Name: purification of the dendritic filopodia-rich fraction
Uploaded: 2019-05-02T13:30:00
Description: Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

We bedanken Shigeo Okabe en Hitomi Matsuno voor de lage-densiteit cultuur van hippocampal neuronen, Masayoshi Mishina voor TLCN-deficiënte muizen, Sachiko Mitsui en Momoko Shiozaki voor technische assistentie, en leden van het Yoshihara laboratorium voor nuttige discussies . Dit werk werd gesteund door JSPS KAKENHI Grant NOS. JP20700307, JP22700354 en JP24500392 en MEXT KAKENHI Grant NOS. JP23123525 naar YF en JP20022046, JP18H04683, en JP18H05146 naar YY.

Alle hier beschreven methoden zijn goedgekeurd door het institutioneel Dierenzorg-en gebruiks Comité van RIKEN WAKO.
1. cultuur van Hippocampal neuronen
<ol><li>Bereiding van kweekmedium
	<ol>
<li>Bereiding van 200x vitamine mengsel. Los 100 mg van D-Pantotheenzuur hemicalcium zout, 100 mg choline chloride, 100 mg foliumzuur, 180 mg i-inositol, 100 mg niacinamide, 100 mg pyridoxal HCl, en 100 mg thiamine HCl in 500 mL ultrapuur water met behulp van een magnetische roerder. ...

<ol>
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We ontwikkelden een zuiveringsmethode voor de dendritische filopodia-rijke fractie met behulp van affiniteit tussen de celadhesie molecule TLCN en het extracellulaire matrix eiwit vitronectine. Vergeleken met de PSD-Fractie, kan het mogelijk zijn om de synaptische eiwitten te identificeren die op de onrijpe Synapse van de dendritische filopodia-rijke fractie optreden. De bestanddelen van de dendritische filopodia-Rich fractie verschillen dus van die van de PSD-fractie met 74%. Anders dan PSD-Fractie, gebruikten we gekwee...

Dendritische filopodia wordt beschouwd als voorlopers van stekels. Actin filamenten in de dendritische filopodia reguleren hun verlenging en terugtrekking1,2,3. Na contact met een axon beginnen geselecteerde dendritische filopodia hun rijping in stekels en wordt een synaps gevormd op4,5. Componenten van stekels zijn vastgesteld aan de hand van een uitgebreide analyse van postsynaptische dichtheids fracties6,7, terwijl de componenten van dendritische filopodia grotendeels onbekend blijven. Het is aangetoond dat telencephalin (tlcn), ERM, syngap, ras, PI3 kinase, Akt, mtor, Polo-achtige kinase 2, camkii, syndecan-2, paralemin-1, ARF6, en ephb reguleren dendritische filopodia vorming5,8,9 ,10,11, terwijl een methode niet is ontwikkeld voor de uitgebreide analyse van moleculen die in de dendritische filopodia aanwezig zijn.
Tlcn (ICAM-5) wordt specifiek uitgedrukt door stekelige neuronen in het meest rostrale migratoire hersen segment, de telencephalon12. TLCN heeft 9 Ig-achtige domeinen in de extracellulaire regio, een transmembraan regio, en een cytoplasmische staart13. Tlcn bindt aan vitronectine (VN) en LFA-1 integrine in zijn extracellulaire regio, aan presenilin in zijn transmembraan regio, en aan fosho-ERM en α-actinine in de cytoplasmische regio5,8,14,15 ,16. Tlcn bindt aan de actine cytoskelet via fosho-ERM op de uiteinden van dendritische filopodia en α-actinine in stekels en dendritische assen8,16.
We toonden aan dat overexpressie van tlcn verbeterde dendritische filopodia-formatie en de reversie van stekels veroorzaakte tot filopodia10. De constitutieve actieve vorm van Ezrin gebonden aan de TLCN cytoplasmatische regio en verbeterde dendritische filopodia formatie8. Zo reguleert TLCN de vorming van dendritische filopodia door middel van actin-bindende eiwitten. Esselens et al. heeft aangetoond dat microbeads de accumulatie van TLCN op gekweekte neuronen17induceerde. We toonden aan dat de fagocytische beker structuren werden gevormd op neuronale dendrites rond VN-gecoate microbeads in een TLCN-afhankelijke manier15. Bestanddelen van dendritische filopodia lijken op die van de phagocytische beker. Het is moeilijk om dendritische filopodia te verzamelen, maar het is relatief gemakkelijker om de phagocytische beker te verzamelen met behulp van magnetische microbeads. Zo ontwikkelden we een methode om de fagocytische beker te zuiveren in plaats van dendritische filopodia18. Hier beschrijven we de zuiveringsmethode voor de dendritische filopodia-rijke Fractie.

