Ringraziamo N. Schwedhelm-Domeyer per l&#39;eccellente assistenza tecnica, C. Martello e S. Papiol per un aiuto con analisi statistiche. Il nostro lavoro è finanziato dalla Max Planck Society, la Deutsche Forschungsgemeinschaft, il Center for Nanoscale Microscopia e Fisiologia Molecolare del cervello (CNMPB), Göttingen, Germania e dal GGNB Junior Gruppo Stipend dell&#39;Università di Göttingen.

CGN possono essere preparati sia dal primo giorno post-natale (P) 5 cuccioli di topo o cuccioli di ratto P6. Seguiamo un protocollo, descritto da Bilimoria e colleghi, che utilizza un inibitore mitotico per selezionare per CGN postmitotic 13. Etica dichiarazione: Tutti gli esperimenti che coinvolgono animali vivi sono stati condotti secondo il protocollo degli animali, approvato dal &quot;Verbraucherschutz und Lebensmittelsicherheit&quot; della Bassa Sassonia, in Germania. Nel test in vitro: 1. Preparazione di plasmidi DNA, Media e buffer per il fosfato di calcio Metodo Transfezione <ol><li> Sciogliere plasmide DNA in acqua privo di endotossine sterile; DMEM (alti livelli di glucosio); fanno 2,5 M CaCl 2; fare 2x HBSS (sciogliere 4 g NaCl, 0,1775 g KCl, 0.095 g di Na 2 HPO 4 • 7H 2 O, 0,675 g di glucosio e 2,5 g HEPES in 250 ml di acqua ultrapura e aggiustare il pH a 7.05, 7.08 e 7.11). Nota: Quando si preparala soluzione HBSS 2x, pH test che dà i migliori risultati per quanto riguarda l&#39;efficienza di trasfezione di data combinazione di plasmidi. </li></ol> 2. Trasfezione di colture di neuroni <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig1highres.jpg" src="/files/ftp_upload/51070/51070fig1.jpg" /> Figura 1. Diagramma di flusso di assoni in vitro e dendrite saggio di crescita. Coltivate CGN (piastra da 24 pozzetti con vetrini), isolato da P6 cuccioli di ratto, sono trasfettati in DIV 0 o 1 con precipitato DNA contenente un marcatore fluorescente trasfezione (es. GFP). Dopo la fissazione e immunocitochimica, i neuroni vengono esposte in modo cieco. Le immagini vengono importati in ImageJ e processi sono misurati. Le misurazioni vengono quindi elaborati utilizzando un programma statistico. <ol><li> CGN Seed (20 x 10 6 per 24 pozzetti, BME, siero di vitello al 10%, 2 mM penicillina-Streptomycin-Glutammina (PSG), 25 mM KCl) in acido nitrico-lavato, polyornithine rivestite 12 millimetri vetrini in una piastra da 24 pozzetti con 500 microlitri di media per pozzetto. </li><li> Il giorno in vitro (DIV) 0 (almeno 8 ore dopo la placcatura) o DIV 1, raccogliere i media di crescita e di mantenere a 37 ° C. Lavare i neuroni due volte con 500 ml di DMEM preriscaldata e aggiungere 500 ml di DMEM. </li><li> Inserire neuroni in incubatore (37 ° C, 5% CO 2) per 45 min. </li><li> Preparare 40 ml precipitato di DNA per ogni ben mescolando: DNA (2-2,5 mg / bene, il 10% del DNA totale dovrebbe essere un marcatore di transfezione es GFP per visualizzarne i neuroni trasfettati), acqua (fino a 18 ml), aggiungere 2 ml di 2,5 M CaCl 2, mescolare bene e aggiungere 20 ml di 2x HBSS. <ol><li> Incubare DNA precipitato per 5 minuti a temperatura ambiente. </li></ol></li><li> Aggiungi precipitato DNA a ciascun pozzetto e incubare i neuroni per 18 min in incubatrice. </li><li> Rimuovere mix DMEM / DNA e lavare i neuroni due volte con 500 ml di preriscaldata DMEM. </li><li> Aggiungere raccolta multimediale dal punto 2.2 torna ai neuroni. Se i neuroni saranno in coltura per più di 3 giorni, i media supplemento con glucosio 25 mm a DIV 3 per ricostituire fonte di carbonio. </li><li> Dopo 1-5 giorni, neuroni sottoposti a immunocitochimica utilizzando anticorpi GFP. </li><li> Immagine almeno 30 neuroni individuali a condizione in modo cieco utilizzando un microscopio a fluorescenza. </li></ol> 3. Misurare assoni e dendriti con NeuronJ, un NIH ImageJ Plugin Importante: Assicurarsi che le immagini vengono ridimensionate correctluy utilizzando appropriate pixel: rapporto micron a seconda di ingrandimento e la risoluzione dell&#39;immagine. <ol><li> Converti immagini a 8-bit con ImageJ: Apri immagine, selezionare &#39;Immagine&#39; -&gt; &#39;Tipo&#39; -&gt; &#39;8 bit &#39;-&gt;&#39; Salva immagine &#39;. </li><li> Eseguire NeuronJ plug-in e aprire l&#39;immagine a 8 bit. