This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

NOTA: Tutte le procedure sugli animali stanno seguendo le linee guida e sono stati approvati dalla cura e uso degli animali Comitato Istituzionale (IACUC) presso l&#39;Università del Connecticut. NOTA: Per questi esperimenti costitutive NG2cre 18 (JAX # 008533) e inducibile NG2creER 16 (JAX # 008538) topi transgenici attraversato dalla A alla Z / EG 19 (JAX # 003920) o gtRosa26: YFP giornalista 20 (JAX # 006148) linee, rispettivamente, sono stati utilizzati di immagine cellule NG2 e la loro progenie. Per immagini oligodendrociti maturi, PLPDsRed topi transgenici 21 sono stati utilizzati. Per la sopravvivenza coerente, fette possono essere isolati da topi fino a 10 giorni dopo la nascita sia dal prosencefalo e il cervelletto. 1 fetta Preparazione <ol><li> In una cappa cultura, posizionare inserti di coltura di tessuti in sei pozzetti e aggiungere 1 ml di fetta di terreno di coltura (ricetta nella sezione reagenti). Mettere le piastre nell&#39;incubatore coltura cellulare a 37 ° C con 5% di CO 2 di almeno 2 ore prima della dissezione. </li><li> Sterilizzare tutti gli strumenti e chopper tessuto con il 70% di etanolo. </li><li> Tampone Bubble dissezione (ricetta nella sezione reagenti) con il 95% O 2, 5% di CO 2 in ghiaccio per almeno 15 minuti prima dell&#39;inizio della dissezione. </li><li> Anestetizzare cuccioli di topo mettendoli in ghiaccio per 5-10 minuti secondo protocolli approvati animali. Accertare la profondità appropriata di anestesia confermando che i topi non rispondono alla coda e pizzicare punta. </li><li> Decapitare animali utilizzando forbici affilate seguenti protocolli di animali. Rimuovere rapidamente il teschio sopra il proencefalo e cervelletto facendo prima un taglio sagittale seguita da incisione laterale, utilizzando strumenti sterilizzati. Tagliare nervi cranici dalla superficie ventrale del cervello e cervelletto rotolando attentamente il tessuto al lato. </li><li> Posizionare il tessuto in un piatto sterile 35 millimetri contenente tampone dissezione ossigenato ghiacciato. </li><li> Utilizzando una lama di rasoio, tagliare il cervelletto e proencefalo. Poi fare un taglio medio-sagittale through proencefalo e cervelletto, che separa gli emisferi. </li><li> Tagliare 300 micron di spessore sezioni coronali e sagittali del proencefalo e cervelletto con un elicottero del tessuto manuale. NOTA: Un elicottero tessuto manuale permette una rapida trasformazione da dissezione alla cultura incubazione senza la necessità di incorporamento agarosio. Un vibratome può anche essere usato come descritto in precedenza 9. </li><li> Trasferire le fette separate in appena bolle buffer di dissezione a freddo su ghiaccio. </li><li> Utilizzare piccola dissezione o di peso spatole per separare singole sezioni e trasferire loro di preincubato sei piatti e con inserti di cultura. </li><li> Rimuovere eventuali buffer di dissezione in eccesso dalla superficie dell&#39;inserto cultura con una pipetta di trasferimento monouso o un suggerimento P 200 pipetta. Da due a tre proencefalo o tre fettine di cervelletto può essere collocata in un inserto cultura. NOTA: Per i risultati coerenti con le culture del prosencefalo, è ideale per prendere le fette che attraversa il anteriore un terzo del corpo calloso(Figura 1A) al fine di studiare entrambe le regioni materia grigia e bianca nella stessa sezione. Ogni animale di solito produce 6 fette prosencefalo e 6 fette cervelletto. </li><li> Posizionare le sei pozzetti in incubatrice e sostituire il supporto fetta con 1 ml di terreno fetta 1 giorno dopo la preparazione fetta e poi a giorni alterni fino fissazione fetta. </li><li> A seconda dell&#39;esperimento, utilizzare fette dopo 5-7 giorni di coltura. Utilizzare solo le fette che diventano trasparenti dopo i primi giorni e scartare quelli che hanno le regioni opache irregolari. NOTA: le regioni opache all&#39;interno fette sono visibili ad occhio e appaiono come un grumo di cellule scuri sotto a contrasto di fase. Dopo che la tecnica è stato masterizzato, fette opache morti si verificano raramente, in meno del 1-5% di fette. </li></ol> 2 Time-Lapse Imaging di divisione delle cellule NG2 e Oligodendrocyte Differenziazione NOTA: Per eseguire lasso di tempo di imaging della proliferazione delle cellule NG2 e differenziazione usiamo NG2cre: ZEG topi 16che esprimono EGFP in cellule NG2 e la loro progenie (Figura 1C-E). Reporter espressione in questa linea è sufficientemente