<table>
	<tbody>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml Low Binding MCT</td>
			<td>Sorenson BioScience</td>
			<td>39640T</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>2530149</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic cell culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-actin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-Actinin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T-9026</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ezrin</td>
			<td>Sigma-Aldrich</td>
			<td>clone3C12, SAB4200806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Galphaq</td>
			<td>Santacruz</td>
			<td>sc-393</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>Chemicon</td>
			<td>clone AP20, MAB3418</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Moesin</td>
			<td>Sigma-Aldrich</td>
			<td>clone 38/87, M7060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PLCbeta1</td>
			<td>Santacuz</td>
			<td>sc-5291</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PSD95</td>
			<td>MA2</td>
			<td>ABR</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Spectrin beta</td>
			<td>Chemicon</td>
			<td>MAB1622</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Merck</td>
			<td>1.08122.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chrolide, hydrous</td>
			<td>Wako</td>
			<td>038-19735</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine beta-D-arabinofuranoside</td>
			<td>Sigma-Aldrich</td>
			<td>C-6645</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase-I</td>
			<td>Sigma-Aldrich</td>
			<td>DN-25</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P5155</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Thermo</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Prime Western Blotting Detection Reagent</td>
			<td>GE</td>
			<td>RPN2232</td>
			<td></td>
		</tr>
		<tr>
			<td>e-PAGEL 5-20% SDS-PAGE gradient gel</td>
			<td>ATTO</td>
			<td>E-T520L</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8758</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr>
			<td>i-Inositol</td>
			<td>Sigma-Aldrich</td>
			<td>I7508</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-1000 mini</td>
			<td>Fuji Film</td>
			<td>LAS-1000 mini</td>
			<td>For detection of luminescence from WB membrane</td>
		</tr>
		<tr>
			<td>Magnetic polystyrene microbeads</td>
			<td>Sperotech</td>
			<td>PM-20-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acid solution</td>
			<td>Gibco</td>
			<td>11130-051</td>
			<td>30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H2O, 20 mM L-Isoleucine, 20 mM L-Leucine, 19.8 mM L-Lysine hydrochloride, 5.1 mM L-Methionine, 10 mM L-Phenylalanine, 20 mM L-Threonine, 2.5 mM L-Tryptophan, 10 mM L-Tyrosine, and 20 mM L-Valine</td>
		</tr>
		<tr>
			<td>Mini-slab size electrophoresis system</td>
			<td>ATTO</td>
			<td>AE-6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Niacinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin / Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP phosphatase inhibitor cocktail</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Nacali</td>
			<td>28360-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P6155</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Sigma-Aldrich</td>
			<td>R9504</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain 2 Kit wako</td>
			<td>Wako</td>
			<td>291-5031</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-rad</td>
			<td>1703940JA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water</td>
			<td>MilliQ</td>
			<td></td>
			<td>For production of ultra pure water</td>
		</tr>
	</tbody>
</table>