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot1.jpg" width="500px" /></li><li> Utilizzare l&#39;opzione &#39;Aggiungi&#39; tracciati per monitorare l&#39;assone: click il pulsante sinistro del mouse una volta all&#39;inizio dell&#39;assone e spostare il mouse lungo il processo. Fare doppio click sulla punta dell&#39;assone se traccia corrisponde a forma assonale. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot2.jpg" width="500px" /> Nota: Se la traccia suggerita differire dalla forma assonale, fare clic una volta sul processo assonale per ancorare traccia, fare doppio clic sulla punta dell&#39;assone. </li><li> Clicca su &quot;tracciati misura&quot;, scegliere il &#39;Display misurazioni tracciamento&#39; opzione e premere &#39;Esegui&#39;. Misurazioni Axon sono tutti visualizzati in una nuova finestra. Per i dendriti, scegliere l&#39;opzione &#39;misure gruppo di visualizzazione&#39; e premere &#39;Esegui&#39;. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot3.jpg" width="500px" /> Misurazioni dendriti totali sono tutti visualizzati in una nuova finestra. Salvarli come file separato che può essere aperto in qualsiasi programma di foglio di calcolo. </li><li> In alternativa, per il tracciamento manuale, utilizzare il software Fiji: clic destro su °e l&#39;opzione &#39;retta&#39;, scegliere &#39;la linea a mano libera&#39;, <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot4.jpg" width="500px" /> mantenere il tasto sinistro del mouse e tracciare manualmente il processo, premere &#39;Ctrl + M&#39; per misurare. </li><li> Calcola lunghezza / assonale media dendritiche per condizione e utilizzare test statistico appropriato. In elettroporazione in vivo: </li></ol> 1. Attrezzature e Preparazione dei reagenti <ol><li> Avete bisogno di 30 aghi G, distanziale (1-2 mm), siringa, riduttore di volume morto (DVR), elettroporatore e tweezertrodes, piastra elettrica o lampada di calore a raggi infrarossi, lampada a collo d&#39;oca e isoflurano. </li><li> Mettere DVR in dell&#39;ago, quindi collegare l&#39;ago alla siringa, e, infine, mettere distanziale sulla ago (Figura 2). <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig2highres.jpg" src="/files/ftp_upload/51070/51070fig2.jpg" /> Figura2. Preparazione dell&#39;ago. DVR è tagliata una punta di pipetta 200 microlitri e collocato all&#39;interno dell&#39;ago per ridurre il volume morto. Spaziatore è derivato da una punta di 200 microlitri di carico e viene posizionato sull&#39;estremità dell&#39;ago per regolare la profondità di penetrazione nel cervelletto di circa 2 mm. Unità righello: cm </li><li> Sciogliere DNA in PBS/0.03% Verde veloce. Nota: Come marcatore trasfezione, è vantaggioso utilizzare una proteina fluorescente che è sotto un promotore neurone-specifica (ad es Synapsin) per visualizzare solo i neuroni. 25% del totale plasmide dovrebbe essere il plasmide codificante il marcatore trasfezione. </li><li> Effettuare il 70% di etanolo. </li><li> Miscelare volumi uguali di ottobre e del 30% di saccarosio disciolto in PBS. </li><li> Riempire la siringa con 4 ml di DNA (4 mg / mL di DNA plasmidico in PBS/0.03% rapidi verde). </li></ol> 2. IVE di Rat Pups Diagramma di flusso di IVE: si veda la Figura 3 <p class="jove_content" fo:keep-together.within-Page = &quot;always&quot;&gt; <img alt="Figura 3" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig3highres.jpg" src="/files/ftp_upload/51070/51070fig3.jpg" /> Figura 3. Diagramma di flusso di elettroporazione in vivo. P4 cuccioli di ratto sono anestetizzati con isoflurano e DNA plasmidico codificante un marcatore fluorescente trasfezione (es. GFP) viene iniettato nel cervelletto, seguito da esposizione a 5 impulsi elettrici. Cinque giorni dopo, isolato cerebella GFP-positive sono sezionato e sottoposto a immunoistochimica. Le