Supported by grants from CNRS and INSERM. PM acknowledges support from the Fondation pour la Recherche M&#233;dicale en France (Equipe FRM 2009). SC acknowledges support from the Fondation de France.

Die in dieser Arbeit verwendeten Mäuse wurden gepflegt und in Übereinstimmung mit den Richtlinien des Rates der Europäischen Union (86/609 / EU) für den Einsatz von Labortieren behandelt. 1. Herstellung der erforderlichen Lösungen <ol><li> Stammlösungen <ol><li> 10x Poly-L-Ornithin (PLO) Lösung: wiegen 10 mg PLO Hydrobromid (Molekulargewicht = 30,000-70,000) und in 70 ml sterilem Wasser auflösen. Die Lösung wird mit 0,2 um Spritzenfilter, aliquoten, und bei -20 ° C. </li><li> 30% Glucose-Lösung: wiegen 30 g D (+) - Glucose und Auflösen in sterilem Wasser auf ein Gesamtvolumen von 100 ml. Filter, aliquoten, und bei 4 ° C. </li><li> 5x Dulbecco-modifiziertes Eagle-Medium (DMEM) / Nährstoff-Mischung F-12 Ham: abwiegen 6 g DMEM / F-12 Ham Pulver und lösen sich in sterilem Wasser auf ein Gesamtvolumen von 100 ml. Filter, aliquoten, und bei 4 ° C. </li><li> 7,5% Natriumbicarbonat (NaHCO 3) Solutiauf: wiegen 7,5 g NaHCO 3 und lösen sich in sterilem Wasser auf ein Gesamtvolumen von 100 ml. Filter, aliquoten, und bei 4 ° C. </li><li> 1 M HEPES-Lösung: Man wiegt 23,8 g HEPES und Auflösen in sterilem Wasser auf ein Gesamtvolumen von 100 ml. Filter, aliquoten, und bei 4 ° C. </li><li> 70% (v / v) Ethanol: Verdünnen mit 70 ml absolutem Ethanol mit 30 ml sterilem Wasser. </li></ol></li><li> Phosphatgepufferte Kochsalzlösung mit Glucose und Antibiotika (PBS-GAB): 10 ml 30% ige Glucoselösung und 5 ml Penicillin-Streptomycin-Lösung zu 500 ml Dulbeccos phosphatgepufferter Kochsalzlösung. Lagerung bei 4 ° C. </li><li> Inaktiviertes fötales Rinderserum (IFB) auftauen Rinderserum über Nacht bei 4 ° C inaktiviert bei 56 ° C für 30 min. Aliquot in 50 ml und bei -20 ° C. </li><li> Kulturmedium: in sterilem Wasser, Mischung nacheinander mit 40 ml 5x DMEM / F12-Ham, 1 ml 1 M HEPES, 2 ml von 200 mM L-Glutamin, 2 ml Penicillin-Streptomycin, 4 ml 30% Glucose einnd 3 ml 7,5% iger NaHCO 3 auf ein Endvolumen von 180 ml. Zu filtern und verwenden am selben Tag. </li><li> Hormon-Mix <ol><li> Planen 180 ml Kulturmedium. Man löst 200 mg des apo-Transferrin in diesem Medium. </li><li> Man wiegt 50 mg Insulin und in 2 ml 0,1 M HCl zu lösen. Fügen Sie langsam 8 ml sterilem Wasser unter Mischen sanft. Verdünne die Lösung in dem Kulturmedium. </li><li> Wiegen 19,3 mg Putrescin-dihydrochlorid und in 10 ml sterilem Wasser auflösen. Fügen Sie die 10 ml-Lösung auf das Hormon-Mix. </li><li> Bereiten Sie eine 3 mM Natriumselenit-Lösung in sterilem Wasser und 2 mM Progesteron-Lösung in absolutem Ethanol. Mit 20 ul jeder Lösung auf das Hormon-Mix. </li><li> Filtern Sie die Hormon-Mix, Aliquot in 10 ml und bei -20 ° C. </li></ol></li></ol> 2. Herstellung von Kulturplatten und Instrumente <ol><li> FBS Beschichtung der Kulturplatten: Der Tag vor der Dissektion, bereiten 180 ml Kulturmedium. 10 mlIFB auf 90 ml Kulturmedium. Geben Sie 300 ul / Kulturmedium / 10% IFBS in Poly-D-Lysin-Fertig 24-Well-Platten. Inkubieren über Nacht bei 37 ° C aufweist. Bewahren Sie die übrigen Medien (mit und ohne IFBS) bei 4 ° C. </li><li> Sterilisieren die Instrumente und Pasteur-Pipetten. Sammeln Sie die in der Materialliste aufgeführt als Class II Laminarströmungshaube und einem CO 2-Inkubator Ausrüstung. </li></ol> 3. Präparation der Maus Mesencephalon <ol><li> Opfern eine E13.5 schwangere Maus (gemäß gültiger Richtlinien). Reinigen Sie den Bauch der Maus mit 70% Ethanol und öffnen Sie die Bauchdecke. Sammeln