robusto per ottenere immagini in diretta di GFP + cellule in fettine subito dopo il sezionamento per un periodo di tempo di almeno quattro settimane. Le immagini più chiare sono ottenute dopo il periodo iniziale assottigliamento della fetta, che si verifica durante i primi 3-5 giorni di coltura. <ol><li> Durante l&#39;esperimento, mantenere le fette in un incubatore di coltura cellulare a 37 ° C e rimuovere solo per brevi periodi di tempo (meno di 15 min per fetta), mantenendo il coperchio sulla piastra ben sei durante l&#39;imaging. </li><li> Utilizzando un microscopio a fluorescenza invertito dotato di una fotocamera digitale, catturare immagini al primo punto temporale utilizzando un obiettivo 10X. NOTA: Primi punti di tempo può variare da esperimento e può essere il giorno in cui sono state preparate le fette o in qualsiasi punto nel tempo dopo. </li><li> Cattura immagini da più regioni in punto una volta in entrambe le zone grigie e bianche materia della fetta. Nota the regione ripreso da punti di riferimento specifici all&#39;interno la fetta e mappando la posizione dell&#39;immagine su uno schema che può essere utilizzato per le sessioni di imaging successive. Punti di riferimento utili possono includere bordi delle fette e / o l&#39;orientamento dei tratti di sostanza bianca. Immagine 4-8 posizioni su ogni fetta in ogni punto. </li><li> Cattura immagini successive con un intervallo di 4-6 ore se l&#39;esame di divisione delle cellule NG2 e la differenziazione degli oligodendrociti, idealmente più di 5-7 giorni. </li><li> Catturare un&#39;immagine finale di tutte le regioni e fissare le fette immediatamente. Aggiungere 1 ml di fissativo (4% PFA con 0.1M L-lisina 0,01 M di sodio meta-periodato) al fondo dell&#39;inserto cultura, quindi aggiungere altre 1 ml sopra le fette. Fare attenzione a non spruzzare il fissativo direttamente sulla fetta come si può staccare dalla membrana. Fissare il tessuto per 30 minuti e procedere con immunoistochimica utilizzando marcatori specifici stadi di sviluppo, come descritto nella sezione 4. </li><li> Fette Lavare con 0,2 M di sodio tampone fosfato 3 x 10 m. Se necessario, memorizzate fette a 4 ° C in 0.2 M tampone fosfato di sodio per 1-3 giorni prima di immunoistochimica ma idealmente eseguire colorazione entro 24 ore. Procedere con immunoistochimica come descritto di seguito nella sezione 4 per determinare la percentuale di cellule che si sono differenziate in oligodendrociti (Figura 2D-F). </li></ol> 3 Fate Mappatura di Progeny cellule NG2 Utilizzando topi inducibile Reporter transgenici nelle culture fetta NOTA: Per monitorare il destino delle cellule NG2 da un particolare punto di tempo nella cultura, inducibili NG2creER: possono essere utilizzati YFP topi transgenici. <ol><li> Indurre Cre ricombinazione e di espressione giornalista con l&#39;aggiunta di 100 4OHT nM disciolto in etanolo al mezzo di coltura. NOTA: cellule positive Reporter dovrebbero apparire entro 1-2 giorni ad una efficienza di induzione di ~ 20-25% YFP + NG2 + / NG2 + cellule ed a questo punto di tempo saranno tutti cellule in fase di cellule NG2 (Figura 2A-C) </li><li> Fissare e lavare la fettas in diversi momenti dopo varie manipolazioni (descritti nelle sezioni 2.5 e 2.6). </li></ol> 4 Slice Immunoistochimica <ol><li> Tagliare le membrane con le fette allegate fuori degli inserti utilizzando un bisturi. </li><li> Trasferimento fette ai singoli pozzetti di una soluzione salina 24 piatto contenente ben tamponata con fosfato (PBS) utilizzando un set di pinzette, facendo attenzione a non interrompere la composizione della fetta al momento del ritiro la membrana. </li><li> Trasferire le fettine su un piatto ben 24 con la soluzione bloccante contenente siero 5% di capra normale (NGS) e 0,1% Triton-X100 in PBS per 1 ora. </li><li> Incubare le fette in anticorpo primario O / N a 5% NGS in PBS a 4 ° C. Per identificare le cellule in fase di cellule NG2, utilizzare anticorpi anti NG2 e PDGFRα. Per identificare oligodendrociti differenziati, utilizzare un anticorpo per la poliposi adenomatosa dell&#39;antigene coli (APC; clone CC1). </li><li> Al secondo giorno fette di lavaggio in PBS 3 x15 min, poi incubare in Antibo secondariamuore in 5% NGS in PBS per 1 ora a temperatura ambiente. Lavare le fette in PBS 3 x15 min e montare su vetrini. Montare con le fette su e membrana di fronte alla diapositiva in modo che la membrana non sarà tra l&#39;obiettivo e il tessuto. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

