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

Dette arbeidet ble støttet av midler fra National Institutes of Health (tilskudd R21 NS45037 og R01 ES014901).

1. Utarbeidelse av Kultur Medium (C2 medium) <ol><li> Legg til følgende, i den rekkefølgen, til en steril, disponibel 500 ml erlenmeyerkolbe: <table border="1"><tbody><tr><td> Beløp </td><td> Component </td><td> Endelig konsentrasjon </td></tr><tr><td> 190 ml </td><td> DMEM (lav glukose) </td><td> N / A </td></tr><tr><td> 10 ml </td><td> fettsyre gratis BSA (20mg/ml i DMEM) </td><td> 50 mikrogram / ml </td></tr><tr><td> 2,8 ml </td><td> L-Glutamin </td><td> 1,4 mM ...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+pathology:+an+overview+of+Golgi+studies+in+man.">Dendritic pathology: an overview of Golgi studies in man.</a> Can. J. Neurol. Sci. 16, 41-50 (1989).</li><li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+I.+Conditions+under+which+neurons+form+axons+but+not+dendrites.">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites.</a> Dev. Biol. 128, 324-336 (1988).</li><li>Tropea, M., Johnson, M. I., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+promote+dendritic+development+in+rat+sympathetic+neurons+in+vitro.">Glial cells promote dendritic development in rat sympathetic neurons in vitro.</a> Glia. 1, 380-392 (1988).</li><li>Lein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+extracellular+matrix+and+osteogenic+protein-1+on+the+morphological+differentiation+of+rat+sympathetic+neurons.">The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons.</a> Int. J. Dev. 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I., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 177-295 (1991).</li><li>Beck, H. N., Drahushuk, K., Jacoby, D. B., Higgins, D., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-5+(BMP-5)+promotes+dendritic+growth+in+cultured+sympathetic+neurons.">Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons.</a> BMC Neurosci. 2, 12-12 (2001).</li><li>Guo, X., Rueger, D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+protein-1+and+related+bone+morphogenetic+proteins+regulate+dendritic+growth+and+the+expression+of+microtubule-associated+protein-2+in+rat+sympathetic+neurons.">Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons.</a> Neurosci. Lett. 245, 131-134 (1998).</li><li>Lein, P. 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A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+II.+Serum+promotes+dendritic+growth.">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth.</a> Dev. Biol. 128, 337-348 (1988).</li></ol>

Fordelene med denne modellen inkluderer: (a) de kulturene består av en homogen befolkning av sympatiske nevroner blottet for andre celletyper 17,23, (b) de vekstfaktor kravene i sympatiske nevroner er godt etablert, som tillater bruk av definert medium, og en rekke definerte medier, og underlag fungerer godt for dyrking og opprettholde sympatiske nevroner 23, (c) nevroner i disse kulturene reagerer likt for den dendrite-fremme aktivitet Matrigel, BMP-7 og andre fil fra BMP av DPP og 60A underfamil...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Glass Dekkglass </td><td> Bellco </td><td> 1943-10012 </td><td> 12 mm runde dekkglass nr. 1 Tykkelse, tysk glass </td></tr><tr><td> Bent Tips pipets </td><td> Bellco </td><td> 1273-50003 </td><td></td></tr><tr><td> Poly-D-lysin </td><td> Sigma </td><td> P0899-100mg </td><td> Stamoppløsning 1 mg / ml </td></tr><tr><td> Destillert vann </td><td> Gibco </td><td> 15230 </td><td></td></tr><tr><td> Fin pinsett Dumont nei. 4 og 5 </td><td> Fin Science Verktøy (FST) </td><td> 11252-30 11241-30 </td><td></td></tr><tr><td> 20-gauge nåler </td><td> Becton Dickinson </td><td> 305176 </td><td></td></tr><tr><td> Leibovitz har 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> Hank balanserte salt løsning (-Ca,-Mg) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> DMEM Lav glukose </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> L-Glutamin </td><td> Invitrogen </td><td> 25030 </td><td> 20 mm </td></tr><tr><td> Insulin / Transferrin / Selen </td><td> Invitrogen </td><td> 51500-056 </td><td> 100X </td></tr><tr><td> Fatty-syrefri BSA </td><td> Calbiochem </td><td> 126609 </td><td></td></tr><tr><td> NGF </td><td> Harlan Bioprodukter </td><td> 5017 </td><td></td></tr><tr><td> Ham F-12 næringsstoff medium (F-12) </td><td> Invitrogen </td><td> 11765 </td><td&gt; </td></tr><tr><td> Cytosin arabinoside </td><td> Sigma </td><td> C1768 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 356234 </td><td> Unngå gjentatte fryse-tine sykluser </td></tr><tr><td> BMP-7 </td><td> R &amp; D Systems Curis, Inc. </td><td> 345-BP BMP7-07H </td><td> 4 fil fra BMP, 5 og 6 fungerer like bra som BMP-7 og er noen ganger lettere å få tak i kommersielt </td></tr><tr><td> Paraformaldehyde </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Triton X-100 </td><td> Fisher </td><td> BP151-500 </td><td></td></tr><tr><td> Mus anti-rotte MAP2 antistoff </td><td> Sternberg Monoclonals Inc. </td><td> SMI52 </td><td> 1/5000 </td></tr><tr><td> Alexa Fluor 546 geit-anti-mus </td><td> Invitrogen </td><td> A11003 </td><td> 1/10000 </td></tr><tr><td> Mounting media </td><td> Forleng Antifade kit molekylære prober </td> &lt;td&gt; P7481 </td><td></td></tr></tbody></table> Tabell 1. Materialer

