Name: ex vivo culturing of whole, developing drosophila brains
Uploaded: 2012-07-27T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Vi vil gerne takke alle de medlemmer af vores laboratorium for deres råd, nyttige diskussioner og kommentarer til manuskriptet. Vi vil også en særlig tak til Marta Koch og Bassem Hassan for den fremragende forslag om, at vi vente 1,5 time APF før dissekere hjerner til dyrkning i remodeling eksperimenter. Vi vil også gerne takke NHGRI imaging mulighed for brug af deres konfokal mikroskop. Dette arbejde blev støttet af Basic Neuroscience Program for NINDS Egne Research Program, NIH (Z01 NS003106).

A. Fremstilling af medier <ol><li> Fremstille komplette medier med filter-steriliserende Schneider Drosophila medium suppleret med 10% kalvefosterserum og 1% penicillin / streptomycin. </li><li> Medier skal fremstilles frisk hver gang. Alternativt kan en frisk aliquot af præ-frosne medier optøs før brug. </li><li> Forud for dyrkning, og der tilsættes 10 ug / ml insulin og 2 ug / ml ecdyson til mediet. Lægemidler blev fremstillet som koncentrerede lagre og fryses i små portioner. Optøede alikvoter blev ...

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<html> Kroniske genetiske manipulationer af væv struktur og funktion, herunder mutationer og ekspressionen af ​​transgener, typisk producere en kombination af umiddelbare effekter og sene indirekte konsekvenser, der kan være meget svært at adskille. Som supplement til genetiske metoder med farmakologi kan lette søgning i tidsforløbet bag en fænotype. For eksempel har dette været en stærk tilgang til dissekere bidrag forskellige cytoskeletale komponenter under spermatogenesen i Drosophila 10.</su...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognummer </td><td> Slutkoncentration </td></tr><tr><td> Schneiders Drosophila medier </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> Føtalt bovint serum (varmeinaktiveret) </td><td> Invitrogen </td><td> 10082-147 </td><td> 10% </td></tr><tr><td> Penicillin / streptomycin </td><td> Invitrogen </td><td> 15140-122 </td><td> 1% </td></tr><tr><td> Insulin </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdyson </td><td> Sigma-Aldrich </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitine </td><td> Sigma-Aldrich </td><td> R7772 </td><td> 100 uM </td></tr><tr><td> Olomoucin </td><td> Sigma-Aldrich </td><td> O0886 </td><td> 100 uM </td></tr><tr><td> Normalt gedeserum </td><td> Invitrogen </td><td> 50062Z </td><td> 1% </td></tr><tr><td> Bovint Serum Albumin </td><td> Fisher Scientific </td><td> M9501 </td><td> 1% </td></tr><tr><td> Formaldehyd </td><td> Sigma-Aldrich </td><td> D4551 </td><td> 4% </td></tr><tr><td> Triton-X 100 </td><td> Fisher Scientific </td><td> BP151 </td><td> 0,5% </td></tr><tr><td> Phosphatbufret saltvand </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> H1000 </td&gt; <td></td></tr><tr><td> Millicell Cell Culture Inserts </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish, 4 og </td><td> Thermo Scientific </td><td> 176740 </td><td></td></tr><tr><td> Dumont # 5 Tang </td><td> Gode ​​Science Tools </td><td> 11251-20 </td><td></td></tr><tr><td> Microscope Slides </td><td> Fisher Scientific </td><td> 12-544-7 </td><td></td></tr><tr><td> Cover Glass </td><td> Fisher Scientific </td><td> 12 til 548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

Beskriver vi en fremgangsmåde til ex vivo dyrkning af hele Drosophila hjerner. Dette kan bruges som et kontrapunkt til kroniske genetiske manipulationer for at undersøge cellebiologi og udvikling af centrale hjernens strukturer ved at tillade akutte farmakologiske interventioner og levende billeder af cellulære processer. Som et eksempel på en teknik, fungerer inden i vores laboratorium 1 har vist, at en hidtil ukendt subcellulær rum ligger mellem de axonale og somatodendritisk rum axoner i Drosophila centrale hjernen. Udviklingen af dette rum, benævnt axon første segment (AIS) 2, viste genetisk at afhænge af neuron-specifikke cyclin-afhængig kinase, cdk5. Vi viser her, at ex vivo behandling af vildtype-Drosophila larvestadium hjerner med cdk5-specifikke farmakologiske inhibitorer roscovitine og olomoucin 3 bevirker akutte ændringer i actin organisation, ogi lokalisering af celleoverfladeprotein fasciclin 2, som efterligner de ændringer, der ses i mutanter, der mangler cdk5 aktivitet genetisk. Et andet eksempel på den ex vivo kulturen teknik er i henhold til ombygning af forbindelserne i embryonale champignon kroppen (MB) gamma neuroner under forvandling fra larve til voksen. Det champignon krop er centrum for olfaktoriske indlæring og hukommelse i fluen 4, og disse gamma neuroner beskære deres axonale og dendritiske grene under puppe udvikling og derefter igen udvider grene på et senere tidspunkterne at etablere voksne innervation mønster 5. Beskæring af disse neuroner i MB har vist sig at forekomme via lokal nedbrydning af neurit grene 6, ved en mekanisme, der udløses af ecdyson, et steroid hormon, der virker ved den ecdyson-receptoren B1 7, og som er afhængig af aktiviteten af ubiquitin-proteasom system 6. Vores fremgangsmåde til ex vivo dyrkning kan Be bruges til at afhøre yderligere mekanisme udviklingsmæssige remodellering. Fandt vi, at i ex vivo-kultur indstilling gamma neuroner i MB gentaget processen udviklingsmæssige beskæring med et tidsforløb der svarer til in vivo. Det var imidlertid væsentligt, at afvente 1,5 timer efter puparium dannelse før eksplantation vævet, for at cellerne forpligte irreversibelt til metamorfose, dissektion af dyr ved begyndelsen af ​​pupariation ført til ringe eller ingen metamorfose i kultur. Således, med passende modifikation, kan ex vivo-kultur fremgangsmåde anvendes til at studere dynamisk samt steady state aspekter centrale hjerne biologi.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

Denne artikel beskriver en fremgangsmåde, ved hvilken man kan efterligne<em&gt; In vivo</em&gt; Udvikling af<em&gt; Drosophila</em&gt; Champignon kroppen i en<em&gt; Ex vivo</em&gt; Kultursystem.

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We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

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