The authors declare that they have no competing financial interests

Mielinizzazione nel sistema nervoso centrale è essenziale per un&#39;efficiente comunicazione neuronale e la sopravvivenza assonale 22. Cellule NG2 continuano a generare oligodendrociti mielinizzanti in età adulta, pur mantenendo una popolazione residente nella maggior parte delle regioni del cervello 16,23 - 25. Alcuni meccanismi genetici e molecolari che regolano il differenziamento di queste cellule sono state descritte, ma resta ancora molto da scoprire. Culture fetta organotipica sono un com...

Culture fetta Organoytpic del sistema nervoso centrale, hanno dimostrato di essere estremamente utile per lo studio dei neuroni e della biologia delle cellule gliali in un sistema semiintact 1 - 4. Queste culture sono relativamente semplici da adottare e mantenere molti benefici di colture primarie di cellule dissociate, come manipolazione dell&#39;ambiente extracellulare e di facile accesso per i ripetuti a lungo termine imaging cellulare dal vivo e registrazioni elettrofisiologiche 5-9. Inoltre, culture fetta mantengono citoarchitettura tessuto 3-dimensionale, connettività neurale regionale, e la maggior parte dei tipi di cellule principali sono presenti nel sistema. Queste proprietà fanno di queste culture un sistema unico e conveniente per studiare il comportamento di singole cellule e fisiologia con interazioni cellulari e ambientali. Cellule NG2 sono una popolazione di cellule gliali nel sistema nervoso centrale dei mammiferi che continuano a proliferare e generare myelinating oligodendrociti durante lo sviluppo embrionale e postnatale 10. Essi sono stati ampiamente studiati in colture cellulari primarie dissociate, e recente sviluppo di linee di topi transgenici con espressione specifica delle cellule NG2 di proteine ​​fluorescenti ha facilitato nella mappatura vivo destino e registrazioni elettrofisiologiche in fettine acute. Anche con questi studi, poco si sa circa le dinamiche temporali di proliferazione delle cellule NG2 e la differenziazione degli oligodendrociti. Sebbene coltura cellulare dissociata è ampiamente usato per la relativa facilità di manipolazioni farmacologiche e genetiche, non è adatto per interrogare differenze funzionali di queste cellule in differenti regioni cerebrali in particolare quando è desiderabile mantenere contesto del microambiente cellulare. Culture fetta forniscono una semplice alternativa che è suscettibile di manipolazioni farmacologiche e sono stati utilizzati per indagare oligodendrociti mielinizzazione 11,12, cellulerisposta colare dopo lisolecitina (LPC) o di anticorpi indotta demielinizzazione 13,14, e l&#39;induzione di rimielinizzazione tramite trattamento farmacologico 15. Un metodo per indagare ed eseguire l&#39;imaging dal vivo e tessuto fissato (o postfixation) analisi della proliferazione delle cellule NG2 e la differenziazione degli oligodendrociti in culture fetta organotipica prelevati sia dal prosencefalo e il cervelletto è descritto. Questo è un metodo potente che può essere utilizzato per studiare il destino delle cellule di singole cellule NG2 dopo la divisione 16 e per scoprire le differenze o una regione e di età-dipendente in fattore di crescita NG2 indotta proliferazione cellulare 17. Questo relativamente semplice tecnica è ampiamente accessibile di approfondire intrinseca e / o ambientale cella meccanismi che regolano la fisiologia di queste cellule gliali e la loro risposta all&#39;attività neuronale o danneggiamento della mielina.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