inducing dendritic growth in cultured sympathetic neurons

Formen på dendrittiske arbor avgjør det totale synaptiske inngang et nevron kan motta 1-3, og påvirker hvilke typer og fordeling av disse inngangene 4-6. Endrede mønstre av dendrittiske vekst og plastisitet er assosiert med nedsatt neurobehavioral funksjon i eksperimentelle modeller 7, og er tenkt å bidra til kliniske symptomer observert i begge nevrologisk lidelser 8-10 og nevrodegenerative sykdommer 11-13. Slike observasjoner understreker den funksjonelle betydningen av nettopp å regulere dendrittiske morfologi, og foreslår at identifisering mekanismene som styrer dendrittiske veksten ikke bare vil fremme forståelse for hvordan neuronal tilkobling er regulert under normal utvikling, men kan også gi innsikt om nye terapeutiske strategier for ulike nevrologiske sykdommer. Mekanistiske studier av dendrittiske veksten ville bli mye lettere å tilgjengeligheten ofa modell system som lar nerveceller kan eksperimentelt gått over fra en tilstand der de ikke strekker dendritter til en der de utarbeide en dendrittiske arbor sammenlignbar med deres in vivo kolleger. Primære kulturer av sympatiske nerveceller dissosiert fra overordnet cervical ganglier (SCG) av perinatale gnagere gi en slik modell. Når dyrket i definert medium i fravær av serum og ganglionic gliacellene, sympatiske nevroner forlenge en enkelt prosess som er aksonal, og dette unipolar tilstanden vedvarer i uker til måneder i kultur 14,15. Imidlertid utløser tillegg av enten Benmorfogenetiske protein-7 (BMP-7) 16,17 eller Matrigel 18 til kulturen medium disse nevronene å utvide flere prosesser som oppfyller de morfologiske, biokjemiske og funksjonelle kriterier for dendritter. Sympatiske nevronene dissosiert fra SCG av perinatale gnagere og voksne under definerte betingelser er en homogen befolkning av nevroner 19som reagerer likt for den dendrite-fremme aktivitet Matrigel, BMP-7 og andre fil fra BMP av decapentaplegic (DPP) og 60A underfamilier 17,18,20,21. Viktigere, oppstår Matrigel-og BMP-indusert dendrite formasjon i fravær av endringer i celle overlevelse eller aksonal vekst 17,18. Her beskriver vi hvordan du setter opp dissosiert kulturer sympatiske nerveceller avledet fra SCG av perinatale rotter slik at de er lydhøre for den selektive dendrite-fremme aktiviteten Matrigel eller fil fra BMP.

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

Inducing Dendritic Growth in Cultured Sympathetic Neurons

Vi beskriver en protokoll for å bruke Benmorfogenetiske protein-7 (BMP-7) eller Matrigel å selektivt indusere dendrittiske vekst i primærnæringene sympatiske nevroner dissosiert fra overordnet cervical ganglier (SCG) av perinatale rotter.

Inducing dendrittiske Veksten i dyrkede sympatiske Neurons

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 ...

Research

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