immagini sono state acquisite utilizzando un microscopio confocale e analizzati utilizzando il software Imaris. I dati sono trattati con un programma statistico. <ol><li> Utilizzare P4 cuccioli di ratto dal ceppo albino (Wistar o Long Evans). </li><li> Anestetizzare cuccioli (uno dopo l&#39;altro) con isoflurano in piccola scatola (ad esempio P1000 box puntale) con 200 ml di isoflurano (imbevuto in tessuto) per 1-2 minuti fino a quando cucciolo non è più in movimento. Fate attenzione che i cuccioli non entrare in contatto wesima Iiquido isoflurano. Monitorare il tempo strettamente individuali cuccioli rispondono diversamente ad anestesia. </li><li> Sterilizzare posteriore della testa del cucciolo con il 70% di etanolo. </li><li> Fissare capo del pup tra il pollice e l&#39;indice e utilizzare la lampada a collo d&#39;oca per individuare cervelletto del pup albino. Il seno trasverso delimita nettamente il mesencefalo (collicolo superiore ed inferiore) a partire da emisferi corticali (Figura 3). Il cervelletto si trova adiacente al mesencefalo e appare in una tonalità più scura. Utilizzare un pennarello indelebile per indicare il cervelletto con un punto. Importante: Conservare pup in posizione fissa! Nota: In caso di anestesia indossare durante questa procedura, esporre cucciolo di isoflurano prima di iniettare il DNA. </li><li> Inserire l&#39;ago (figura 3) e iniettare lentamente 3 ml di DNA nel cervelletto. </li><li> Lasciate che la soluzione di DNA diffusa per 30-60 sec. <ol><li> Inserire la testa del cucciolo tra tweezertrodes in modo che il polo negativo a contatto con la parte posteriore della testa (cerebellaregione r) e che il polo positivo contro il lato opposto della testa (Figura 3). </li><li> Oggetto pup a 5 impulsi elettrici. Regolare la tensione al peso dei cuccioli di garantire una buona efficienza elettroporazione senza compromettere la loro sopravvivenza (Tabella 1). <table border="0"><tbody><tr><td> Peso </td><td> Tensione </td><td> Impulso </td><td> Intervallo </td></tr><tr><td> 8-9 g </td><td> 160 V </td><td> 50 msec </td><td> 950 msec </td></tr><tr><td> 9-10 g </td><td> 165 V </td><td> 50 msec </td><td> 950 msec </td></tr><tr><td> &gt; 10 g </td><td> 170 V </td><td> 50 msec </td><td> 950 msec </td></tr></tbody></table> Tabella 1. Elettroporazione di P4 cuccioli di ratto. </li><li> Lasciate cuccioli recuperare il pad riscaldato o sotto una lampada a raggi infrarossi. Cuccioli di diga di ritorno. Importante: Assicurarsi che fonte di riscaldamento non infliggere ustioni. </li></ol></li><li> Sacrifica cuccioli 5 ​​giorni dopo l&#39;elettroporazione mettendoli in CO 2, seguita da decapitazione. <ol><li> Isolare il cerebella e lo schermo per quelli GFP-positive cerebella utilizzando un microscopio a fluorescenza. </li></ol></li><li> Fix cerebella in 4% PFA O / N a 4 ° C, poi incubare in saccarosio al 30% a 4 ° C fino cerebella lavello a fondo della provetta. </li><li> Incorpora cerebella in OCT/30% di saccarosio e tagliare 40 micron sezioni coronali utilizzando un criostato. Nota: Tratto ogni cervelletto in modo cieco. </li><li> Sezioni soggette a immunoistochimica con l&#39;anticorpo GFP. Controcolorare con colorante nucleare (DAPI o Hoechst 33258) e determinare la localizzazione di almeno 200 neuroni trasfettati per animale. </li><li> Per un&#39;analisi approfondita, suddividere il IGL in due metà, con un conseguente IGL superiore di fronte alla ML e una IGL minore di fronte alla materia bianca e contare i neuroni GFP-positive che risiedono in ogni tempo. Nota: Count neuroni GFP-positive di ogni sezione in modo cieco. </li&gt; </ol> 3. Misurare Dendrite intera, Acquisire le immagini della sezione in x, y, z aereo utilizzando un microscopio confocale Nota: per esempio, utilizzare 40 immagini per una sezione di 40 micron con una z-stwp di 1 micron. <ol><li> Serie di immagini Open in software, Imaris, per generare una immagine 3D di dendriti. </li><li> Clicca su modalità &#39;Superare&#39; per vedere il neurone in 3D. