Name: assessing neurodegenerative phenotypes in drosophila dopaminergic neurons by climbing assays and whole brain immunostaining
Uploaded: 2013-04-24T13:00:00
Description: Watch this Scientific Journal Video about Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining at JoVE.com

Vi takker Leo Pallanck for flyve aktier, Christine Sommers for teknisk assistance og Gerasimos P. Sykiotis for nyttige diskussioner. Dette arbejde blev finansieret af NIH træning tilskud T32CA009363 til MCB

1.. Startle-induceret Negativ Geotaxis Assay <ol><li> Vælg fluer i den ønskede genotype, adskille dem efter køn, tilfældigt gruppere dem i kohorter af 20-30 dyr og vend dem i nye fødevarer hætteglas hver 2-3 dage, test hvert sæt af fluer (dvs. aldersmatchede kohorter, en kohorte / genotype) mindst en gang om ugen. </li><li> Tredive minutter før den negative geotaxis test, bedøver fluerne med CO 2 og placere dem i præ-mærkede klare plastrør lavet med to tomme fødevarer hætteglas (se <s...

<ol>
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Drug Targets. 9, 504-523 (2010).</li><li>Lundell, M., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spacial+development+of+serotonin+and+dopamine+neurons+in+the+Drosophila+CNS.">Temporal and spacial development of serotonin and dopamine neurons in the Drosophila CNS.</a> Dev. Biol. 165, 385-396 (1994).</li><li>Mao, Z., Davis, R. 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Assayene beskrevet her give en nyttig metode til at undersøge betydningen af specifikke gener, signalveje eller små forbindelser i vedligeholdelse af DA neuroner i aldring samt i forskellige sygdomme forbundne genetiske baggrunde (revideret i 5). Den startle-induceret negative geotaxis adfærd Drosophila har været flittigt brugt som en funktionel læse-out for funktionaliteten af DA neuroner i forskellige genetiske baggrunde, især i tilstedeværelse af PD-linked mutat...

Specificiteten af assays beskrevet her bygger på anvendelsen af en Gal4 fluelinen som udnytter de regulatoriske sekvenser af tyrosinhydroxylase-genet for at opnå specifik ekspression i dopaminerge neuroner 3. Tyrosinhydroxylase katalyserer den første og hastighedsbegrænsende trin i dopaminsyntese. TH immunreaktivitet i den voksne flyve hjernen overlapper dopamin, hvilket TH en god kandidat til at identificere DA neuroner in vivo (se eksempel 6). Desuden ekspressionsmønsteret af THGal4 er mere specifik end andre Gal4 linjer såsom DdcGal4, som indeholder regulatoriske sekvenser fra dopadecarboxylase genet og driver transgen ekspression ikke kun i DA neuroner, men også i serotonerge neuroner og delsæt af gliaceller 3 . Feany og Bender (2000) først observeret, at pan-neuronal udtryk for PD-bundet human α-synuclein genet accelererer fremadskridende tab af startle-induceret negative geotaxis adfærd i Drosophila. Vi har observeret lignende resultater ved hjælp af DA-neuron specifik THGal4 linje at drive expressionof en α-synuclein transgen 10 og brugt dette funktionelle udlæsning at studere neuroprotektive rolle Nrf2 vej i denne Drosophila model af PD 14.. Den specifikke assay beskrives her er tilpasset fra 2. og 3.. Drosophila Hjernen indeholder mere end 100 DA neuroner, arrangeret i mindst 5 forskellige klynger (PPL1, PPL2, PAL, PPM1 / 2, PPM3), der kan visualiseres i hele hjernen mounts ved konfokal mikroskopi (se for eksempel 11 og 7). Det er stadig kontroversielt, hvorvidt antallet af DA neuroner i Drosophila hjernen falder med alderen 9, 11, men er aldersafhængig neurodegeneration observeret i fluer hvor PD-bundne gener er blevet muteret / overudtrykt at modellere menneskelige sygdomme5.. Den ekspression af human α-synuclein i DA neuroner har en sådan virkning, og dermed er blevet brugt som model for PD. Her beskriver vi en analyse for DA neuroner tælle i hele hjernen mounts bruger TH som en celle markør. Denne analyse er blevet tilpasset fra 13 og 10..