Sie die Gebärmutterhörner, öffnen Sie sie mit zarten Schere, sezieren jedem Embryo von der Gebärmutterhörner, und entfernen Sie das Frucht Membranen. Legen Sie die Embryonen in einer 100 mm Petrischale mit sterilem PBS-GAB und waschen Sie sie durch Übertragung in 3 aufeinanderfolgenden gleichen Bäder mit einer Pinzette. Für die Präparation, legen Sie die Embryonen von 3 in einer 60 mm sterilen Petrischale. </li><li> Move die letzten Petrischale unter dem Stereomikroskop. Enthaupten nicht die Embryonen. Verwendung Vannas Schere, die Gehirne herauszuschneiden. <ol><li> Entfernen und entsorgen den Vordergrund und Hinterhirn Regionen. Um rostralen Seite, schneiden in der Nähe des Thalamus-Region, an der Schwanzseite Schnitt an der Landenge Region, entfernen Sie den Colliculus superior. </li><li> Sobald der ventralen Mittelhirn isoliert ist, sorgfältig Hirnhäute mit ultra feinen Pinzette entfernen. Sammeln Sie die Segmente zerlegt, ohne die Hirnhäute, in einer sterilen 13-ml-Tube mit PBS-GAB gefüllt. </li></ol></li><li> Reinigen Sie das Rohr mit dem mesencephala gründlich mit 70% Ethanol und bringen sie unter der Haube. </li></ol> 4. Zelldissoziation <ol><li> Wasch mesencephala mit sterilem PBS-GAB 3x. Erlauben Gehirn Fragmente zwischen jedem Wasch absetzen und vermeiden das Gewebe zu beeinträchtigen. Nach dem letzten Wasch, entfernen Sie sorgfältig, wie viel Lösung wie möglich und Inkubation Gehirn Segmente in 3 ml Trypsin / EDTA für 15 min bei 37 ° C in eineCO 2-Inkubator. </li><li> Feuer-Politur die Pasteur-Pipetten, um das Überleben der Neuronen als das Ende eines nicht-polierten Glaspipette zu maximieren ist scharf und kann die Zellen während der Dissoziation Schritte beschädigen. Reduzieren Sie die Extremität Durchmesser (bis 0,5 mm) des Pasteur-Pipette, während Feuer-polieren. </li><li> So viel Trypsin / EDTA-Lösung entfernen Sie vorsichtig wie möglich und mit 10 ml Kulturmedium / 10% IFBS. Wasch Gehirn Segmente 3x mit Kulturmedium / 10% IFBS. </li><li> Verwendung der feuerpolierten Pasteurpipette beginnen Dissoziation mesencephala in 6 ml Kulturmedium / 10% IFBS. Man reibt 10x (Luftblasen vermeiden), lassen die Stücke zu regeln, dann sammeln das Medium in ein neues steriles 13-ml-Tube. </li><li> 6 ml Kulturmedium, wiederholen Sie den vorherigen Schritt einmal zu sammeln und das Medium mit dissoziierten Zellen in der gleichen 13-ml-Tube. </li><li> Zentrifuge Zellen bei 160 g für 5 min. Überstand verwerfen und Resuspendieren der Zellen in Kulturmedium, ergänzt mit10% Hormon-Mix. </li></ol> 5. Handy-Überzug <ol><li> Zählen von Zellen in Suspension in einem Malassez Zelle und die Lautstärke von mittleren bis zu einer Konzentration von 600.000 Zellen / ml. Rund 1.700.000 Zellen können aus einer Maus Hirn gewonnen werden. </li><li> Entfernen FBS-haltigen Beschichtungsmedium von 24-Well-Platten und fügen Sie in jede Vertiefung 1 ml Zellsuspension (600.000 Zellen / Well, 2-4% der TH + Zellen). </li><li> Inkubieren der Platte bei 37 ° C und 5% CO 2/95% Luft. Kein Medium Veränderung ist notwendig. Dopaminergen Neuronen sind ausgereift nach etwa 5-7 Tagen in vitro (in Übereinstimmung Ausdruck der Dopamin-Transporter). Zellen in Kultur zu halten bis zu 15 Tage ohne Mediumwechsel. </li></ol> 6. Immunfluoreszenz-Protokoll <ol><li> Reinigung der Deckgläser <ol><li> Am Tag vor der Präparation, 10 ml 1 M HCl in eine Petrischale. Lag an der Oberfläche der Flüssigkeit 12-15 Deckgläser und warten Sie 15 min. </li><li> Sinken die Abdeckungschlüpfen in die Flüssigkeit und warten Sie 15 min. </li><li> HCl zu entfernen und waschen 3x mit Wasser. </li><li> Schnell