This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

NOTA: Gli animali sono stati mantenuti e gestiti in conformità con le linee guida istituzionali e tutte le procedure di animali sono stati approvati dal benessere degli animali del Imperial College e Ethical Review Body (AWERB) e il Ministero degli Interni e la Harvard University Istituzionale Animal Care and Use Committee (IACUC), in conformità alle normative federali e statali. 1. reagente e installazione attrezzature <ol><li> Thaw, aliquota e conservare 1 mg / ml soluzione laminina a -80 ° C. Sciogliere 2 microlitri in 1 ml DMEM / F12, ottenendo una concentrazione di rivestimento di 1-2 mg / cm 2. NOTA: Thaw laminina lentamente sul ghiaccio e si dissolvono in freddo DMEM / F12 e aggiungerlo ai piatti / coprioggetti immediatamente. </li><li> Diluire 75 ml di 0,01% Poly-L-ornitina in 425 ml di PBS e conservare a 4 ° C. NOTA: La durata della soluzione di lavoro è fino a un mese. </li><li> Sciogliere 25% (peso / volume) BSA in PBS, pH 7.4, e conservare a 4 ° C per un massimo di un year. </li><li> Preparare 50% FBS (in HBSS) mezzi di disattivazione. </li><li> Preparare terreno completo aggiungendo 50 U / ml di penicillina e streptomicina, 1x N2 supplemento, 5% (vol / vol) FBS, 0,36% D - (+) - Glucosio (peso / volume), 0,25% di BSA (peso / volume) di DMEM / F12 e conservare a 4 ° C. NOTA: La durata è fino a 10 giorni. </li><li> Luogo coprioggetto in ebollizione il 70% (vol / vol) di etanolo per almeno 30 minuti per rimuovere eventuali tracce di grasso e di autoclave. </li><li> Luogo coprioggetto in piastre da 24 pozzetti e aggiungere 500 microlitri soluzione Poli-L-ornitina in ciascun pozzetto. Incubare 1 ora sotto il cofano di coltura tissutale a temperatura ambiente e poi, lavare 3 volte con 500 microlitri acqua. Aggiungere la soluzione laminina 500 microlitri per ogni piatto e incubare O / N in umidificata coltura tissutale incubatore a 37 ° C. NOTA: Anche se non sono necessari lavaggi dopo il trattamento laminina, lavare i poli-L-ornitina meno di tre volte provoca la morte delle cellule 24 ore dopo coltura. </li><li> Lucidare le punte delle pipette di vetro oltre a gas naturale o torcia fzoppo. NOTA: Dopo questo passo il diametro della punta dovrebbe essere circa la metà della sua dimensione normale. </li><li> Tagliare i tappi di provette da 1,5 ml microcentrifuga, utilizzando una forbice autoclave e autoclave. </li></ol> 2. dissezione embrionali ventrale Mesencefalo <ol><li> Narcotizzare gravide temporizzato (E12.5) dighe di CO 2 inalazione e quindi eutanasia da dislocazione cervicale. </li><li> Spruzzare l&#39;addome con il 70% di etanolo e fare un&#39;incisione addominale fino a quando le sacche uterine sono tutti esposti. Uso pinze tagliare l&#39;attaccamento vaginale e rimuovere l&#39;utero. Inserire l&#39;utero in ghiacciata HBSS, in una piastra di Petri 100 mm. </li><li> Rimuovere embrioni dall&#39;utero e sacco amniotico, usando pinze (Figura 1C). Luogo embrioni in HBSS ghiacciata fresca. NOTA: Il rettangolo in figura 1D indica la posizione del mesencefalo ventrale embrionale. </li><li> Mettere gli embrioni al microscopio dissezione (10x) e sezionare il mesencefalicaarch tagliando il cervello al istmo e mesencefalica-diencephalic regione di confine, con il bioscissor e pinza (Figura 1E, si prega di fare riferimento alla Figura 1A per le linee di incisione). </li><li> Dopo aver preso l&#39;intera mesencefalo, fare un taglio nel mesencefalo mediodorsale (Figura 1F). </li><li> Rimuovere meningi, utilizzando due pinze (Figura 1H). NOTA: PUNTO CRITICO: Questo passaggio è fondamentale per la resa ottimale neuronale e la sopravvivenza. Poiché le condizioni di coltura sono ottimali per le cellule cerebrali, la maggior parte delle cellule del tessuto circostante muoiono dopo placcatura e il segnale apoptotico da questo tessuto può danneggiare l&#39;integrità delle culture. </li><li> Appiattire il tessuto mesencefalo sul piatto Petri per osservare la forma a farfalla e tagliare circa la metà di ogni ala, utilizzando una lama da barba sterilizzato (Figura 1H, I). NOTA: Figura 1J descrive isolato mesencefalo ventrale. </li><li> Trasferire ilpezzo di mesencefalo ventrale in ghiaccio-freddo HBSS in un