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot5.jpg" width="500px" /></li><li> Selezionare &#39;Aggiungi nuovo filamento&#39; e cliccare su &#39;Skip creazione automatica&#39; per avviare l&#39;analisi semiautomatica. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot6.jpg" width="500px" /> Nota: Analizzare ogni immagine 3D in modo cieco </li><li> Selezionare la scheda &#39;Draw&#39; e &#39;AutoPath&#39;. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot7.jpg" width="500px" /></li><li> Spostare il cursore del mouse sul ccorpo ell e Maiusc + tasto destro del mouse per selezionare il corpo cellulare. Nota: autocalculation dal software potrebbe richiedere alcuni minuti. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot8.jpg" width="500px" /></li><li> Aggiungi percorsi per il filamento (dendriti) usando Shift + click del mouse a sinistra. Nota: I percorsi possono essere visualizzati in tempo reale. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot9.jpg" width="500px" /></li><li> Vai alla finestra delle statistiche ad incandescenza e clicca su &quot;valori specifici, &#39;&#39; dettagliate &#39;e&#39; la lunghezza del filamento dendrite (somma) &#39;per somma di lunghezza totale dendrite. <img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot10.jpg" width="500px" /></li><li> Utilizzare appropriati test statistici per analizzare i dati. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII-NeuroD+Signaling+Pathway+Specifies+Dendritic+Morphogenesis.">CaMKII-NeuroD Signaling Pathway Specifies Dendritic Morphogenesis.</a> Neuron. 41, 229-241 (2004).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cdc20-APC+ubiquitin+signaling+pathway+regulates+presynaptic+differentiation.">A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation.</a> Science. 326, 575-578 (2009).</li><li>Litterman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+OBSL1-Cul7Fbxw8+ubiquitin+ligase+signaling+mechanism+regulates+Golgi+morphology+and+dendrite+patterning.">An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.</a> PLoS Biol. 9, (2011).</li><li>Kim, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centrosomal+Cdc20-APC+pathway+controls+dendrite+morphogenesis+in+postmitotic+neurons.">A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons.</a> Cell. 136, 322-336 (2009).</li><li>Shalizi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-regulated+MEF2+sumoylation+switch+controls+postsynaptic+differentiation.">A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.</a> Science. 311, 1012-1017 (2006).</li><li>Vadhvani, M., Schwedhelm-Domeyer, N., Mukherjee, C., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Centrosomal+E3+Ubiquitin+Ligase+FBXO31-SCF+Regulates+Neuronal+Morphogenesis+and+Migration.">The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration.</a> PLoS One. 8, (2013).</li><li>Puram, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CaMKIIbeta+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nat. Neurosci. , (2011).</li><li>Jia, Y., Zhou, J., Tai, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+promote+cerebellar+granule+neuron+survival.">TRPC channels promote cerebellar granule neuron survival.</a> Nat. Neurosci. 10, 559-567 (2007).</li><li>Huynh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isoform-specific+SnoN1-FOXO1+repressor+complex+controls+neuronal+morphogenesis+and+positioning+in+the+mammalian+brain.">An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.</a> Neuron. 69, 930-944 (2011).</li><li>de la Torre-Ubieta, L., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+neuronal+polarity+and+morphogenesis+in+the+mammalian+brain.">Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain.</a> Neuron. 72, 22-40 (2011).</li><li>Calissano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+insulin-like+growth+factor+I+exerts+a+trophic+action+and+confers+glutamate+sensitivity+on+glutamate-resistant+cerebellar+granule+cells.">Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8752-8756 (1993).</li></ol>