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rabbit anti-tyrosine hydroxylase antibody</td>
			<td>Millipore</td>
			<td>AB152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey TRITC-labeled anti rabbit IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-026-152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Sigma</td>
			<td>D2522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Polystyrene vials</td>
			<td>VWR</td>
			<td>89092-734</td>
			<td>28.5 x 95 mm</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Canon</td>
			<td>PowerShot A3100IS (model)</td>
			<td>12.1 Megapixels 4X Optical Zoom</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 1.5</td>
			<td>VWR</td>
			<td>48366 205 48393 252</td>
			<td>Sizes: 18 x 18 mm, 24 x 60 mm</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 2</td>
			<td>VWR</td>
			<td>48368-040</td>
			<td>Size: 18 x 18 mm</td>
		</tr>
		<tr>
			<td>Dissection dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-01</td>
			<td>Size: 30 mm O.D.</td>
		</tr>
		<tr>
			<td>Dumostar No. 5 tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>72705-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000 (model)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP2 or SP5 (model)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Materials Table.

assessing neurodegenerative phenotypes in drosophila dopaminergic neurons by climbing assays and whole brain immunostaining

Drosophila melanogaster er en værdifuld model organisme for at studere aldring og patologiske degenerative processer i nervesystemet. Fordelene ved fluen som et eksperimentelt system omfatter dens genetiske sporbarhed, kort levetid og mulighed for at observere og kvantitativt analysere komplekse adfærd. Udtrykket for sygdom-bundne gener i specifikke neuronale populationer af Drosophila hjernen, kan bruges til at modellere humane neurodegenerative sygdomme som Parkinsons og Alzheimers 5.. Dopaminerge (DA) neuroner er blandt de mest sårbare neuronale populationer i den aldrende menneskelige hjerne. Ved Parkinsons sygdom (PD), den mest almindelige neurodegenerative bevægelsesforstyrrelser, fremskyndet tab af DA neuroner fører til en progressiv og irreversibel nedgang i lokomotorisk funktion. Ud over alder og udsættelse for miljøgifte, er tab af DA neuroner forværres af specifikke mutationer i kodningeneller promotorområder flere gener. Identifikation af sådanne PD-associerede alleler giver den eksperimentelle grundlag for brug af Drosophila som en model til at studere neurodegeneration af DA-neuroner in vivo. For eksempel. Ekspressionen af PD-bundet human α-synuclein genet i Drosophila DA neuroner rekapitulerer nogle funktioner i den menneskelige sygdomme, fx fremadskridende tab af DA neuroner og faldende bevægeapparatet funktion 2 Følgelig har denne model er blevet anvendt med succes til at identificere potentielle terapeutiske mål i PD 8.. Her beskriver vi to assays, der almindeligvis er blevet anvendt til at studere aldersafhængig neurodegeneration af DA neuroner i Drosophila: en klatring assay baseret på startle-inducerede negativ geotaxis respons og tyrosinhydroxylase immunfarvning af hele den voksne hjerne monteringer at overvåge antallet af DA neuroner på forskellige alderstrin. I begge tilfælde in vivo ekspression af UAS transgenes specifikt i DA neuroner kan opnås ved anvendelse af et tyrosin hydroxylase (TH)-promotor-Gal4 driver linie 3, 10.

Vi har brugt assayene beskrevet her at undersøge den rolle, stress beskyttende Nrf2 vej i en Drosophila model af PD 14.. Denne model bygger på ekspressionen af det humane α-synuclein genet i DA neuroner ved hjælp af en TH Gal4 driver 2, 10. Figur 1 viser et repræsentativt resultat af et startle-inducerede negative geotaxis (klatring) assay i mandlige fluer udtrykker forskellige transgener under kontrol af TH Gal4 driveren. Alle genotyper t...

Watch this Scientific Journal Video about Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining at JoVE.com

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Her beskriver vi to analyser, som er blevet oprettet for at studere aldersafhængig neurodegeneration af dopaminerge (DA) neuroner i<em&gt; Drosophila</em&gt;: Klatring / startle-induceret negativ geotaxis assay, der gør det muligt at studere de funktionelle virkninger af DA neuroner degeneration og tyrosinhydroxylase immunfarvning som bruges til at identificere og tælle DA neuroner i hele hjernen mounts.

Vurdere Neurodegenerative fænotyper i<em&gt; Drosophila</em&gt; Dopaminerge neuroner ved at klatre assays og Whole Brain-immunfarvning

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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Neuroscience

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

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