Cellule NG2 che esprimono (polydendrocytes, cellule precursori degli oligodendrociti) sono la quarta maggiore popolazione di cellule gliali nel sistema nervoso centrale. Durante lo sviluppo embrionale e postnatale che proliferano attivamente e generano oligodendrociti mielinizzanti. Queste cellule sono state comunemente studiate in colture primarie dissociate, co-colture di neuroni, e nel tessuto fisso. Utilizzo di recente disponibili linee di topi transgenici fetta sistemi di coltura possono essere utilizzati per studiare la proliferazione e la differenziazione delle cellule oligodendrociti lineage in entrambe le regioni grigi e bianchi questione di proencefalo e cervelletto. Culture fetta sono preparati dai primi topi postnatale e sono tenuti in coltura per fino a 1 mese. Queste sezioni possono essere esposte più volte nel corso del periodo di coltura per studiare il comportamento e le interazioni cellulari. Questo metodo permette la visualizzazione di divisione cellulare NG2 e le fasi che portano alla differenziazione degli oligodendrociti consentendo un&#39;analisi dettagliata della regione dipendeent cellule NG2 e oligodendrociti eterogeneità funzionale. Questa è una tecnica potente che può essere usato per studiare i segnali intrinseci ed estrinseci che influenzano queste cellule nel tempo in un ambiente cellulare che si avvicina a quella trovata in vivo.

Esempi di dati rappresentativi sono riportati qui di seguito, che sono stati ottenuti con culture fetta sia dal proencefalo e nel cervelletto di P8 NG2cre: ZEG, NG2creER: YFP e PLPDsRed linee di topi transgenici. Cellule NG2 possono essere esposte nei giorni in prosencefalo e cervelletto fette (Figura 1B-D, video 1) e il fenotipo di queste cellule può essere determinata dopo la fissazione e immunoistochimica con anticorpi NG2 e CC1 (Figura 1E). Oltre a vivere l&#39;imaging, la prolifer...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Research

JoVE Journal

Neuroscience

18.2K Views.  University of Connecticut. A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Video: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Il organotipica ippocampale Cultura Modello Slice per l&#39;esame di danno neuronale

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Ricerca

Neuroscienze

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipica Culture Slice ippocampale

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotipica Culture cerebellare: sfide apoptotiche e rilevamento

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Registrazione intera cella da un Preparazione Tagliare organotipica della neocorteccia

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-array di elettrodi Recordings Valanghe neuronali in Culture organotipica

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivazione di Arricchito Culture oligodendrociti e degli oligodendrociti / Neuron mielinizzanti co-culture di tessuti murini post-natale

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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.

L&#39;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

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

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

L&#39;analisi di neurovascolare ritocca in Entorhino-ippocampali Culture Organotipica Slice

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...