Gli autori dichiarano interessi finanziari concorrenti.

Vantaggi e limitazioni del descritto in vitro e in vivo: CGN Colta da mouse e ratti sono ugualmente adatti per le analisi morfologiche. A causa della dimensione più grande di un cervelletto di ratto, la resa di CNGS da cuccioli di ratto supera quello dei cuccioli di topo 3-4x. A parte CGN, neuroni corticali e ippocampali possono essere utilizzati come sistema cultura. Il metodo di fosfato di calcio si traduce in un basso (0,01-5%) efficienza di trasfezione, che è desidera...

Il cervelletto è un ottimo sistema per studiare i meccanismi di crescita degli assoni e la migrazione. Il cervelletto è stata oggetto di studi anatomici fin dagli albori delle neuroscienze 1. Microscopia moderna e tecniche di immunoistochimica hanno notevolmente ampliato e perfezionato le scoperte iniziali di Santiago, Ramon e Cajal 2-4. Genetica del topo e studi molecolari scoperti fattori di crescita e di trascrizione essenziali nel controllo dello sviluppo del cervelletto, che ha portato ad una maggiore comprensione degli eventi cruciali necessari per il corretto cablaggio dei diversi tipi di neuroni, tra cui neuroni granulari del cervelletto (CGN) 5-7. Il cervelletto è un derivato di rhombomere 1 della hindbrain sviluppo 8. Il labbro rombico, che è parte del tetto del 4 ° ventricolo, dà origine a progenitori neuronali dei granuli cerebellari, che costituiranno la più numerosa popolazione neuronale nelcervelletto adulto 9. Dopo la migrazione rostrale, si depositano nel anlage cerebellare. Qui, mitosi di precursori neuronali dei granuli porta alla drammatica espansione dello strato granulare esterno (EGL), che avviene dopo la nascita nei roditori. Dal EGL, i neuroni iniziano la migrazione verso l&#39;interno attraverso lo strato molecolare (ML), oltre lo strato di cellule di Purkinje a prendere in ultima analisi, la residenza nello strato granulare interno (IGL 2). Durante questo processo migratorio, acquisiscono una forma bipolare con due assoni si estende nella ML. Su ulteriore migrazione, il corpo cellulare migra distanti assoni ei due processi fondono per formare uno biforcato, assone forma di T 10. Successivamente, questi assoni fasciculate e sono indicate come fibre parallele. Stabilitosi nel IGL, CGN crescere dendriti, che formano artigli dendritiche stabilire sinapsi con le fibre di muschio. Per esaminare processi fondamentali nel cervelletto sviluppo, un combinato in vitro e in vivo approach consente di ottenere risultati affidabili e conclusioni. CGN sono non solo più numerosi neuroni del cervelletto ma dell&#39;encefalo e possono essere coltivate a elevata purezza 11-13. Nella cultura, questa popolazione neuronale molto omogenea diventa rapidamente postmitotico e acquisisce una morfologia polare con assoni e dendriti facilmente identificabili. CGN coltura hanno dimostrato di essere estremamente utile per studiare i vari aspetti dello sviluppo neuronale, tra cui la proliferazione progenitore, la differenziazione, assonale e lo sviluppo dei dendriti, migrazione neuronale, l&#39;apoptosi e le proprietà elettrofisiologiche (14-19 e molti altri). L&#39;uso della manipolazione genetica ha ampliato la versatilità di CGN colti e consentito per una visione più meccanicistica nei suddetti eventi. La trasfezione di neuroni in coltura con bassa efficienza fosfato di calcio o metodi lipofile seguiti da immunocitochimica con marcatori polarità o software supportato da analisi di facilitareTATI la valutazione ad esempio la morfologia dei singoli neuroni in coltura neuronale denso. Con questo approccio, il ruolo delle proteine ​​di interesse in assoni e dendriti crescita può essere studiato 20-25,26-28. Questo sistema di coltura però è meno utile per analizzare migrazione neuronale, come la migrazione è molto limitata nelle culture ad alta densità e richiederebbe co-colture. L&#39;analisi in vitro di assoni e dendriti crescita consente anche per l&#39;esame di proteine ​​interconnessi di una via di segnalazione utilizzando combinazioni di RNA interference (i), sovra-espressione o piccole molecole. Per stabilire la rilevanza della proteina di interesse in assoni e dendriti regolazione della crescita o la migrazione neuronale, l&#39;elettroporazione in vivo (IVE) tecnica permette l&#39;analisi in via di sviluppo corteccia cerebellare. Per il fatto che lo sviluppo cerebellare nei roditori estende strada nelle prime due settimane dopo la nascita, il cervelletto rappresenta un accessibstruttura del cervello per le manipolazioni genetiche per esaminare lo sviluppo di assoni e dendriti, migrazione neuronale, sinaptogenesi e apoptosi 20-24,29,30,26,27,31-34. Inoltre, questo modello di sistema è utile anche per altri aspetti dello sviluppo neuronale che richiedono la corteccia cerebellare intatta come assone pathfinding, il cablaggio e la connettività dei neuroni e delle interazioni neuroni-glia Preso insieme, questo protocollo prevede in vitro e in vivo le tecniche per affrontare un approccio complementare per quanto riguarda la morfogenesi neuronale e la migrazione.

