Name: a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system
Uploaded: 2018-05-05T14:30:00
Description: Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

We thank Jessica Goldshteyn for technical support. Work in the Song lab is funded by the NIH grant R00NS088211, and the Intellectual and Developmental Disabilities Research Center (IDDRC) New Program Development Award.

1. Preparation of Culture Plates and Bottles
<ol>
	<li>Preparation of grape juice agar plates
	<ol>
		<li>Add 10 g of agar powder, 200 mL grape juice, and 192 mL ddH2O into a beaker and microwave for about 4-5 min, stirring intermittently until the agar is completely dissolved.</li>
		<li>In a fume hood, cool down the solution to approx. 60 &#176;C. Add 4.2 mL 95% ethanol and 4.0 mL glacial acetic acid. Adjust the total volume of the solution to 400 mL with ddH2O. Mix well.</li>
		<li>For each 35-...

<ol>
	<li>Yakura, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recovery following spinal cord injury. , (1996).</li><li>Harel, N. Y., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+regenerating+axons+recapitulate+developmental+guidance+during+recovery+from+spinal+cord+injury?.">Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury?.</a> Nature reviews. Neuroscience. 7, 603-616 (2006).</li><li>Jurewicz, A., Matysiak, M., Raine, C. 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P., Cafferty, W. B., Budel, S. O., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+regulators+of+axonal+growth+in+the+adult+central+nervous+system.">Extracellular regulators of axonal growth in the adult central nervous system.</a> Philos Trans R Soc Lond B Biol Sci. 361, 1593-1610 (2006).</li><li>Liu, K., Tedeschi, A., Park, K. K., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+intrinsic+mechanisms+of+axon+regeneration.">Neuronal intrinsic mechanisms of axon regeneration.</a> Annu Rev Neurosci. 34, 131-152 (2011).</li><li>Schwab, M. E., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nogo+limits+neural+plasticity+and+recovery+from+injury.">Nogo limits neural plasticity and recovery from injury.</a> Curr Opin Neurobiol. 27, 53-60 (2014).</li><li>He, Z., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+Control+of+Axon+Regeneration.">Intrinsic Control of Axon Regeneration.</a> Neuron. 90, 437-451 (2016).</li><li>Park, K. K., 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=Promoting+axon+regeneration+in+the+adult+CNS+by+modulation+of+the+PTEN/mTOR+pathway.">Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.</a> Science. 322, 963-966 (2008).</li><li>Geoffroy, C. G., Hilton, B. 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M., 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=The+Drosophila+cell+corpse+engulfment+receptor+Draper+mediates+glial+clearance+of+severed+axons.">The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons.</a> Neuron. 50, 869-881 (2006).</li><li>Song, Y., 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=Regeneration+of+Drosophila+sensory+neuron+axons+and+dendrites+is+regulated+by+the+Akt+pathway+involving+Pten+and+microRNA+bantam.">Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam.</a> Genes Dev. 26, 1612-1625 (2012).</li><li>Song, Y., 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=Regulation+of+axon+regeneration+by+the+RNA+repair+and+splicing+pathway.">Regulation of axon regeneration by the RNA repair and splicing pathway.</a> Nat Neurosci. 18, 817-825 (2015).</li><li>Kato, K., Forero, M. G., Fenton, J. C., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+regenerative+response+to+central+nervous+system+injury+is+enabled+by+pros-notch+and+pros-NFkappaB+feedback.">The glial regenerative response to central nervous system injury is enabled by pros-notch and pros-NFkappaB feedback.</a> PLoS Biol. 9, e1001133 (2011).</li><li>Fang, Y., Soares, L., Teng, X., Geary, M., Bonini, N. 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J., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+axon+regeneration+in+Drosophila.">Models of axon regeneration in Drosophila.</a> Exp Neurol. 287, 310-317 (2017).</li><li>Hao, Y., Collins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+mechanisms+for+axon+regeneration:+insights+from+injured+axons+in+Drosophila.">Intrinsic mechanisms for axon regeneration: insights from injured axons in Drosophila.</a> Curr Opin Genet Dev. 44, 84-91 (2017).</li><li>Galbraith, J. A., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+damage+in+thick+specimens+by+multiphoton+excitation.">Controlled damage in thick specimens by multiphoton excitation.</a> Mol Biol Cell. 14, 1808-1817 (2003).</li><li>Sugimura, K., 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=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Yanik, M. F., 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=Neurosurgery:+functional+regeneration+after+laser+axotomy.">Neurosurgery: functional regeneration after laser axotomy.</a> Nature. 432, 822 (2004).</li><li>Wu, Z., 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=Caenorhabditis+elegans+neuronal+regeneration+is+influenced+by+life+stage,+ephrin+signaling,+and+synaptic+branching.">Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching.</a> Proc Natl Acad Sci U S A. 104, 15132-15137 (2007).</li><li>Stone, M. C., Nguyen, M. M., Tao, J., Allender, D. L., Rolls, M. 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B., 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=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Buss, A., 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=NG2+and+phosphacan+are+present+in+the+astroglial+scar+after+human+traumatic+spinal+cord+injury.">NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury.</a> BMC Neurol. 9, 32 (2009).</li><li>McKeon, R. J., Jurynec, M. J., Buck, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chondroitin+sulfate+proteoglycans+neurocan+and+phosphacan+are+expressed+by+reactive+astrocytes+in+the+chronic+CNS+glial+scar.">The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.</a> J Neurosci. 19, 10778-10788 (1999).</li><li>Raabe, I., Vogel, S. K., Peychl, J., Tolic-Norrelykke, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+nanosurgery+and+cell+enucleation+using+a+picosecond+laser.">Intracellular nanosurgery and cell enucleation using a picosecond laser.</a> J Microsc. 234, 1-8 (2009).</li><li>Hutson, M. S., Ma, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+cavitation+dynamics+during+pulsed+laser+microsurgery+in+vivo.">Plasma and cavitation dynamics during pulsed laser microsurgery in vivo.</a> Phys Rev Lett. 99, 158104 (2007).</li><li>Venugopalan, V., Guerra, A., Nahen, K., Vogel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laser-induced+plasma+formation+in+pulsed+cellular+microsurgery+and+micromanipulation.">Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation.</a> Phys Rev Lett. 88, 078103 (2002).</li><li>Bourgeois, F., Ben-Yakar, A. <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+nanoaxotomy+properties+and+their+effect+on+axonal+recovery+in+C.+elegans.">Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans.</a> Opt Express. 16, 5963 (2008).</li><li>O'Brien, G. 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When setting up fly crosses, the number of females and males used can vary depending on the genotypes and the number of larvae needed for specific experiments. For WT flies, typical cross uses 10 females and 5 males. The collection window may be narrowed, depending on the accuracy of the larvae age required. For example, a 2-h collection period will yield larvae of a more homogenous population. In this case, using 20 or more virgin females to set up the crosses will help yield sufficient eggs. The yield from the first da...