tubo da 15 ml e procedere con il successivo dell&#39;embrione. NOTA: i passaggi 2,1-2,8 non dovrebbe prendere più di 1 ora. Mantenere gli embrioni di là di questo lasso di tempo sul ghiaccio diminuisce vitalità cellulare in modo significativo. </li></ol> 3. La dissociazione delle cellule ventrale Midbrain <ol><li> Trasferire i pezzi di mesencefalo ventrale sotto una cappa a flusso laminare. Rimuovere il HBSS e aggiungere 1 ml di pre-riscaldato (37 ° C) 0,05% tripsina-EDTA nel tubo conico (volumi sufficienti per un massimo di 12 pezzi di mesencefalo ventrale). Incubare il tessuto a 37 ° C per 5-10 minuti. </li><li> Rimuovere tripsina-EDTA sotto il cofano e aggiungere 1 ml di mezzo di disattivazione (siero disattiverà tripsina) al tessuto. </li><li> Rimuovere il mezzo disattivazione e lavare il tessuto in 1 ml di mezzo completo due volte. NOTA: evitare di rimuovere tutto il supporto dal tubo e pipettando il tessuto, per evitare che scartando le cellule dissociate. </li><li> Aggiungere 1 ml terreno completo e triturato il tessuto con un bicchiere pipetta fuoco lucido. Evitare la formazione di bolle e continuare triturazione fino al raggiungimento sospensione di cellule singole. NOTA: Di solito, 8-10 passa attraverso la punta ristretta sono necessari per la completa dissociazione. </li><li> Centrifugare le cellule a 400 xg per 5 minuti a RT e rimuovere il supporto. </li><li> Risospendere il pellet in 1 ml di media completo lentamente pipettando su e giù per un massimo di quattro volte. </li></ol> 4. Cellule placcatura ventrale Midbrain <ol><li> Miscelare 10 ml di sospensione cellulare con 90 microlitri Trypan blue soluzione e contare il numero di cellule con un emocitometro. NOTA: la vitalità delle cellule può essere controllato in questa fase. </li><li> Mettere tappi provetta sterilizzati in 100 millimetri piastre di Petri e trasferire i coprioggetti Poly-L-ornitina / laminina rivestite, utilizzando pinze, una alla volta sulla parte superiore dei tappi. NOTA: Non lavare i coprioggetti ed evitare l&#39;essiccazione della laminina, poiché questo può causare uneveculture n e diminuzione sopravvivenza delle cellule. </li><li> Regolare il volume a 1500 cellule per 1 ml aggiungendo mezzo completo e aggiungere 100 microlitri (totale di 150.000 cellule) della sospensione cellulare sopra l&#39;coprioggetto (il volume è ottimale per coprire la superficie del vetrino senza perdite). Chiudere le piastre di Petri e incubare per 1 ora in un incubatore umidificato coltura tissutale (37 ° C, 5% CO 2). NOTA: PUNTO CRITICO: Questo passo è necessario per migliorare il collegamento e la vitalità delle cellule e per aumentare il numero di lamelle per dell&#39;embrione. In alternativa, le cellule possono essere trasferiti direttamente nei pozzetti (contenenti coprioggetto), ma il numero di celle deve essere aumentata a 350.000 per pozzetto (invece di 150.000). In altre parole, con questo metodo, 8-10 coprioggetto possono essere acquisiti, mentre il trasferimento diretto produce circa 3-4 vetrini a causa delle cellule, che si attaccano alle piastre (accanto o sotto i coprioggetti). La viabili mediaty delle culture, con questo metodo è stato di circa il 90%. </li></ol> 5. Cultura crescita e manutenzione <ol><li> Dopo 1 ora di incubazione, trasferire accuratamente i coprioggetti compreso il terreno in piastre da 24 pozzetti, contenente 400 ml di pre-riscaldato (a 37 ° C) terreno completo (volume totale: 500 ml). Incubare le cellule O / N a 37 ° C. </li><li> Entro 24 ore dopo incubazione dei pozzetti, aggiungere delicatamente 500 microlitri terreno completo in ogni pozzetto. NOTA: Le prime ore è la fase più critica per la sopravvivenza dei neuroni dopaminergici e il numero di cellule sopravvissute non cambia significativamente oltre questo punto. </li><li> Non aggiungere supporti supplementare alle colture per le prime due settimane o finché il terreno diventa gialla. Se coltura per più di due settimane o cambiamenti di colore medio al giallo, cambio la metà dei mezzi di comunicazione (500 ml) con mezzi freschi completi pre-riscaldato (tipicamente ogni due settimane). NOTA: i neuroni dopaminergici nelculture sarebbero sopravvissuti per più di 6 settimane in queste condizioni. </li></ol>