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			<td height="21" style="height:21px;width:160px;">BME</td>
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			<td style="width:88px;">41010-026</td>
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			<td height="21" style="height:21px;width:160px;">Insulin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
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			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
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			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
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			<td style="width:168px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:160px;">ImageJ</td>
			<td style="width:125px;">NIH</td>
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			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
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			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
			<td style="width:125px;">GraphPad Software, Inc.</td>
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			<td height="22" style="height:22px;width:160px;">MS Excel</td>
			<td style="width:125px;">Microsoft</td>
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genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration

Eventi dello sviluppo del cervello, tra cui la morfogenesi neuronale e migrazione sono processi altamente orchestrati. In vitro e in vivo analisi consentono di una caratterizzazione approfondita per identificare meccanismi coinvolti in questi eventi. Neuroni granulari cerebellari (CGN) che sono derivati ​​dal cervelletto di sviluppo sono un sistema modello ideale che consente analisi morfologiche. Qui, descriviamo un metodo di come manipolare geneticamente CGN e come studiare axono-e dendritogenesis dei singoli neuroni. Con questo metodo gli effetti dell&#39;interferenza RNA, sovraespressione o piccole molecole possono essere confrontati per controllare neuroni. Inoltre, la corteccia cerebellare roditore è facilmente accessibile in sistema vivo a causa del suo sviluppo postnatale predominante. Vi presentiamo anche una tecnica di elettroporazione in vivo per manipolare geneticamente il cerebella sviluppo e descrivere la successiva cerebellare analisi per valutare la morfologia neuronale unand migrazione.

Per analizzare la morfologia della CGN in risposta a differenti condizioni di coltura, abbiamo trasfettato i neuroni sul DIV 0 come descritto sopra. Dopo trasfezione, abbiamo messo una serie di neuroni in pieno mezzo (BME, siero di vitello al 10%, 2 PSG mM, 25 mM KCl) e un&#39;altra serie in minima quantità di terreno contenente insulina (BME, glucosio 25 mM, 2 PSG mm, 10 mg / insulina ml). Abbiamo sottoposto i neuroni a immunocitochimica utilizzando l&#39;anticorpo GFP a DIV 1, 2, e 3, seguita da assoni e dendriti di ...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Morfogenesi neuronale e la migrazione sono eventi cruciali alla base un corretto sviluppo del cervello. Qui, descriviamo i metodi per manipolare geneticamente colture di neuroni granulari del cervelletto e del cervelletto in via di sviluppo per la valutazione della morfologia e delle caratteristiche migratorie dei neuroni.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

genetic-manipulation-cerebellar-granule-neurons-vitro-vivo-to-study

Research

JoVE Journal

Neuroscience

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

13.6K Views.  Max Planck Institute of Experimental Medicine.  Morfogenesi neuronale e la migrazione sono eventi cruciali alla base un corretto sviluppo del cervello. Qui, descriviamo i metodi per manipolare geneticamente colture di neuroni granulari del cervelletto e del cervelletto in via di sviluppo per la valutazione della morfologia e delle caratteristiche migratorie dei neuroni. 

Video: Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Analisi automatizzata Sholl di Digitized morfologia neuronale a scale multiple

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Ricerca

Neuroscienze

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus-mediata manipolazione genetica e la visualizzazione dei neuroni sensoriali olfattivi In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

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

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection di roditori neuroblasti per studiare neuroblasti migrazione<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Il sistema olfattivo come modello per studiare gli schemi di crescita assonale e Morfologia<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging di postnatale cerebellare granuli migrazione cellulare Utilizzando confocale macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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.

Analizzando dendritiche Morfologia in colonne e strati

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 Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Valutazione della vitalità neuronale utilizzando la fluoresceina diacetato-propidium ioduro a doppia colorazione nel granulo cerebrale Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...