablación de una sola interneurona demuestra la competencia sináptica entre un par bilateral de neuronas que puede compensar la pérdida de una neurona y restaurar las respuestas normales al circuito de escape.

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.

Autor destacado: Investigación de los mecanismos de ensamblaje de circuitos neuronales y formación de sinapsis en Drosophila

La ablación celular con láser en larvas intactas de Drosophila revela una competencia sináptica

We are trying to understand the mechanisms underlying neural circuit assembly during development, and we use simple nervous systems like drosophila to study this question. One of these mechanisms is the role of synaptic competition in circuit assembly, and the present paper is focused on this issue. One exciting new technology in neuroscience is called optogenetics.It uses light sensitive ion channels to turn neurons on or off using light pulses. These new methods allow us to control neural circuitry in new ways and link the circuits to behavior. Ablation of a single neuron in the living animal has allowed us to characterize the role of competition in the assembly of simple neural circuits.Now we can screen for the molecular machinery that underlies this widespread phenomenon. We demonstrate a competitive interaction between the two giant neurons for synaptic contact with their target motor neurons. This result has paralleled throughout the animal kingdom, especially in the vertebrate visual system where it was first discovered.This paves the way for investigating the molecules that regulate this competition in a genetically tractable organism like drosophila. We will use this new method of optogenetics to enhance our understanding of the neural circuit underlying the escape behavior. We will also use our understanding of the neural circuit to better understand how these new optogenetic tools work.

Este método se puede utilizar para manipular el desarrollo de redes neuronales específicas en el sistema nervioso de Drosophila. La principal pregunta de investigación aquí fue la formación de conexiones sinápticas. La eliminación de la GF presináptica o la TTMn postsináptica permitió la investigación de la sinaptogénesis reactiva en esta sinapsis central y los mecanismos moleculares cruciales para la función y el desarrollo sináptico. Como se describe en el protocolo, se realizó una ablación cel...

Este protocolo demuestra la ablación celular con láser de neuronas individuales en larvas intactas de Drosophila . El método permite estudiar el efecto de la reducción de la competencia entre neuronas en el sistema nervioso en desarrollo.

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

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 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.

Investigación

Neurociencias

Laser Ablation of Neurons in Intact Drosophila Third Instar Larva for Neuronal Manipulation

To begin, take a glass dish with a tightly-fitting lid. Place a cotton ball in the dish. Under a fume hood, add three to five milliliters of ethyl ether to the cotton ball.Immediately cover the dish with the lid. Use a paintbrush to pick up the third instar larvae of drosophila from fly vials. Transfer one larva into a small, open container.Place the container in the glass dish with ethyl ether, and immediately close the dish tightly. Use a dissection microscope to check the larva for mobility every 30 seconds. Transfer the anesthetized larva onto a glass microscope slide.Submerge the larvae in a drop of insect saline. Under the dissection microscope. Remove most of the saline with a paper tissue.Position the larva dorsal side up for giant fiber ablation, or the ventral side up for TTMn ablation. Then slowly lower a glass cover slip onto the larva. Add saline to the side of the cover slip.To fill-in the space between the glass slide and the cover slip. For giant fiber ablation, check the positioning of the brain under high-magnification on the dissection microscope. Ensure the brain is lying level and is visible through the cuticle.To displace any fat tissue covering the brain, use forceps to apply slight pressure to the cover slip and move the cover slip from side to side. Place the sample on a multi-photon microscope stage. Use the GFP filter to locate the sample in epifluorescence mode.For giant fiber ablation, focus on the cells of interest, then switch to two photon mode. Set the laser to 870 nanometers and adjust the detector gain to view the GFP expressing cells with the Galvano scanner, define the area for ablation using a circular region of interest. Next, proceed to set up the ablation protocol in the software, to do so, sequentially set one frame of acquisition, stimulation, followed by another frame of acquisition.Start the stimulation laser power at a lower value and run the stimulation protocol. If the ablation was not successful and only bleach the cell, increase the laser power by increments of 5%or the number of loops one at a time, and run the protocol again. Do this until successful cell ablation is seen.After successfully ablating the cell, remove the cover slip. With a paintbrush, gently pick up the larvae from the slide, then transfer the larvae into a food vial. In giant fiber ablation samples.The ablation was verified through the absence of a GFP-labeled soma on the ablated side of the brain. For the TTMn ablated larvae. The absence of TTMn on one side was easily recognized due to missing soma and dendrites.