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No conflict of interest declared.

Neuroni dopaminergici nel mesencefalo sono la principale fonte di dopamina nel sistema nervoso centrale. Sono divisi in tre gruppi, pars compacta della sostanza nera (SNPC), area ventrale tegmentale (VTA) e campo retrorubral (RRF) 10, 11. I neuroni in SNPC e VTA danno luogo a grandi vie dopaminergiche, mesocorticali, mesolimbico e nigrostriatale, coinvolte in funzioni come il controllo delle emozioni, la motivazione e il comportamento del motore. Scomparsa dei neuroni in SNPC e disgregazione funzionale del sistema nigrostriatale è la principale caratteristica patologica della seconda malattia neurodegenerativa più importante, il morbo di Parkinson (PD) 12,13. Approcci terapeutici attuali affrontare i sintomi della malattia e non arrestare o rallentare la degenerazione e le cause della lenta, progressiva perdita di cellule sono ancora sconosciute 14-16. Pertanto, lo sviluppo di in vivo e in vitro per lo studio tecniche del DEVELOPMEntale, biochimico, e le caratteristiche fisiologiche di questa popolazione neuronale è indispensabile per comprendere l&#39;eziologia di PD e allo sviluppo di nuovi approcci terapeutici. Qui si descrive un metodo ottimizzato per la dissezione, la dissociazione e coltura di neuroni primari di topo e ratto embrionale mesencefalo ventrale, che si traduce in-lungo termine in vitro sopravvivenza dei neuroni dopaminergici di questa regione, consentendo studio dei processi quali la differenziazione, assonale escrescenza e formazione di sinapsi. Diversi vantaggi di questo protocollo includono l&#39;aumento del numero di coprioggetto per dell&#39;embrione, indipendenza da fattori di crescita e la lunghezza della sopravvivenza in vitro, massimizzando sviluppo di neuroni 4. Questo metodo può integrare gli studi in vivo nel permettere per la comprensione dei meccanismi sottostanti normali funzioni fisiologiche dei neuroni dopaminergici mesencefalo e progressiva degenerazione questo neuroneAl popolazione in modelli citotossici e genetici della malattia di Parkinson. Uno dei principali svantaggi di questo protocollo è la mancanza di distinzione tra SNPC e VTA neuroni dopaminergici. Questo problema esiste perché al momento della coltura (E12.5 in topi o ratti in E14.5) differenziazione terminale dei neuroni e la loro regionalizzazione non è completa e mentre è possibile l&#39;uso di embrioni anziani, riduce significativamente la sopravvivenza neuroni a causa di assotomia. Distinguendo le due popolazioni sarebbe possibile mediante immunocitochimica, utilizzando anticorpi contro marcatori specifici per ogni popolazione come GIRK2 (per identificare neuroni SNPC) e calbindin (per identificare neuroni VTA) 17. La placcatura ottimizzato descritto al punto 4 diminuisce la quantità di embrioni per esperimento di oltre il 50% e quindi contribuisce a ridurre l&#39;impiego di animali nella ricerca. Le fasi più critiche per la sopravvivenza del cultoUres sono il tempo tra euthanization delle dighe e completa dissezione dei midbrains embrionali (passo 2,1-2,8), nonché la rimozione completa delle meningi (passo 2.6, Figura 1H).

