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><tbody><tr><td colspan="4">&lt;strong&gt;Slice Culture Medium&lt;/strong&gt;</td></tr><tr><td>Minimum Essential Medium</td><td>Invitrogen</td><td>11090</td><td>50%</td></tr><tr><td>Hank&amp;rsquo;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 colspan="4">Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection Buffer&lt;/strong&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>1.25mM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td>4.004mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7902</td><td>2mM</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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 colspan="4">Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O&lt;sub&gt;2&lt;/sub&gt; 5%CO&lt;sub&gt;2&lt;/sub&gt; before use</td></tr><tr><td colspan="4">&lt;strong&gt;Other Reagents and Supplies&lt;/strong&gt;</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&amp;mu;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% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt; 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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Colture organotipiche Slice di studi di post-natale neurogenesi

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Ricerca

Biologia dello sviluppo

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 ...

Neuroscienze

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunologia e infezioni

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. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Visualizzazione e Live Imaging del Oligodendrocyte organelli in fettine di cervello Organotypic utilizzando Virus Adeno-associati e microscopia confocale

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Preparazione e Immunostaining di su colture organotipiche Myelinating fetta cerebellare

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Ex Vivo Oculomotor Slice Coltura da embrionico GFP-Expressing Mice per Time-Lapse Imaging di Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Generazione di Oligodendrociti e Mezzo Condizionato oligodendrocito per Esperimenti Co-Culture

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

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