Name: three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells
Uploaded: 2015-10-10T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006; SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry. We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work.

1. Neuronal Kultur Bemærk: Fibroblast omprogrammering i pluripotente stamceller, engagement i dorsale telencephalon afstamning, afledning, forstærkning, og bankvæsen af sene kortikale stamceller (LCP), blev beskrevet i Boissart et al 4. Neuronal differentiering af LCP-lignende celler blev også udført ifølge Boissart et al 4 med mindre ændringer. Der er udviklet andre procedurer til direkte omprogrammering af fibroblaster i inducerede pluripotente ...

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Den kvantificering af de morfologiske træk af pyramideformede neuroner påberåbt softwaren. Glødetråden Tracer grænsefladen blev anvendt til segmentering af neuroner og pigge, og XT modulet blev anvendt til analysen. For at analysere nøjagtigheden af vores teknik, vi først sammenlignet de målte morfologiske parametre (længde, areal, og total rygsøjlen volumen, når det er relevant), med dem, der offentliggøres ved hjælp af rotte modne pyramideformede neuroner i kultur 6, 7</...

Dendritiske spidser af corticale pyramideneuroner er små og tynde fremspring, som er fordelt langs basale og apikale dendritter af disse neuronale undertyper i gnavere, primat, og den menneskelige hjerne. De er de steder, hvor de fleste excitatoriske synapser og vise nøglefunktioner i læring og kognitive processer. De detaljerede strukturer af humane dendritiske spines er teknisk studeret ved elektronmikroskopi 2. Men sådan tilgang er tidskrævende og repræsenterer tung arbejdsbyrde. For nylig er en tredimensional (3D) rekonstruktion af morfologien af dendritiske spidser er rapporteret i human hjernebark via bestemte software kombineret til store manuel rygsøjlen analyse 3. Grøn fluorescens protein (GFP) teknologi koblet til immunfluorescens repræsenterer et præcist værktøj til identifikation ryg og måling form ved fluorescensmikroskopi. Denne fremgangsmåde kan let anvendes til dyrkede neuroner. However, har ingen data er indberettet på analysen af ​​rygsøjlen modning og morfologi på humane neuroner afledt af inducerede pluripotente stamceller (IPSC). Formålet med denne undersøgelse var at beskrive en protokol, som gør det muligt dendritisk rygsøjlen billeddannelse fra dyrkede humane neuroner in vitro. GFP mærkning, konfokal mikroskopi og 3D-analyse med Filament Tracer modul Imaris software blev anvendt i den foreliggende protokol. Kultur trin, der er nødvendige for at opnå corticale neuroner glutamaterge af lag II til IV fra neurale stamceller (NSC) er også kort beskrevet her. Hele protokol for menneskelige NSC produktion er allerede blevet offentliggjort andre steder 4.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>PD-PBS (1X), sans Calcium, Magnesium et Phenol Red</td>
			<td>Gibco/ Life Technologies</td>
			<td>14190169</td>
			<td></td>
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			<td>Poly-L-Ornithine Solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>P4957</td>
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			<td>Mouse laminin</td>
			<td>Dutscher Dominique</td>
			<td>354232</td>
			<td></td>
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			<td>N2 Supplement</td>
			<td>Gibco/ Life Technologies</td>
			<td>17502048</td>
			<td></td>
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		<tr>
			<td>B-27 Supplement w/o vit A (50X)</td>
			<td>Gibco/ Life Technologies</td>
			<td>12587010</td>
			<td></td>
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		<tr>
			<td>DMEM/NUT.MIX F-12 W/GLUT-I</td>
			<td>Gibco/ Life Technologies</td>
			<td>31331028</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Med SFM</td>
			<td>Gibco/ Life Technologies</td>
			<td>21103049</td>
			<td></td>
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			<td>2-mercaptoethanol</td>
			<td>Gibco/ Life Technologies</td>
			<td>31350-010</td>
			<td></td>
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		<tr>
			<td>Pen-Steptomycin</td>
			<td>Gibco/ Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Rabbit Serum Polyclonal Antibody</td>
			<td>Gibco/ Life Technologies</td>
			<td>A-6455</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco/ Life Technologies</td>
			<td>16050130</td>
			<td></td>
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		<tr>
			<td>Alexa Fluor 488 Goat Anti-Rabbit&#160;</td>
			<td>Gibco/ Life Technologies</td>
			<td>A11034</td>
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			<td>Polyclonal Anti-betaIII tubulin antibody</td>
			<td>Millipore</td>
			<td>AB9354</td>
			<td></td>
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			<td>Coverglass 13 mm</td>
			<td>VWR</td>
			<td>631-0150</td>
			<td></td>
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		<tr>
			<td>Prolong Gold Antifade Reagent avec DAPI</td>
			<td>Gibco/ Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween(R) 20 Bioextra, Viscous Liquid</td>
			<td>Sigma Aldrich Chimie</td>
			<td>P7949</td>
			<td></td>
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		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich Chimie</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Fibroblasts</td>
			<td>Coriell Cell Line Biorepository</td>
			<td>GM 4603 and GM 1869</td>
			<td>Coriell Institute for Medical Research, Camden, NJ, USA</td>
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			<td>Confocal laser scanning microscope</td>
			<td>Zeiss (Germany)</td>
			<td>LSM 700</td>
			<td></td>
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		<tr>
			<td>Imaris Software</td>
			<td>Bitplane AG, Zurich</td>
			<td>6.4.0 version</td>
			<td>Filament Tracer and Imaris XT modules are necessary</td>
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		<tr>
			<td>Huygens Software</td>
			<td>Huygens software, SVI, Netherlands</td>
			<td>Pro version</td>
			<td>Optional (for deconvolution testing)</td>
		</tr>
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three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