La perdita di neuroni dopaminergici della substantia nigra pars compacta conduce ai sintomi motori cardinali della malattia di Parkinson (PD), la seconda malattia neurodegenerativa più diffusa. La causa della morte di questa popolazione neuronale mesencefalica non è nota. Per studiare i meccanismi biochimici responsabili dello sviluppo e modulando le proprietà neurofisiologiche e la sopravvivenza dei neuroni mesDA, diverse colture cellulari e sistemi modello animali sono stati utilizzati. Linee cellulari immortalizzate, compreso il ratto dopaminergica linea cellulare 1RB 3 AN 27 (N27), il dopaminergico umano linea cellulare di neuroblastoma SH-SY5Y, la linea cellulare di ibridoma del mouse dopaminergica MN9D e mesencefalico umana LUHMES cellule sono state usate per gli studi meccanicistici biochimici e limitate 1 -5. Per lo studio della perdita di neuroni specifica mesDA, vari neurotossina-based e modelli genetici sono stati sviluppati 6-8. Culture mesencefalo ventrale primarie, Fornire uno strumento indispensabile per lo studio delle proprietà neuronali e sinaptiche dei neuroni dopaminergici e i meccanismi coinvolti nella patogenesi di questa malattia comune. Qui vi presentiamo un protocollo dettagliato per l&#39;isolamento dei neuroni dopaminergici mesencefalici, che contiene le modifiche con conseguente maggiore sopravvivenza e una maggiore resa di vetrini per dell&#39;embrione. L&#39;uso di pre-maturo mesencefalo del mouse E12.5 (E14.5 nel ratto) migliora la sopravvivenza. A questa età i neuroni non hanno ancora sviluppato assoni, che lascia intatte le cellule durante la dissezione e riduce al minimo lo stress incrementando in modo significativo la redditività. Inoltre, attenta dissezione del mesencefalo ventrale, come descritto nella sezione 2 del presente protocollo, migliora ulteriormente la capacità di sopravvivenza. Per aumentare il numero di vetrini per embrione, un metodo alternativo di placcatura è presentato nella sezione 4 del presente protocollo. Questo porta ad un rendimento di fino a 10 vetrini per dell&#39;embrione rispetto a 4 coprioggetti sottocondizioni standard di placcatura riducendo così la quantità di animali per esperimento. I neuroni in coltura mostra conseguenza di assoni e dentrites, formano connessioni sinaptiche e rivelano la presenza di marcatori neuronali e sinaptiche rendono queste culture adatto per l&#39;imaging cellulare dal vivo, immunocitochimica e studi elettrofisiologici. Inoltre, l&#39;uso di colture neuronali facilita la manipolazione genetica e farmacologica. Escrescenza dei neuriti dal giorno 2 in vitro consente per gli studi di sviluppo. Inoltre, la sopravvivenza a lungo termine di culture (fino a sei settimane) li rende adatti per lo studio della lenta, progressiva degenerazione dei neuroni questi.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:364px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified Eagle medium nutrient mixture F-12</td>
			<td>Invitrogen</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td>Invitrogen</td>
			<td>24020-117)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum, heat-inactivated (FBS)</td>
			<td>Invitrogen</td>
			<td>16140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td>17502-048)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose solution (45% (wt/vol) in water</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin BSA</td>
			<td>Sigma</td>
			<td>A9430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% (wt/vol)</td>
			<td>Invitrogen</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA) cell culture tested</td>
			<td>Sigma&#160;</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Tyrosine hydroxylase (Th) antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td>P60101-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-ornithine, 0.01% solution</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td>
			<td>Millipore</td>
			<td>MAB3418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Synapsin-1 antibody</td>
			<td>Millipore</td>
			<td>AB1543P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-rabbit IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-sheep IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-11016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 donkey anti-mouse IgG&#160;&#160; antibody</td>
			<td>Molecular Probes</td>
			<td>A-21203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue solution (0.4% (wt/vol)</td>
			<td>Biowhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted phase contrast microscope</td>
			<td>Carl Zeiss</td>
			<td>Axiovert 40 C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>May-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover Slip Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11251-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two Dumont #45 Forceps &#8211; Dumoxel</td>