purification of the dendritic filopodia-rich fraction

Dendritische filopodia zijn dunne en lange uitsteeksels op basis van de actine filament, en ze verlengen en intrekken alsof ze zoeken naar een doel-axon. Wanneer de dendritische filopodia contact leggen met een doel-axon, beginnen ze te rijpen in stekels, wat leidt tot de vorming van een synaps. Telencephalin (TLCN) is overvloedig gelokaliseerd in dendritische filopodia en wordt geleidelijk uitgesloten van stekels. Overexpressie van TLCN in gekweekte hippocampal neuronen induceert dendritische filopodia vorming. We toonden aan dat telencephalin sterk bindt aan een extracellulair matrix molecuul, vitronectine. Vitronectin-gecoate microbeads geïnduceerde fagocytische beker vorming op neuronale dendrieten. In de fagocytische beker worden TLCN, TLCN-bindende eiwitten zoals gefosforyleerd Ezrin/Radixin/Moesin (phospho-ERM) en F-actin verzameld, wat suggereert dat componenten van de phagocytische beker vergelijkbaar zijn met die van dendritische filopodia. Zo ontwikkelden we een methode voor het zuiveren van de fagocytische beker in plaats van dendritische filopodia. Magnetische polystyreen kralen werden bekleed met vitronectine, die overvloedig aanwezig is in het cultuurmedium van hippocampal neuronen en die fagocytische beker vorming op neuronale dendrieten induceert. Na 24 uur incubatie werden de fagocytische bekers mild gesolubiliseerd met wasmiddel en verzameld met behulp van een Magneet separator. Na het wassen van de kralen werden de bindende eiwitten geeluteerd en geanalyseerd door zilver kleuring en Western blotting. In de bindende fractie waren TLCN en actine overvloedig aanwezig. Bovendien werden veel eiwitten die uit de Fractie werden geïdentificeerd, gelokaliseerd in de dendritische filopodia; zo noemden we de bindende fractie als de dendritische filopodia-rijke Fractie. Dit artikel beschrijft de details met betrekking tot de zuiveringsmethode voor de dendritische filopodia-Rich Fractie.

In gekweekte hippocampal neuronen, TLCN was overvloedig gelokaliseerd aan de dendritische filopodia, schacht, en Soma en cogelokaliseerd met F-actin (Figuur 1a, B). Wanneer polystyreen microbeads werden toegevoegd aan gekweekte hippocampal neuronen, werden de parels automatisch bekleed met vitronectine (VN) afgeleid van foetaal runderserum (FBS) in het kweekmedium; ze waren voornamelijk gebonden aan dendrites, en ze veroorzaakte de vorming va...

Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

Purification of the Dendritic Filopodia-rich Fraction

In dit protocol introduceren we een methode voor het zuiveren van de dendritische filopodia-rijke Fractie van de phagocytische beker-achtige uitsteeksel structuur op gekweekte hippocampal neuronen door gebruik te maken van de specifieke en sterke affiniteit tussen een dendritische filopodial adhesie molecuul, TLCN, en een extracellulaire matrix molecuul, vitronectine.