The inability of axons to regenerate after an injury to the central nervous system (CNS), may lead to permanent disabilities in patients, and also plays a role in the irreversible neurological deficits in neurodegenerative diseases1,2,3,4,5. The CNS environment, as well as the intrinsic growth ability of neurons, determines whether axons are able to regenerate after trauma. Extracellular factors from oligodendrocyte, astroglial, and fibroblastic sources have been shown to impede neuronal growth4,6,7,8, but the elimination of these molecules only allows for limited sprouting5. Intrinsic regeneration signals can influence regenerative success5,9 and represent potential therapeutic targets, but these processes are still not well-defined at the molecular level. Increases in trophic factor signaling or elimination of endogenous brakes, such as the Pten phosphatase10, can result in axonal regeneration in certain circumstances. Combinations of different methods found to be individually effective also only provide limited overall recovery to date11,12,13,14. Therefore, there is a desperate need to identify additional pathways for targeted therapy. In addition to the initiation of axon regrowth, whether and how axons re-wire to the correct target, reform synapse specificity, and achieve functional recovery are important unanswered questions.
In summary, current understanding of the machinery dictating axon regeneration is still very fragmentary. Part of the problem is the technical difficulty of studying axon regeneration in mammals in real-time, an approach that is costly, time-consuming, and challenging for conducting large-scale genetic screens. Drosophila melanogaster, on the other hand, has proven to be an exceptionally powerful system for the study of complex biological questions. The fruit fly has been instrumental in defining genes and signaling pathways that are strikingly conserved in humans and has been a successful model for the study of human conditions, such as neurodegenerative diseases, through the vast molecular genetics tools available to manipulate gene function15. In particular, fruit flies are considered to be an ideal tool for the discovery of genes involved in neural injury and regrowth15,16. Several fly neural injury models have been developed, including adult-head or larval ventral nerve cord (VNC) stabbing with needles, larval VNC or nerve crush with forceps, larval neuron laser axotomy, olfactory receptor neuron removal, brain explants injury, and peripheral nerve lesion by wing severance15,17,18,19,20,21,22,23. Excitingly, recent work utilizing Drosophila injury models have&#160;advanced our understanding of the cellular and genetic pathways used by the nervous system to respond to neural injuries, some of which have been shown to be conserved in mammals24,25. Again, this emphasizes the utility of this model organism for identifying novel mechanisms of neural repair.
Described here is a two-photon laser-based Drosophila larval sensory neuron injury model. A two-photon laser was first used to cut axons in zebrafish in vivo in 200326. In the same year, the first laser dendriotomy was performed in Drosophila using a pulsed nitrogen laser27. Shortly afterward, several C. elegans labs used femtosecond lasers to establish models of axon regeneration28. In 2007, Wu and colleagues compared and reported the differences between laser injuries in C. elegans induced by various types of lasers29. In 2010, axon regeneration after laser axotomy was first shown to occur in Drosophila30. Building on this extensive laser injury literature, we have developed a fly neural injury model using the two-photon laser, which allows precise induction of injury to targeted sites with minimal perturbation of neighboring tissues, providing a relatively clean system to study both the intrinsic and extrinsic properties of neuroregeneration with single-cell resolution. Specifically, we have established a set of injury methods for dendritic arborization (da) sensory neurons in both the peripheral nervous system (PNS) and CNS. Da neurons can be grouped into four distinct classes distinguished primarily by their dendrite branching complexities: class I to IV31. Our published work shows that da neuron regeneration resembles mammalian injury models at the phenotypic and molecular level: da neurons display class specific regeneration properties, with class IV but not class I or III da neurons exhibiting regeneration in the PNS; class IV da neuron axons regenerate robustly in the periphery, but their regenerative potential is dramatically reduced in the CNS, thus resembling dorsal root ganglion (DRG) neurons in mammals; enhancing mTOR activity via Pten deletion or Akt overexpression enhances axon regeneration in the fly CNS19. Utilizing this injury model, we have been performing genetic screens and have identified the RNA processing enzyme Rtca as an evolutionarily conserved inhibitory factor for axon regeneration, linking axon injury to cellular stress and RNA modification20.
In the presented paradigm, the injury is induced via laser axotomy/dendriotomy of larval class IV or III da neurons, labeled by ppk-CD4-tdGFP or 19-12-Gal4, UAS-CD4-tdGFP, repo-Gal80, respectively. The injury is performed on 2nd to 3rd instar larvae at around 48 - 72 h after egg laying (h AEL). For PNS axotomy the lesion is targeted to the section of axon ~20 - 50 &#181;m away from the cell body, for CNS axotomy to an area of ~20 &#181;m in diameter at the commissure junction in the VNC, and for dendriotomy to the primary dendritic branch points. The same neuron is imaged at 8 - 24 h after injury (AI) to confirm complete transection, and at 48 - 72 h AI to assess regeneration. Through time-lapse confocal imaging, the degeneration and regeneration of individual axons/dendrites that have been injured in vivo can be monitored over time.