			<td>Fine Scientific Tools</td>
			<td>11245-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blade Holder/Breaker Flat Grip - 11cm</td>
			<td>Fine Scientific Tools</td>
			<td>10052-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Iris Scissors - Straight 11.5cm</td>
			<td>Fine Scientific Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber optic halogen illuminator</td>
			<td>Nikon</td>
			<td>MKII</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Proscitech</td>
			<td>SVZ2NIOU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m sterile filter units</td>
			<td>Nalgene</td>
			<td>NL-CE-156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100x20mm Petri dishes</td>
			<td>BD Biosciences</td>
			<td>351005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Cover Slip #1 Thickness German Glass 12mm</td>
			<td>Bellco Glass</td>
			<td>1943-10012&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Immunoistochimica contro tirosina idrossilasi (TH) mostra che tra 0,5-1% delle cellule in coltura sono dopaminergica. Proiezioni neuronali sembrano in 2 ore dopo la placcatura e il primo giorno, assoni e dendriti sono distinguibili (figura 2), utilizzando la tirosina idrossilasi (TH) e proteina 2 (Map2) anticorpi associata ai microtubuli (Figura 3). I neuroni sopravvivono per più di sei settimane e mostrano un&#39;ampia conseguenza. Il rapporto neurone-glia nelle colture è direttamente correlata alla concentrazione di siero nel mezzo, come indicato in precedenza. Eliminando FBS dalle culture possono produrre colture neuronali relativamente puri, noi e altri abbiamo trovato che l&#39;aggiunta di siero aumenta sopravvivenza dei neuroni dopaminergici nella cultura 9. <img alt="Figura 1" src="/files/ftp_upload/52475/52475fig1.jpg" /> Figura 1. Procedimento per l&#39;isolamento diembrionale mesencefalo ventrale. La vista schematica di cervello di topo E12.5, confini mesencefalo (linee tratteggiate), e la posizione approssimativa della regione di interesse, mesencefalo ventrale, all&#39;interno del rettangolo rosso (A) e gli strumenti per la dissezione del mesencefalo ventrale , un bioscissor, foreceps, e un supporto lama (B) vengono visualizzati. Gli embrioni sono prese dal sacco amniotico per rottura (C). La regione di interesse, mesencefalo ventrale, è indicata dal rettangolo (D). Mesencefalo è isolato tagliando presso l&#39;istmo (a sinistra del pezzo tagliato) e il confine mesencefalo-prosencefalo (a destra, E). La parte mediodorsale del mesencefalo viene tagliato (F) ed è appiattita, per assomigliare ad una forma a farfalla (G, H). Poi, meningi sono dissociate dal cervello (H) e mesencefalo ventrale (rettangolo) è isolato dalla parte dorsale tagliando metà delle ali, utilizzando una lama da barba <strong&gt; (I). Mesencefalo ventrale (J) viene trasferito in un tubo da 15 ml e tenuta in ghiaccio, fino dissociazione. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52475/52475fig2.jpg" /> Figura 2. Rappresentante immagini brightfield di culture mesencefalo ventrale. Le immagini sono state scattate a DIV1 (a sinistra), DIV2 (al centro), e DIV15 (a destra). Scala bar:. 50 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52475/52475fig3.jpg" /> Figura 3. La sopravvivenza a lungo termine, conseguenza di processi e sinaptogenesi in due settimane oculture ld ventrali mesencefalo. neuroni dopaminergici Midbrain, identificati da anticorpi tirosina idrossilasi crescono vaste assoni e dendriti, identificati da anticorpi Map2, nelle culture mesencefalo ventrale due settimane dopo dissociazione da embrioni E12.5. Dendriti si distinguono per sovrapposizione di Th e processi Map2-positivi, dove solo Th-positive fibre rappresentano assoni. Anticorpi stati usati ad una diluizione di 1: 200. Scala bar:. 20 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.9K Views.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Video: Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e cultura primaria di neuroni embrionali di topo ippocampale e corticale

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e la coltura di neuroni ippocampali da topi prenatali

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primario di topo neuroni dopaminergici

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura della dissociato neuroni sensoriali da embrioni di gallina

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Un metodo semplificato per Ultra-Low Density, Long-Term primaria ippocampale Neuron Cultura

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Isolamento e cultura dei neuroni del midollo spinale da topi neonatali

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generazione e manutenzione a lungo termine di nervi liberi<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...