Zuivering van de dendritische Filopodia-rijke Fractie

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

In vergelijking met psd-fractie kan het mogelijk zijn om het synaptische eiwit te identificeren dat werkt op de onrijpe synaps rond de dendritische filopodia-rijke fractie. We gebruikten levend neuron om fagocytische cap vorming te induceren. Het belangrijkste voordeel van deze techniek is dat actief eiwit in kunstmatige ontstaan kan worden geïdentificeerd uit de dendritische filopodia rijke fractie.Om te beginnen, bereid 200x vitamine mix door het oplossen van 100 mg vitamine B5, 100 mg Choline Chloride, 100 mg foliumzuur, 180 mg I-inositol, 100 mg vitamine B3, 100 mg vitamine B6chloride hydro, en 100 mg thiaminechloride in 500 ml ultrazuiver water met behulp van een magnetische roerder. Meng aliquot voorzichtig in buizen van 50 ml en bewaar het op 20 graden Celsius. Voor het bereiden van riboflavine oplossing, los 100 mg riboflavine in 500 mg ultrazuiver water met behulp van een magnetische roerder.Meng aliquot voorzichtig in buizen van 50 ml en bewaar op 20 graden Celsius. Voor te bereiden een molaire calciumdichloride oplossen 7,35 gram calciumchloride dihydraat in 50 ml ultrazuiver water met behulp van een magnetische roerder. Los voor minimum essential medium preparaat 400 mg kaliumchloride 6 op; 800 mg sodumchloride, 2, 200 mg natriumbicarbonaat, 158 mg natriumfosfaatmonobasisch dihydraat, 7, 000 mg D-glucose en 200 mg magnesiumsulfaat heptahydraat en 950 ml ultrazuiver water, met behulp van een magnetische roerder.Gebruik vervolgens een pipet van 1 ml met een constante agitatie op een magnetische roerder om 1,8 ml calciumdichloride van 1 molaire calcium in een druppel voor druppel aan het MEM toe te voegen. Voeg vervolgens zoutzuur toe om de pH van het MEM aan te passen aan pH 7,25. Voeg vervolgens 5 ml vitaminemix van 200x en 200 microliter riboflavine aan het MEM toe.Stel het volume van de oplossing in op 1000 ml met ultrazuiver water. Filter de oplossing met een filtersysteem van 0,22 micron en bewaar deze op 4 graden Celsius. Voor de bereiding van 10x DNase-1 voorraadoplossing los 100 mg Dnase-1 op in 12,5 ml HBSS.Filtreer door een filter van 0,22 micron, aliquot in buizen van 1,5 ml en bewaar de buizen op 20 graden Celsius. Los voor Ara-C voorraadoplossing 25 mg Ara-C op in 8,93 ml ultrazuiver water. Filtreer door een filter van 0,22 micron, aliquot in 1,5 ml buizen en bewaar op 20 graden Celsius.Ter voorbereiding van het Plating Medium, meng 1 ml MEM Aminozuuroplossing, 750 microliter van 1 molaire HEPES, 1 ml B27, 125 microliter 200 millimolar glutamine, 250 microliter penicilline-streptomycine, 2,5 ml Fetal Bovine Serum en 44,375 ml MEM in een buis van 50 ml. Om het stopmedium voor te bereiden, meng je 1 ml MEM-aminozuuroplossing, 750 microliter van 1 molaire HEPES, 5 ml FBS en 43,25 ml MEM in een buis van 50 ml. Voor de bereiding van HBSS, aangevuld met 15 millimolar HEPES, meng 49,25 ml HBSS en 750 microliters van 1 kies HEPES.Voor poly-l-lysine gecoate gerechten bereiding, jas 35 mm plastic cel kweek gerechten met 0,2 mg per ml poly-l-lysine hydrobromide voor een dag op 25 graden Celsius. Was vervolgens de afwas met 2 ml ultrazuiver water drie keer en broed ze uit met 1,5 ml Stop Medium bij 25 graden Celsius tot gebruik. Incubeer de ontleed hippocampi in een mengsel met 500 microliters elk van 2,5% trypsine en 10X Dnase-1 in HBSS aangevuld met 15 millimolar HEPES, pH 7.2, bij 37 graden Celsius gedurende 15 minuten met agitatie om de 3 minuten.Breng de hippocampi vervolgens 5 minuten over op 10 ml van het stopmedium en broed op 4 graden Celsius om trypsine te inactiveren. Vervolgens, incubeer de ontleed hippocampi in 10 ml vers stopmedium bij 4 graden Celsius gedurende 5 minuten. Verplaats hippocampi in 10 ml vers stopmedium en incubbate nog 5 minuten bij 4 graden Celsius.Verplaats de hippocampi vervolgens in 900 microliter van het stopmedium en 100 microliter van 10X Dnase-1 in een buis van 15 ml. Gebruik een pipet van 1 ml om de hippocampi te scheiden in geïsoleerde neuronen door 20 keer te pipetten. Voeg 9 ml beplatingsmedium toe en filtreer door een 70 micron celzeef in een buis van 50 ml.Tel het aantal cellen met behulp van een hemocytometer en een aanpassing tot 3,5 keer 10 tot de vierde cellen per milliliter in beplating medium. Vervolgens, aspirate het stopmedium van de poly-l-lisine gecoate gerechten. Plaat 2 ml per schotel van de cellen op de gecoate gerechten en incubeer onder 5%kooldioxide bij 37 graden Celsius gedurende 60-64 uur.Voeg na de incubatie 2 microliters Ara-C-voorraadoplossing toe aan de neuronen en schud de schotel langzaam. Bewaar de kweekgerechten in een bevochtigde doos zonder het kweekmedium onder de 5% kooldioxide te veranderen bij 37 graden Celsius. Zuiver de dendritische filopodia rijke fractie na 13 dagen in vitro door eerst drie miljoen magnetische polystyreen microbeads per gerecht toe te voegen aan 20 gerechten met de gekweekte neuronen.Na een dag, was de neuronen in 1 ml