Name: a simple neuronal mechanical injury methodology to study drosophila motor neuron degeneration
Uploaded: 2017-07-19T15:00:00
Description: Watch this Scientific Journal Video about A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration at JoVE.com

Vi vil gerne takke alle medlemmer af Keller og Magie Labs på Quinnipiac University for nyttige forslag. Vi vil især takke Barron L. Lincoln II for udvikling af denne skadeanalyse inden for Keller Lab. Vi vil også gerne takke Quinnipiac University College of Arts og Science Grant-In-Aid tildelt LC Keller.

1. Fremstilling af reagenser og udstyr <ol><li> Forberede 1x Dissektion Buffer (70 mM NaCI, 5 mM KCI, 0,02 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 115 mM saccharose, 5 mM trehalose, 5 mM HEPES, pH 7,2). </li><li> Forbered 1x phosphatbufferet saltvand (PBS). </li><li> Forbered 1x PBT ved anvendelse af 1x PBS med 0,01% Triton X-100. </li><li> Forbered frugtjuice agarplader. 14 Kort fortalt blandes 30 g agar i 700 ml H2O og autoklave. Opløs 0,5 g methylpar...

<ol>
	<li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophilia,+the+golden+bug,+emerges+as+a+tool+for+human+genetics.">Drosophilia, the golden bug, emerges as a tool for human genetics.</a> Nat. Rev. Genet. 6 (1), 9-23 (2005).</li><li>Ugur, B., Chen, K., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+tools+and+assays+for+the+study+of+human+diseases.">Drosophila tools and assays for the study of human diseases.</a> Dis Model Mech. 9 (3), 235-244 (2016).</li><li>McGurk, L., Berson, A., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+an+in+vivo+model+for+human+neurodegenerative+disease.">Drosophila as an in vivo model for human neurodegenerative disease.</a> Genetics. 201 (2), 377-402 (2015).</li><li>Shin, J. E., Cho, Y., Beirowski, B., Milbrandt, J., Cavalli, V., 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=Dual+leucine+zipper+kinase+is+required+for+retrograde+injury+signaling+and+axonal+regeneration.">Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.</a> Neuron. 74 (6), 1015-1022 (2012).</li><li>Xiong, X., Wang, X., Ewanek, R., Bhat, P., Diantonio, A., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+turnover+of+the+Wallenda/DLK+kinase+regulates+a+retrograde+response+to+axonal+injury.">Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury.</a> J Cell Biol. 191 (1), 211-223 (2010).</li><li>Xiong, X., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditioning+lesion+protects+axons+from+degeneration+via+the+Wallenda/DLK+MAP+kinase+signaling+cascade.">A conditioning lesion protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade.</a> J Neurosci. 32 (2), 610-615 (2012).</li><li>Mishra, B., Ghannad-Rezaie, M., Li, J., Wang, X., Hao, Y., Ye, B., Chronis, N., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidics+chips+for+live+imaging+and+study+of+injury+responses+in+Drosophila+larvae.">Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae.</a> J Vis Exp. (84), e50998 (2014).</li><li>Eaton, B. A., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynactic+is+necessary+for+synapse+stabilization.">Dynactic is necessary for synapse stabilization.</a> Neuron. 34 (5), 729-741 (2002).</li><li>Eaton, B. A., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+Kinase1+controls+synaptic+stability+downstream+of+the+type+II+BMP+receptor.">LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor.</a> Neuron. 47 (5), 695-708 (2005).</li><li>Pielage, J., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+spectrin+is+essential+for+synapse+stabilization.">Presynaptic spectrin is essential for synapse stabilization.</a> Curr Biol. 15 (10), 918-928 (2005).</li><li>Pielage, J., Cheng, L., Fetter, R. D., Carlton, P. M., Sedat, J. W., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+presynaptic+giant+Ankyrin+stabilizes+the+NMJ+through+regulation+or+presynaptic+microtubules+and+transsynaptic+cell+adhesion.">A presynaptic giant Ankyrin stabilizes the NMJ through regulation or presynaptic microtubules and transsynaptic cell adhesion.</a> Neuron. 58 (2), 195-209 (2008).</li><li>Massaro, C. M., Pielage, J., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+that+enhance+synapse+stability+despite+persistent+disruption+of+the+spectrin/ankryin/microtubule+cytoskeleton.">Molecular mechanisms that enhance synapse stability despite persistent disruption of the spectrin/ankryin/microtubule cytoskeleton.</a> J Cell Biol. 187 (1), 101-117 (2009).</li><li>Lincoln, B. L., Alabsi, S. H., Frendo, N., Freund, R., Keller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuronal+injury+follows+a+temporal+sequence+of+cellular+events+leading+to+degeneration+at+the+neuromuscular+junction.">Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction.</a> J Exp Neurosci. 9, 1-9 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+apple+juice-agar+plates.">Drosophila apple juice-agar plates.</a> Cold Spring Harb Protoc. , (2011).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+immunohistochemistry.">Drosophila larval NMJ immunohistochemistry.</a> J Vis Exp. (25), (2009).</li><li>Smith, R., Taylor, 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=Dissection+and+imaging+of+active+zones+in+the+Drosophila+neuromuscular+junction.">Dissection and imaging of active zones in the Drosophila neuromuscular junction.</a> J Vis Exp. (50), (2011).</li><li>Jan, L., Jan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+horseradish+peroxidase+as+specific+neuronal+markers+in+Drosophila+and+in+grasshopper+embryos.">Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos.</a> Proc. Natl. Acad. Sci. 79 (8), 2700-2704 (1982).</li><li>Lahey, T., Gorczyca, M., Jia, X., Budnik, V. <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+tumor+suppressor+gene+dlg+is+required+for+normal+synaptic+bouton+structure.">The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure.</a> Neuron. 13 (4), 823-835 (1994).</li></ol>

