Name: inducing dendritic growth in cultured sympathetic neurons
Uploaded: 2012-03-21T12:00:00
Description: Watch this Scientific Journal Video about Inducing Dendritic Growth in Cultured Sympathetic Neurons at JoVE.com

Dit werk werd ondersteund door de financiering van de National Institutes of Health (subsidies R21 NS45037 en R01 ES014901).

1. Voorbereiding van Cultuur Medium (C2 medium) <ol><li> Voeg de volgende, in de aangegeven volgorde, in een steriele, disposable 500 ml Erlenmeyer: <table border="1"><tbody><tr><td> Bedragen </td><td> Bestanddeel </td><td> Eindconcentratie </td></tr><tr><td> 190 ml </td><td> DMEM (lage glucose) </td><td> N / A </td></tr><tr><td> 10 ml </td><td> vetzuur vrij BSA (20mg/ml in DMEM) </td><td> 50 ug / ml </td></tr><tr><td> 2,8 ml </td><td> L-Glutamine </td><td> 1,4 mM </t...

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De voordelen van dit model omvatten: (a) de culturen uit een homogene populatie van sympathische neuronen zonder andere celtypen 17,23, (b) de groeifactor eisen van sympathische neuronen gevestigde, waardoor het gebruik van beschreven medium, en een verscheidenheid aan bepaalde media, en substraten werken goed voor het kweken en onderhouden van sympathische neuronen 23; (c) neuronen in deze culturen uniform te reageren op de dendriet-bevorderend activiteit van Matrigel, BMP-7 en andere BMP&#39;s va...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> Dekglaasjes </td><td> Bellco </td><td> 1.943 tot 10012 </td><td> 12 mm Ronde Coverslip # 1 Dikte, Duits glas </td></tr><tr><td> Bent tip pipetten </td><td> Bellco </td><td> 1273 tot 50.003 </td><td></td></tr><tr><td> Poly-D-lysine </td><td> Sigma </td><td> P0899-100mg </td><td> Voorraadoplossing 1 mg / ml </td></tr><tr><td> Gedistilleerd water </td><td> Gibco </td><td> 15230 </td><td></td></tr><tr><td> Fijn tang Dumont niet. 4 en 5 </td><td> Fine Science Tools (FST) </td><td> 11252-30 11241-30 </td><td></td></tr><tr><td> 20-gauge naalden </td><td> Becton Dickinson </td><td> 305176 </td><td></td></tr><tr><td> Leibovitz&#39;s L-15-medium </td><td> Invitrogen </td&gt; <td> 11415 </td><td></td></tr><tr><td> Collagenase </td><td> Worthington </td><td> 4176 </td><td> 1 mg / ml </td></tr><tr><td> Dispase </td><td> Roche </td><td> 04942078001 </td><td> 5 mg / ml </td></tr><tr><td> Balanced Salt Hank&#39;s oplossing (-Ca, Mg-) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> DMEM laag glucose </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> L-Glutamine </td><td> Invitrogen </td><td> 25030 </td><td> 20 mM </td></tr><tr><td> Insuline / Transferrine / Selenium </td><td> Invitrogen </td><td> 51500-056 </td><td> 100X </td></tr><tr><td> Vetzuren vrij BSA </td><td> Calbiochem </td><td> 126609 </td><td></td></tr><tr><td> NGF </td><td> Harlan Bioproducts </td><td> 5017 </td><td></td></tr><tr><td> Ham F-12 voedingsbodem (F-12) </td><td> Invitrogen </td><td> 11765 </td><td&gt; </td></tr><tr><td> Cytosinearabinoside </td><td> Sigma </td><td> C1768 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 356234 </td><td> Vermijd herhaaldelijk vries-dooi cycli </td></tr><tr><td> BMP-7 </td><td> R &amp; D Systems Curis, Inc </td><td> 345-BP BMP7-07H </td><td> BMP 4, 5 en 6 werken even goed als BMP-7 en soms beter commercieel te verkrijgen </td></tr><tr><td> Paraformaldehyde </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Triton X-100 </td><td> Visser </td><td> BP151-500 </td><td></td></tr><tr><td> Muis anti-rat MAP2 antilichaam </td><td> Sternberg monoklonalen Inc </td><td> SMI52 </td><td> 1/5000 </td></tr><tr><td> Alexa Fluor 546 geit-anti-muis </td><td> Invitrogen </td><td> A11003 </td><td> 1/10000 </td></tr><tr><td> Montage media </td><td> Verleng Antifade kit Molecular Probes </td> &lt;td&gt; P7481 </td><td></td></tr></tbody></table>