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			<td height="63" style="height:63px;width:444px;">Diethyl ether, ACS reagent, anhydrous</td>
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			<td style="width:153px;">AC615080010</td>
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			<td height="84" style="height:84px;width:444px;">Halocarbon 27 Oil</td>
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			<td height="21" style="height:21px;">Phosphate buffered saline (PBS), 20x Concentrate, pH 7.5, supplier # E703-1L</td>
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			<td>97062-948&#160;</td>
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			<td height="21" style="height:21px;">Agar powder, Alfa Aesar, 500GM</td>
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			<td style="width:155px;">Zeiss</td>
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			<td height="21" style="height:21px;width:444px;">Zen software</td>
			<td style="width:155px;">Zeiss</td>
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			<td height="42" style="height:42px;width:444px;">Chameleon Ultra II</td>
			<td style="width:155px;">Coherent</td>
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a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

Da neurons show differential regeneration potential between the peripheral and central nervous system, as well as class specificity. This provides unique opportunities to screen for novel factors that are required for axon regeneration (using class IV PNS injury), as well as those that are inhibitory for regeneration (with class IV CNS injury and class III PNS injury).
Axon regeneration in the PNS
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Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

The overall goal of this Drosophila larval sensory neuron injury model is to combine in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox into a platform for screening potential promoters and inhibitors of neuroregeneration. This method will help address key questions in a field of neurogeneration, such as by identifying new intrinsic and extrinsic regulator for neurogeneration, both in a peripheral and central nerve system. The main advantage of this technique is that novel candidates for neuroregeneration can be screened in an easy, fast and cheap way.Though this system can provide insight into neuroregeneration, it can also be applied to other systems, such as neurodegenerative diseases and neuron-glia interactions. Generally, individuals new to this method will struggle because different experimental setups that utilize different types of neurons are used to model axon versus dendritic regeneration. For the larvae collection, prepare culture bottles as follows.Use a blade to punch a 1.5-centimeter hole in one wall of a Drosophila culture bottle, and fill the hole with a cotton ball for ventilation. Then put a dab of yeast paste on a grape juice agar plate, and use the plate to plug the main opening of the bottle. In such a bottle, set up a cross of 10 virgin females and five males, and change the plate daily while culturing at 25 degrees Celsius.Culture the collected plates with a wet tissue soaked in mild propionic acid to prevent contamination. From the cultured plates, harvest larvae at the required stage using forceps. Gently transfer the picked larvae to a new grape juice agar plate without yeast paste.After they have cleaned themselves off by crawling around, they can be imaged. To begin each imaging session, turn on the imaging lasers and the microscope. For the injury, use a two-photon microscope.In the imaging software, set the laser to view GFP at 930 nanometers with a maximum power of 1950 milliwatts. Select Line Scan Mode, and open the pinhole all the way. Then increase the laser intensity to about 20%of full power to make a PNS injury, or to 50%to 100%of full power to make a VNC injury.Next, select 512 square pixel frame for scanning, and use the highest possible scanning speed. Use an average number of one, and a bit depth of eight bits. Then set the gain to about 750, and set the offset to zero.Now save this preset experimental protocol as 2P GFP 930 Ablation, allowing for easy reuse in further experiments. For post-injury imaging, use a confocal microscope. First set up the argon laser at 488 nanometers.Select the Acquisition tab and then select Z Stack. Under Laser, turn on the 488 nanometer argon laser. Next go to Channels, select the 488 nanometer laser, and increase