Name: mass histology to quantify neurodegeneration in drosophila
Uploaded: 2016-12-15T12:30:00
Description: Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. Fastsættelse chefen på Kraver og Indlejring i Paraffin Bemærk: Alle trinene i fiksering processen bør ske i et stinkskab. Methylbenzoat, mens ikke udgør en sundhedsrisiko, har en højt udtalt lugt, som kan være overvældende, hvis ikke håndteres i et stinkskab. <ol><li> Før bedøve fluerne, udgør 50 ml Carnoy ved tilsætning 15 ml chloroform og 5 ml iseddike til 30 ml 99% ethanol (ikke blande chloroform og eddikesyre). Hæld det i en glasbeholder med en flad bund, såsom en crystalizing skålen, fo...

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Den beskrevne fremgangsmåde tilvejebringer et middel til at kvantificere neurodegeneration i hjernen hos Drosophila. Mens andre metoder, såsom at tælle en specifik celletype, kan anvendes til at identificere neurodegeneration, fordelen ved denne fremgangsmåde er, at den kan anvendes mere generelt. Tælle celler kræver, at disse celler kan identificeres pålideligt ved anvendelse af enten et specifikt antistof eller ekspressionen af ​​en cellespecifik markør, som ikke altid er til rådighed. Endvidere ...

Med stigningen i den forventede levetid, har neurodegenerative sygdomme som Alzheimers eller Parkinsons blevet et stigende sundhedstrussel for den almene befolkning. Ifølge National Institutes of Health, 115 millioner mennesker verden over forventes at blive påvirket af demens i 2050. Selv om der er gjort betydelige fremskridt med at identificere gener og risikofaktorer der er involveret i det mindste nogle af disse sygdomme, for mange af dem, den underliggende molekylære mekanismer er stadig ukendt eller ikke godt forstået. Simple hvirvelløse modelorganismer som Caenorhabditis elegans og Drosophila melanogaster tilbyder en bred vifte af eksperimentelle fordele at studere mekanismerne i neurodegenerative sygdomme, herunder en kort livscyklus, stort antal afkom, og tilgængeligheden af veletablerede og til tider unikke genetiske og molekylære metoder 1 -12. Desuden er disse organismer er modtagelige for fordomsfriinteraktion skærme, der kan identificere faktorer, der bidrager til disse sygdomme ved deres skærpende eller lindring af virkninger på neurodegenerative fænotyper. Analyse sådanne genetiske interaktioner og vurdere aldrende effekter kræver kvantitative protokoller til at opdage neurodegeneration og måle dens sværhedsgrad. Denne vurdering kan gøres relativt let ved måling adfærdsmæssige aspekter i Drosophila, såsom olfaktoriske læring, negative geotaxis, eller hurtig phototaxis, som giver en numerisk ydeevne værdi 13-21. Det er også muligt at bestemme virkningerne på neuronal overlevelse ved at tælle neuroner. Men dette er kun muligt, når der fokuseres på en bestemt befolkning, der er klart identificerbare, ligesom de dopaminerge neuroner, der er berørt i PD, og selv da, har resultaterne været kontroversielle 22-24. Protokollen beskrevet her anvender kraven metode til at udføre paraffin serielle snit, en fremgangsmådeder oprindeligt blev udviklet af Heisenberg og Böhl, der brugte det til at isolere anatomiske hjernen mutanter i Drosophila 25. Brugen af kraven metode er efterfølgende blevet tilpasset, herunder kryosektioner, Vibratome sektioner, og plastprofiler 26-28. Her er denne metode anvendes til opnåelse serielle sektioner af hele flue hoved, som derefter kan anvendes til at måle vakuolerne der udvikler sig i fluer med neurodegenerative fænotyper 16,21,29-32. Disse målinger kan udføres på specifikke områder i hjernen eller kan dække hele hjernen; sidstnævnte tilgang tillader en at identificere selv svage degenerative fænotyper, som observeret under aldring. Endelig, når anvendelse af kraverne, op til 20 fluer kan behandles som én præparat, som ikke blot er mindre tidskrævende, men også giver mulighed for analyse af kontrol- og eksperimentelle fluer i det samme præparat, hvilket minimerer artefakter som følge af små ændringer i præparatet.

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			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
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			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&#160;</td>
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			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
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		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
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			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
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			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
			<td></td>
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			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
		<tr>
			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
		</tr>
		<tr>
			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass histology to quantify neurodegeneration in drosophila

Progressive neurodegenerative sygdomme som Alzheimers sygdom (AD) eller Parkinsons sygdom (PD) er en voksende trussel mod menneskers sundhed i hele verden. Selv pattedyr modeller har givet vigtig indsigt i de underliggende mekanismer af patogenicitet, er kompleksiteten af ​​pattedyr systemer sammen med deres høje omkostninger begrænser deres anvendelse. Derfor er den enkle, men veletablerede Drosophila model-system giver et alternativ til at undersøge de molekylære veje, der er berørt i disse sygdomme. Udover adfærdsmæssige afvigelser, er neurodegenerative sygdomme karakteriseret ved histologiske fænotyper såsom neuronal død og axonopathy. For at kvantificere neuronal degeneration og at bestemme, hvordan det påvirkes af genetiske og miljømæssige faktorer, bruger vi en histologisk tilgang, der er baseret på måling af vakuoler i voksne flyve hjerner. For at minimere virkningerne af systematisk fejl og til direkte at sammenligne sektioner fra kontrol- og experimental fluer i en forberedelse, bruger vi &quot;krave&quot; metode til paraffinsnit. Neurodegeneration vurderes derefter ved at måle størrelsen og / eller antallet af vakuoler, der har udviklet i gylp hjernen. Dette kan enten ske ved at fokusere på en bestemt region af interesse eller ved at analysere hele hjernen ved at opnå serielle snit, der spænder over fuldstændige hoved. Derfor er denne metode tillader en at måle ikke blot alvorlig degeneration, men også relativt milde fænotyper, der kun kan påvises i et par afsnit, som forekommer under normal aldring.

Under anvendelse af den beskrevne fremgangsmåde resulterer i serielle snit farvet af øjet pigment 33, der omfatter hele flue hoved. En del af dette er vist i figur 1B, hvor sektionerne fra en individuel hoved er vist fra top til bund. Sektionerne fra forskellige fluer ses venstre til højre i dette eksempel. For at lette orienteringen og identifikation af fluerne, er en eyeless flue (sinus oculis) indsættes som en markør ved position 3 (pil, <stro...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila er almindeligt anvendt som et modelsystem til at studere neurodegeneration. Denne protokol beskriver en metode, hvorved degenerering, som bestemt ved vacuole dannelse i hjernen, kan kvantificeres. Det minimerer også effekter som følge af den eksperimentelle procedure ved behandling og sektionering kontrol og eksperimentelle fluer som en prøve.

Mass Histologi at kvantificere neurodegeneration i<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Methods to Assay Drosophila Behavior

Methods to Assay Drosophila Behavior

Fremgangsmåder til assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualisering af proprioceptorer i<em&gt; Drosophila</em&gt; Larver og pupper

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Udnytte 3D Printing Technology til Flet MRI med Histologi: En protokol for Brain Skæring

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

En ny metode til at modellere kronisk traumatisk encephalopati i<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modellering Amyloid-β42 toksicitet og Neurodegeneration i voksen zebrafisk hjerne

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Kvantitativ analyse af Neuronal dendritiske Arborization kompleksitet i Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...