Dendritiske spidser er små fremspring, der svarer til de post-synaptiske rum af excitatoriske synapser i centralnervesystemet. De er fordelt langs dendritter. Deres morfologi afhænger i høj grad neuronal aktivitet, og de er dynamiske. Dendritiske spines udtrykker glutamaterge receptorer (AMPA og NMDA-receptorer) på deres overflade, og på niveauet af postsynaptiske tætheder. Hver rygsøjlen tillader neuron til at styre sin tilstand og lokal aktivitet uafhængigt af hinanden. Spine morfologier er blevet grundigt undersøgt i glutamaterge pyramidale celler i hjernen cortex, ved hjælp af både in vivo fremgangsmåder og neuronale kulturer opnået fra gnaver væv. Neuropatologiske tilstande kan tilknyttes ændret rygsøjlen induktion og modning, som vist i gnavere dyrkede neuroner og endimensional kvantitativ analyse 1. Den foreliggende undersøgelse beskriver en protokol for 3D-kvantitativ analyse af rygsøjlen morfologier hjælp menneskelige corticAL neuroner afledt fra neurale stamceller (sene kortikale progenitorer). Disse celler blev oprindeligt fremstillet fra inducerede pluripotente stamceller. Denne protokol muliggør analysen af ​​rygsøjlen morfologier ved forskellige kultur perioder, og med mulighed for sammenligning mellem inducerede pluripotente stamceller opnået fra kontrolpersoner med dem, der opnås fra patienter med psykiatriske lidelser.

<html> Den foreliggende undersøgelse beskriver en standardiseret protokol for rygsøjlen kvantificering af dyrkede dendritter af pyramideformede neuroner afledt af iPSC. Denne protokol muliggør analysen af rygsøjlen modning på menneskelige neuroner og dens mulige sammenligning med modning af pigge i standard gnaver neuronkulturer samt i in vivo-dyremodeller. Figur 1A viser et skema over de forskellige trin i kultur, der tillader produktion af corticale pyramidene...

Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritiske spines af pyramideformede neuroner er de steder, hvor de fleste excitatoriske synapser i pattedyrs hjerne cortex. Denne metode beskriver en 3D-kvantitativ analyse af rygsøjlen morfologier i humane kortikale pyramideformet glutamaterge neuroner afledt af inducerede pluripotente stamceller.

Tre-dimensionel Kvantificering af dendritiske Spines fra pyramideneuroner Afledt af menneskeskabte pluripotente stamceller

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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