Name: dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays
Uploaded: 2012-03-23T12:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Kollagenmatrix&#39;en analysen er blevet udviklet og forbedret af arbejdet i mange forskellige forskergrupper i løbet af de seneste to til tre årtier. De beskrevne fremgangsmåder her for dopaminerge og striatale eksplantaterne stor gavn af disse undersøgelser. Desuden ville forfatterne gerne takke Asheeta Prasad for hendes hjælp til opsætning striatale eksplantatkulturer. Arbejde i laboratoriet blev finansieret af Human Frontier Science Program Organization (Career Development Award), Nederlandene Organization of Health Research og udvikling (ZonMW-VIDI og ZonMW-TOP), den Europanian Union (mdDA-NeuroDev, FP7/2007-2011 , Grant 222.999) (til RJP), og Holland Organisation for Videnskabelig Forskning (TopTalent, at ERES).

1. Fremstillinq af rotte Tail Collagen <ol><li> Indsamle 6-10 voksne rotter haler (det er muligt at lagre halerne ved -20 ° C indtil anvendelse). </li><li> Gennemvæde haler i 95% ethanol natten over ved stuetemperatur (RT). </li></ol> Dissektion af rotte haler (i vævskultur hætte): (Hold værktøjer i 70% ethanol når du ikke bruger dem, og sørg for, at alle løsninger, værktøjer og glasvarer, der anvendes i hele denne procedure er sterile) <ol start="3"><li> At indsa...

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Kollagenmatrixen assayet beskrevet her er blevet anvendt, og forbedret af mange forskellige laboratorier i de sidste årtier til at undersøge en række axon vejledning molekyler og neuronale systemer (se f.eks referencer 5-8). Disse undersøgelser har vist, at dette assay er et stærkt værktøj til at studere virkningerne og regulering af axon vejledning molekyler, der udskilles af forskellige (mellemprodukt) målvæv. Imidlertid bør det bemærkes, at kollagenmatrixen er en erstatning for...

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dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

Midthjernen dopamin (mdDA) neuroner projekt via mediale forhjernen bundle mod flere områder i telencephalon, herunder striatum 1. Omvendt medium piggede neuroner i striatum, der giver anledning til striatonigral (direkte) pathway innerverer substantia nigra 2. Udviklingen af disse axon skrifter er afhængig af de kombinatoriske handlinger foretaget af en overflod af axon vækst og vejledning signaler, herunder molekyler, der frigives ved neurites eller (mellemliggende) målregioner 3,4. Disse opløselige faktorer kan studeres in vitro ved dyrkning mdDA og / eller striatale eksplantater i en collagenmatrix, der tilvejebringer en tredimensionel substrat for axoner efterligner ekstracellulære miljø. Desuden tillader kollagenmatrixen til dannelse af relativt stabile gradienter af proteiner frigivet af andre eksplantater eller celler er anbragt i nærheden (se f.eks referencer 5 og 6). Her beskriver vi metoder til purverifikationsprogrammet af rotte hale kollagen, mikrodissektion af dopaminerge og striatale eksplantaterne, deres kultur i kollagengeler og efterfølgende immunhistokemiske og kvantitativ analyse. Første hjernerne af E14.5 museembryoer er isolerede og dopaminerge og striatale eksplantater mikrodissekeres. Disse eksplantater derefter (co) dyrket i kollagengeler på dækglas i 48 til 72 timer in vitro. Efterfølgende aksonal fremskrivninger er visualiseret ved hjælp af neuronale markører (fx tyrosin hydroxylase, DARPP32 eller βIII tubulin) og axon vækst og tiltrækkende eller frastødende axon svar er kvantificerede. Dette neuronal forberedelse er et nyttigt værktøj for in vitro-undersøgelser af de cellulære og molekylære mekanismer mesostriatal og striatonigral axon vækst og vejledning under udvikling. Ved hjælp af dette assay, er det også muligt at vurdere andre (mellemprodukt) mål for dopaminerge og striatale axoner eller at teste specifikke molekylære signaler.

Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Eksplantater fra midthjernen dopaminsystemet og striatum anvendes i en collagenmatrix assay for<em&gt; In vitro</em&gt; Analyse af mesostriatal og striatonigral pathway udvikling. I dette assay axonal udvækst og vejledning kan manipuleres og kvantificeres. Det kan også modificeres til at vurdere andre regioner eller molekylære signaler.

Dissektion og kultur Mouse dopaminerge og Striatale eksplantater i Tredimensionelle kollagenmatrix Analyser

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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