waschen Sie die Deckgläser mit reinem Ethanol einmal, dann fügen reinem Ethanol in die Schale und warten Sie 30 min. </li><li> Unter der Haube entfernen gereinigt Deckgläser aus Ethanol und fügen Sie ein Deckglas pro Vertiefung einer 12-Well-Zellkulturplatte mit sterilisierten Pinzette. </li><li> Waschdeckgläser zweimal mit sterilem Wasser (1 ml / Vertiefung). Dann waschen Sie einmal mit sterilem PBS. </li></ol></li><li> Beschichtung der Deckgläser <ol><li> PBS zu entfernen und fügen Sie 500 ul / 2x PLO (verdünnt in PBS). Inkubation für 4 Stunden bei 37 ° C im CO 2-Inkubator. </li><li> Entfernen Sie die PLO-Lösung und waschen 3x mit sterilem PBS. </li><li> PBS zu entfernen und fügen Sie 500 ul / 20% IFBS / 1 g / ml Laminin. Inkubieren über Nacht bei 37 ° C im CO 2-Inkubator. </li></ol></li><li> Ausplattieren der Zellen für Immunfluoreszenz <ol><li> Entfernen Beschichtungsmedium von 12-Well-Platten. </li><li> Fügen 900,000 Zellen / Vertiefung in 2 ml Volumen und Wachstum der Zellen bei 37 ° C im Brutschrank. Zellen können in Kultur bis zu 15 Tage ohne Mediumwechsel gehalten werden. </li></ol></li><li> Immunfärbung <ol><li> Entfernen Medium von 12-Well-Platten und Waschen mit 500 ul / well DMEM vorgewärmt auf 37 ° C. </li><li> Gib 500 ul / Vertiefung DMEM vorgewärmt auf 37 ° C, dann fügen vorsichtig 160 ul / Vertiefung von 8% Paraformaldehyd (PFA, 2% Endkonzentration) und Inkubation für 10 min bei 37 ° C aufweist. </li><li> PFA entfernen und waschen 3x mit PBS / 0,1 M Glycin für 10 min. </li><li> PBS zu entfernen, fügen Sie 300 ul / PBS / 0,05% Triton X-100/20% Ziegenserum und Inkubation für 30 Minuten bei Raumtemperatur. </li><li> Medium entfernen, werden 300 ul / Vertiefung von primärem Antikörper, verdünnt in PBS / 0,05% Triton X-100/1% Ziegenserum und Inkubation für 2 Stunden bei Raumtemperatur. Primäre Antikörper, die zum Nachweis von dopaminergen Neuronen und Charakterisierung der Kultur verwendet werden können, sind in der Tabelle angegebenen Materialien. Halten1 auch ohne primären Antikörper für Hintergrund der verschiedenen Sekundärantikörper zu bewerten. </li><li> Medium entfernen und waschen 3x mit PBS / 0,2% Gelatine für 10 min. </li><li> Medium entfernen, werden 300 ul / Vertiefung des sekundären Antikörpers, verdünnt in PBS / 0,05% Triton X-100/1% Ziegenserum und Inkubation für 1 h bei Raumtemperatur im Dunkeln. Sekundärantikörper verwendet werden, in der Tabelle angegebenen Materialien. </li><li> Medium entfernen und waschen 3x mit PBS / 0,2% Gelatine für 10 min. </li><li> Medium entfernen und waschen 3x mit PBS. </li><li> Montieren Sie die Deckgläser auf Glasobjektträgern Zellseite nach unten in einem Tropfen Vectashield. Halten Sie die Folien bei Raumtemperatur über Nacht, um die Montage Medium trocknen lassen, dann speichern Sie sie bei 4 ° C. </li><li> Erwerben Sie Bilder mit einem konfokalen Mikroskop oder einem Phasenkontrast Epifluoreszenz ausgestattet. Repräsentative Bilder wurden mit einem Leica SP2 UV konfokalen Mikroskop erfasst. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Dieses Protokoll stellt die Verfahren und Reagenzien notwendig, eine Primärkultur von Mittelhirnneuronen von der embryonalen Maus und der Immunfluoreszenz-Verfahren zu dopaminergen Neuronen zu erkennen vorzubereiten. Kritische Schritte des Verfahrens sind die Dissektion der Embryonen und die mechanische Dissoziation der Fragmente gesammelt Gehirn. Hochwertige Dissektionsinstrumente hilft, die Technik zu beherrschen Dissektion. DA-Neuronen bilden einen kleinen Teil des Mittelhirns. Dementsprechend sammelt den rechten Te...