Den neuronale mekaniske skade, der er beskrevet tidligere og demonstreret her, kan anvendes til at fremkalde skade / stress i segmenternes nerver af Drosophila larver. 4 , 5 , 6 , 14 Denne eksperimentelle teknik er tidligere blevet anvendt til at dissekere den tidsmæssige rækkefølge af hændelser, som fører til neurodegeneration, såvel som at undersøge transkriptionelle ændring...

Neuronal degeneration forekommer under normal udvikling og kan forårsages af den naturlige aldringsproces, skade, stress eller sygdomstilstande. Drosophila melanogaster , den fælles frugtflyve, giver en simpel og kraftfuld modelorganisme til at studere neurodegeneration på grund af de bemærkelsesværdige ligheder i de molekylære mekanismer, der driver degenerationen af ​​neuroner. Disse ligheder er fremhævet af den kendsgerning, at ca. 70% af sygdomsassocierede humane gener har en Drosophila homolog. 1 Derudover er talrige analyser og teknologiske værktøjer til undersøgelse af humane neurodegenerative sygdomme blevet udviklet og anvendt i Drosophila . 2 , 3 Inden for Drosophila giver det neuromuskulære kryds (NMJ) mulighed for analyse af både cellulære og elektrofysiologiske egenskaber og har vist sig at være et vigtigt system til at studere neuromuskulær sygdom på grund af den synlige nEuron-muskelforbindelser. 2 I dette studie beskriver vi et in vivo neuronskadesassay i Drosophila larver, som muliggør reproducerbar skade på segmentale nerver. Denne motoneuron-skade resulterer i en tidssekvens af cellulære hændelser, der resulterer i neurodegenerering ved NMJ 24 h efter skade. Evnen til reproducerbar skade på motoneuroner, der resulterer i neurodegenerering, har forskellige anvendelser, såsom identifikation af specifikke gener, der kræves til degenerative processer, dissekationen af ​​transkriptionelle reaktioner på neuronal skade og analysen af ​​beskyttende signaleringskaskader. 4 , 5 , 6 Denne metode er også blevet anvendt i kombination med mikrofluidika til at studere neuronal degenerering og regenerering hos levende dyr. 7 Vi anvender et etableret kvantitativt assay for at undersøge motor neuron degeneratIon ved Drosophila NMJ efter mekanisk skade. Dette assay er baseret på det faktum, at tab af presynaptisk membran og proteiner går forud for demontering af det subsynaptiske retikulum (SSR) karakteriseret ved postsynaptiske muskelmembranfoldninger. 8 , 9 , 10 , 11 , 12 , 13 Dette assay tillader kvantificering af &quot;synaptiske fodspor&quot;, hvor det præsynaptiske neuron har mistet forbindelsen til den tilstødende postsynaptiske muskel. Den degenerative proces har vist sig at være progressiv gennem larvudvikling 12 og kan ikke regnes for ved ændret synapsudvikling eller spiring. 8 , 9 , 10 , 11 , 12 AnnoncenUdsigten ved at bruge mekanisk skade over tidligere eksisterende mutationer er, at det muliggør dissektion af den tidsmæssige rækkefølge af cellulære hændelser, der fører til neurodegenerering ved NMJ. 13