the laser power to five to 10%For the pinhole, use the option for one to two area units.Then adjust the gain to 650. Now in Acquisition Mode, select the 1024 square pixels as the frame scan. Use the maximum scan speed.Use an average number of two and a bit depth of eight bits. Save this pre-experimental protocol as GFP Imaging. Begin with anesthetizing the larvae.In a fume hood, place a 60-millimeter glass dish into a 15-centimeter plastic petri dish. Then fold a piece of tissue paper and place it in the glass dish. Place the grape agar plate on the tissue, after the diethyl ether is added.Next, onto a glass slide, place one drop of Halocarbon 27 oil at the center, and place a spot of vacuum grease on each of the four corners. Then use forceps to transfer one larva onto the agar plate, and cover the glass dish to anesthetize the larva. As soon as the larva stops moving, carefully transfer it to the Halocarbon oil, with its head upright.Then place a cover slip over the slide and press it down gently until it touches the larva. Next, use gentle force to slide the cover slip to roll the cells to be ablated to where the two-photon laser will most easily hit them. The location will vary, depending on what neurons are being targeted.Now secure the assembly on the two-photon microscope stage, and focus on the cells of interest, using a 40X oil immersion objective. In the software, switch to the scanning mode and load the saved protocol. Make sure the pinhole is opened all the way.Then in Live Mode, get a good image of the region of interest. Next, stop the live scan so that the Crop button will become available. Using the Crop function, adjust the scan window to focus the target area on just the prospective site of injury.Then open a new imaging window. Now reduce the scan speed and increase the laser intensity. Then toggle the Continuous button to start and stop the scan.Watch carefully. As soon as there is a drastic increase in florescence, end the scan. Next switch back to the original imaging window and select the Live Mode, and find the region that was just targeted by adjusting the focus.A good indication of successful injury is the appearance of a small crater, ring-like structure, or localized debris right on the injury site. If the laser power was too high, a large damaged area will be visible, which can be lethal. Now carefully remove the cover slip, and transfer the injured larva onto a new plate with yeast paste.Put the plate into a 60-millimeter dish, along with a propionic acid soaked tissue. Then return the plate to culturing temperature. For subsequent imaging of the larva, make use of the saved confocal setup, and collect Z stack images with a 25X objective.Be sure to include the normalization point, so the regeneration can be quantified. Using the described protocol, regeneration of class three and four DA neurons was investigated. Typically, three or four neurons on the right side of abdominal segments A7 to A2 were injured.Specifically, class three DDAF and class four V Prime ADA neurons were targeted. Larvae were imaged at 24, 48, and 72 hours post-injury. At 24 hours, the distal axons usually completed degeneration, and the axon stem was readily visible.At 48 hours, it was possible to assess regeneration in the class four DA neurons. About 70%of these neurons regenerated beyond the injury site. Even after 72 hours, it was clear that class three DA neurons, however, failed to regrow.This was assessed based on repeated observations of stalled growth cones. After watching this video, you should have a good understanding of how to set up the experiment, perform two-photon injury, and assess the results. Once mastered, this technique takes 15 minutes per larvae, if it's performed properly.While attempting this procedure, it's important to remember to compare the experimental growth with the corresponding controls, side by side. Following this procedure, a method like immunostaining can be performed in order to answer more questions, like whether axon injury can cause protein translocation, resulting in a pathway alteration. Since its development, this technique paved the way for the researchers in the field of neuroscience to explore neuroregeneration in Drosophila larval sensory neurons.Finally, don't forget that working with diethyl ether can be extremely hazardous, and precautions, such as performing larval anesthesia in a fume hood, should always be taken while performing this procedure.

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