Dopamin, eine der wesentlichen Neurotransmitter im Gehirn 1,2, wird hauptsächlich von dopaminergen Mittelhirn (DA) Neuronen freigesetzt. Die Mehrheit der DA Neuronen befinden sich in der ventralen Teil des Mittelhirns 2-6. Mesostriatal, mesolimbischen und mesocorticalen Wege 2,5: schematisch, Mittelhirn-Neuronen DA kann in drei anatomisch und funktionell verschiedene Projektionssysteme aufgeteilt werden. Der Weg wird in nigrostriatale Motorik beteiligt, die mesolimbischen Bahnen spielen eine wichtige Rolle in der Verstärkung, Motivation und Lernen, während die dopaminergen Bahnen zum präfrontalen Kortex vorstehen, sind in Erkenntnis 2 beteiligt. DA-Neuronen in mehreren neurologischen Störungen menschlichen wie Schizophrenie, Aufmerksamkeitsdefizit, Hyperaktivität Erkrankung und Parkinson-Krankheit (PD) 2,4 beteiligt. PD wird durch eine progressive Degeneration der selektiven und DA Neuronen verbinden Substantia nigra gekennzeichnetpars compacta (SNC) zum Striatum. Der Verlust der nigrostriatalen DA Neuronen führt zu einer starken Verarmung Dopamin im Striatum, die verantwortlich für die motorischen Symptome der PD (Bradykinesie, Ruhetremor, und Steifigkeit) 7. Die ursprüngliche Ursache der idiopathischen PD ist nicht nachgewiesen und die aktuellen Behandlungen sind nur symptomatisch, mit dem Ziel der Wiederherstellung Dopaminspiegel im Striatum. Das verschriebene Medikament ist L-Dopa (Levodopa), das natürliche Vorläufer von Dopamin. Obwohl Verabreichung von Levodopa kompensiert den Verlust von Dopamin zu einer bestimmten Zeit auftreten Motor Komplikationen nach einer Langzeitbehandlung (Dyskinesie und / AUS-Zustände) 8,9. Forschung auf dopaminerge Neuronen und PD ist in ständiger Progression und intensive Anstrengungen unternommen werden, um Behandlungen auf Zelltransplantation, Gentherapie, oder neuroprotektive Mittel 10,11 zu entwickeln. Allerdings bleibt ein wichtiges Thema nicht geklärt: Was ist die Ursache für die extreme vulnerability von DA Neuronen? Ein Teil der Antwort kann in der Aktivität von Neuronen DA gefunden werden. Eine Reduktion der elektrischen Aktivität und der Erregbarkeit von DA-Neuronen scheint die Neigung zu degenerieren 12 zu erhöhen. Dennoch ist die Komplexität der PD Pathogenese erfordert weitere Studien, um die Mechanismen in DA Neuronen beteiligt Degeneration 13-15 identifizieren. Primärkulturen sind besonders relevant für die DA Neuron Eigenschaften 16-19 studieren und diese Neuronen auf verschiedene Belastungen für die Bewertung der neuroprotektive Mittel 20-24 herauszufordern. Ratte Kulturmodelle werden häufig verwendet, da die Präparation der Rattenembryohirn ist einfacher, verglichen mit der Maus, und höhere Mengen von Neuronen bei Ratten erhalten werden. Allerdings hat von transgenen Mausmodellen der Krankheit 25 deutlich erhöht das Interesse der Neurowissenschaftler Community für Primärkulturen von der Maus 26-29. Obwohl Kulturen prvon neugeborenen Tieren epared verwendet werden kann, ist es besser, sie von Embryonen im post-mitotischen Stadium (E13.5 für Mesencephalon-Neuronen) herzustellen, wenn Neuronen haben ihre Fähigkeit zur Differenzierung beibehalten. Das folgende Protokoll stellt isoliert Hirn-Neuronen in Primärkultur von Maus-Embryonen (E13.5), die am schwierigsten zu vorzubereiten. Insbesondere stellen wir ein Protokoll mit serumfreiem Kulturmedium für eine bessere Reproduzierbarkeit. Die beiden wichtigsten Schritte in der Kultur-Präparat (Dissektion und mechanische Dissoziation) wird vorsichtig in den zugehörigen Video detailliert werden.

