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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar powder, Alfa Aesar, 500GM</td>
			<td style="width:155px;">VWR</td>
			<td>AAA10752-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grape juice</td>
			<td>Welch&#8217;s</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Ethanol 95% (Reagent Alcohol 95%)</td>
			<td style="width:155px;">VWR</td>
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			<td></td>
		</tr>
		<tr height="42">
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			<td style="width:155px;">Sigma-Aldrich</td>
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			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:444px;">Propionic Acid</td>
			<td style="width:155px;">J.T.Baker</td>
			<td style="width:153px;">U33007</td>
			<td></td>
		</tr>
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			<td height="63" style="height:63px;width:444px;">Cover Glasses: Rectangles</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:153px;">12-544-D</td>
			<td>50 mm X 22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Zeiss LSM 880 laser scanning microscope</td>
			<td style="width:155px;">Zeiss</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Zen software</td>
			<td style="width:155px;">Zeiss</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:444px;">Chameleon Ultra II</td>
			<td style="width:155px;">Coherent</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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