PBS met agiatie drie keer om de medium en ongebonden microbeads te verwijderen. Na het verwijderen van PBS, lyse de neuronen met 500 microliter per schotel van de lyse buffer. Verzamel vervolgens het lysaat met een celschraper en breng het lysaat over in 10 eiwitarme bindende microbuisjes.Zet de buizen op een magneet separater en wacht een minuut. Verzamel de supernatant en gebruik het als de niet-gebonden fractie voor zilver vlekken en westerse vlek analyse. Breng de kralen vervolgens over naar een nieuwe eiwitarme bindende microbuis en zet ze een minuut op een magnetische separater.Verwijder de supernatant volledig en voeg 500 microliter van de lysebuffer toe. Was de kralen met behulp van een Vortex mixer gedurende 15 seconden. Herhaal het wassen van de kralen 10 keer en verwijder de supernatant.Elute eiwitten tot aan de kralen door de toevoeging van 50 microliters van 1X SDS monster buffer en kook de buis op 98 graden Celsius gedurende 5 minuten. Centrifuge de buis op 860xG 3000 RPM gedurende 10 seconden en zet de buis op een magnetische separater voor een minuut. Verzamel de supernatant en gebruik het als de gebonden fractie.Meet de concentraties van de niet-gebonden eiwitfracties door de BCA-eiwittest. Visualiseer protien-oplossingen met bromfylblauw en pas de concentratie aan op 5 nanogram per microliter voor SDS-pagina. Scheid de gebonden en niet-gebonden breuken per SDS-pagina met behulp van een verloopgel van 5-20%.Zilver vlek de gel. Ten slotte, westerse vlek met behulp van de juiste primaire en secundaire antilichamen volgens het manuscript. In gekweekte hippocampal neuronen, TLCN was overvloedig gelokaliseerd om de dendritische filopodia, schacht, en soma en co-gelokaliseerd met F-actine.De gecodeerde kralen waren voornamelijk gebonden aan dendrieten en induceren de vorming van fagocytische bekers door accumulatie van TLCN en F-actin op neuronale dendrieten. Thorecense beelden toonden aan dat fagocytische bekervorming was van cruciaal belang afhankelijk van de aanwezigheid van TLCN in dendrieten. De fagocytische cups werden alleen gevormd op wilde type hippocampal neuronen, maar niet op TLCN defficient hippocampal neuronen.SDS pagina resultaten toonden aan dat de eiwit band patronen waren bijna hetzelfde voor de niet-gebonden en gebonden fracties, maar de intensiteiten op 50 en 70 kilodalton, in de gebonden fractie waren lager dan die in de niet-gebonden fractie. Echter, de band intensiteit was niet duidelijk verschillend tussen de niet-gebonden en gebonden fracties bereid van TLCN defficient cultuur hippocampal neuronen. Uit de westerse vlekanalyse van de niet-gebonden en gebonden fracties bleek dat TLCN en VN vooral in de gebonden fractie werden gedetecteerd.Actin, Ezrin, G alpha q, PLC beta 1, MAP-2 en Spectrin, werden gedetecteerd in zowel de gebonden als niet-gebonden fracties. Moesin, PSD-95, alpha-Actinin en alpha-Tubulin werden in de niet-gebonden fractie gedetecteerd. Als coatingeiwit van de microbeads verandert, kan deze procedure worden toegepast om de interactie met de test te identificeren.Deze techniek kan het eiwit identificeren dat op kunstmatige stoffen werkt. Dus we geloven dat mechanisme kunstmatige ontstaan kan worden gekwantificeerd. Wij geloven dat nieuwe eiwitten geassocieerd met psychische stoornis kan worden geïdentificeerd uit de dendritische filopodia-rijke fractie.En kan worden uitgebreid tot de therapie.

purification-of-the-dendritic-filopodia-rich-fraction

Research

JoVE Journal

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Analyse van de dendritische Spine Morfologie in gekweekte CNS Neuronen

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic 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 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

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 ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

De Analyse van de Purkinje cel dendritische morfologie in organotypische slice culturen

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Snel Micro-iontoforese van glutamaat en GABA: een nuttig instrument om Synaptic Integratie Onderzoek

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Beoordeling van Dendritische arborization in de gyrus dentatus van de hippocampus regio in Muizen

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Zuivering van Mouse Brain Schepen

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Het analyseren van dendrietmorfologie in Kolommen en Lagen

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Beoordeling van Hippocampale Dendritische Complexiteit in Oude Muizen Met behulp van de Golgi-Cox Methode

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

Real-time iontophorese met tetramethylammonium om volume-fractie en tortuositeit van de extracellulaire ruimte van de hersenen te kwantificeren

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Kwantitatieve analyse van neuronale dendritische Arborization complexiteit in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...