<table>
	<tbody>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>CO2 Air Tank</td>
			<td>Tech Air</td>
			<td>UN 1013</td>
			<td>Various tank sizes can be purchased/</td>
		</tr>
		<tr>
			<td>CO2 Anesthetizing Apparatus</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless-steel pins, size 0.1</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SylGard 184 Silicone Elastomer, Base and Curing Agent</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>To pour dissecting plates</td>
		</tr>
		<tr>
			<td>Bouin&#39;s Solution</td>
			<td>Sigma</td>
			<td>HT 10132-1L</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde in PBS</td>
			<td>Affymetrix</td>
			<td>FLY-8030-20</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>Dissecting Stereo MIcroscope</td>
			<td>AmScope</td>
			<td>SM-1BZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>AmScope</td>
			<td>HL150-AY-220V</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- nc82 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82-s</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AF3</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor anti-horseradish peroxidase</td>
			<td>Jackson Immunoresearch</td>
			<td>123-545-021; 123-585-021; 123-605-021</td>
			<td>One can you Alexa Fluor&#174; 488, 594 or 647</td>
		</tr>
		<tr>
			<td>Flystuff Grape Juice Agar Premix</td>
			<td>Genesee Scientific</td>
			<td>47-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Genesee Scientific</td>
			<td>29-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Utility Box</td>
			<td>Fisher Scientific</td>
			<td>03-484C</td>
			<td>Used to create humid chamber for larval recovery</td>
		</tr>
	</tbody>
</table>

a simple neuronal mechanical injury methodology to study drosophila motor neuron degeneration

Degenerationen af ​​neuroner opstår under normal udvikling og som reaktion på skade, stress og sygdom. De cellulære kendetegn ved neuronal degeneration er bemærkelsesværdigt ens i mennesker og hvirvelløse dyr, ligesom de molekylære mekanismer, der driver disse processer. Frugtflugten, Drosophila melanogaster , giver en stærk, men enkel genetisk modelorganisme til at studere de cellulære kompleksiteter af neurodegenerative sygdomme. Faktisk har ca. 70% af sygdomsassocierede humane gener en Drosophila homolog og en overflod af værktøjer og analyser er blevet beskrevet ved hjælp af fluer for at studere humane neurodegenerative sygdomme. Mere specifikt har det neuromuskulære kryds (NMJ) i Drosophila vist sig at være et effektivt system til at studere neuromuskulære sygdomme på grund af evnen til at analysere de strukturelle forbindelser mellem neuron og muskel. Her rapporterer vi om en in vivo motor neuron skade undersøgelse i Drosophila , somReproducerbart inducerer neurodegenerering ved NMJ ved 24 timer. Ved hjælp af denne metode har vi beskrevet en tidssekvens af cellulære hændelser, der resulterer i degenerering af motor neuron. Skadesmetoden har forskellige anvendelser og er også blevet anvendt til at identificere specifikke gener, der kræves til neurodegenerering og dissekere transkriptionelle reaktioner på neuronal skade.

Ved anvendelse af den her viste procedure har vi vist, at mekanisk neuronal skade tillader tidsmæssig dissektion af neurodegenerative hændelser. 14 , 18 Sekvensen af ​​hændelser er tidligere blevet karakteriseret og begynder med en øjeblikkelig forstyrrelse af cytoskelet, efterfulgt af aksonale menneskehandel, en akkumulering af ubiquitinerede proteiner og efterfølgende neurodegenerering 24 timer efter skader. Før skade...

Watch this Scientific Journal Video about A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration at JoVE.com

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Her beskriver vi en enkel og bredt tilgængelig metode til at skade segmentale nerver i Drosophila larver for at visualisere og kvantificere neurodegenerering af motorneuroner ved det neuromuskulære kryds (NMJ) af tredje instar larver.

En simpel neurologisk mekanisk skademetode til undersøgelse<em&gt; Drosophila</em&gt; Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

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A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

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Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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En metode til at påføre lukket hoved traumatisk hjerneskade i<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

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En simpel måde at måle Ændringer i Reward-søger Behavior Brug<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...