inducing dendritic growth in cultured sympathetic neurons

De vorm van de dendritische spil bepaalt de totale synaptische ingang een neuron kan ontvangen 1-3, beïnvloedt de samenstelling en verdeling van deze ingangen 4-6. Veranderde patronen van dendritische groei en plasticiteit worden geassocieerd met een verminderde neuro-functie in experimentele modellen 7 en worden verondersteld bij te dragen aan de klinische symptomen waargenomen bij zowel neurologische stoornissen 8-10 en neurodegeneratieve ziekten 11-13. Dergelijke waarnemingen onderstrepen het functionele belang van precies regelen dendritische morfologie, en suggereren dat het identificeren van mechanismen die dendritische groei te beheersen niet alleen inzicht in hoe neuronale connectiviteit wordt geregeld tijdens de normale ontwikkeling te bevorderen, maar kan ook inzicht te geven op nieuwe therapeutische strategieën voor diverse neurologische aandoeningen. Mechanistische studies van dendritische groei zou aanzienlijk worden vergemakkelijkt door de beschikbaarheid ofa model systeem waarmee neuronen om proefondervindelijk te worden omgeschakeld van een toestand waarin ze niet dendrieten uit te breiden tot een waarin ze een dendritische prieel vergelijkbaar met die van hun in vivo tegenhangers uit te werken. Primaire culturen van sympathische neuronen los worden gezien van de superieure cervicale ganglia (SCG) van perinatale knaagdieren voorziet in een dergelijk model. Wanneer gekweekt in beschreven medium in de afwezigheid van serum en ganglion gliacellen, sympathiek neuronen uit te breiden een proces dat is axonale, en dit unipolaire toestand aanhoudt weken tot maanden in de cultuur 14,15. De toevoeging van een bot morfogenetische proteïnen 7 (BMP-7) 16,17 of Matrigel 18 het kweekmedium activeert deze neuronen te verlengen meerdere processen dat de morfologische, biochemische en functionele criteria voor dendrieten. Sympathische neuronen los van de SCG van perinatale knaagdieren en geteeld onder bepaalde voorwaarden zijn van een homogene populatie van neuronen 19die op uniforme wijze reageren op de dendriet-bevorderend activiteit van Matrigel, BMP-7 en andere BMP&#39;s van de decapentaplegic (DPP) en 60A subfamilies 17,18,20,21. Belangrijk Matrigel en BMP-geïnduceerde vorming dendrietmorfologie treedt in de afwezigheid van veranderingen in celoverleving of axonale groei 17,18. Hier beschrijven we hoe je los culturen van sympathische neuronen afgeleid van de SCG van perinatale ratten, zodat ze kunnen inspelen op de selectieve dendriet-bevorderend activiteit van Matrigel of BMP.

Watch this Scientific Journal Video about Inducing Dendritic Growth in Cultured Sympathetic Neurons at JoVE.com

Inducing Dendritic Growth in Cultured Sympathetic Neurons

We beschrijven een protocol voor het gebruik van botmorfogenetische eiwit-7 (BMP-7) of Matrigel om selectief te induceren dendritische groei van de primaire sympathische neuronen los worden gezien van de superieure cervicale ganglia (SCG) van perinatale ratten.

Het induceren van dendritische groei in gekweekte sympathische neuronen

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

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Neuroscience

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