<table border="0" cellpadding="0" cellspacing="0" width="1291">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">DMEM 4.5g/L Glucose with L-Glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">0.05% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td style="width:139px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 200 mM Solution</td>
			<td>Life Technologies</td>
			<td>25030123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D0547</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Laminin - 1 mg/mL in Tris buffered NaCl</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Ornithine hydrobromide</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin from porcine pancreas</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">I5523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apo-Transferrin human</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Putrescine dihydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sodium selenite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">H4034&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G7126</td>
			<td>Stock solution 1M in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">G9391</td>
			<td>Stock solution 2% (w/v) in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td style="width:139px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% in water</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:139px;">RT 15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">106329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D(+)-Glucose, Monohydrate</td>
			<td>Merck Millipore</td>
			<td>4074-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Hydrochloric acid - c(HCl) = 1 mol/l (1 N) Titripur</td>
			<td>Merck Millipore</td>
			<td style="width:139px;">109057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile water - Aqua B. Braun</td>
			<td style="width:176px;">Braun</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Ethanol absolute NORMAPUR analytical reagent</td>
			<td>VWR</td>
			<td style="width:139px;">20821.321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sterile Petri Dishes</td>
			<td>VWR</td>
			<td>82050-566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Pasteur pipettes plain glass - Wilhem Ulbrich GdbR.</td>
			<td>VWR</td>
			<td>612-2297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Counting chamber Malassez</td>
			<td>VWR</td>
			<td style="width:139px;">631-0975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum Acrodisc Syringe Filter with Supor Membrane, Sterile, GF/0.2 &#181;m, 37 mm</td>
			<td>PALL Life science</td>
			<td style="width:139px;">4525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors - Straight, sharp-sharp, 14.5 cm long</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td>14002-14</td>
			<td>To open the abdominal wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors - Straight, pointed, delicate, 10 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>4877A</td>
			<td>To open the uterine wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Curved, usual, serrated jaws 1 mm</td>
			<td style="width:176px;">MORIA</td>
			<td>2183</td>
			<td>To manipulate embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Vannas Scissors - Curved, pointed, 7 mm blades</td>
			<td style="width:176px;">MORIA</td>
			<td>MC50</td>
			<td>To take out the mesencephalon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Forceps - Curved, delicate, 13 cm long</td>
			<td style="width:176px;">MORIA</td>
			<td>9987</td>
			<td>To remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD BioCoat Poly-D-Lysine 24-well Multiwell Plates</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">356414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Falcon 12-well Cell Culture Plate, flat-bottom with lid</td>
			<td>BD Bioscience</td>
			<td style="width:139px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Microscope Slides, Ground edges 90&#186;</td>
			<td>MENZEL-GL&#196;SER</td>
			<td style="width:139px;">AG00008032E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision cover glasses thickness No. 1.5H circular 18 mm &#216;</td>
			<td>MARIENFELD</td>
			<td>117580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyclonal Rabbit Anti-Microtubule-Associated Protein 2 (MAP2) Antibody</td>
			<td>Chemicon Millipore</td>
			<td>AB5622</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-Glutamate Decarboxylase (GAD67) Antibody, clone 1G10.2</td>
			<td>Chemicon Millipore</td>
			<td>MAB5406</td>
			<td>1/400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Rat Anti-Dopamine Transporter (DAT) Antibody, clone DAT-Nt&#160;</td>
			<td>Chemicon Millipore</td>
			<td>MAB369</td>
			<td>1/500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal Mouse Anti-5-HT Antibody</td>
			<td style="width:176px;"></td>
			<td style="width:139px;"></td>
			<td>1/8,000 - Generous gift from Yves Charnay (Swizerland, Yves.Charnay@hcuge.ch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum, New Zealand Origin</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 405 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:139px;">A-31556</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11001</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 Goat Anti-Rat IgG (H+L) Antibody</td>
			<td style="width:176px;">Life Technologies</td>
			<td>A-11007</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Stereomicroscope</td>
			<td>Carl Zeiss microscopy</td>
			<td>Stemi-2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Bunsen Burner FIREBOY</td>
			<td style="width:176px;">VWR</td>
			<td style="width:139px;">451-0136</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of mouse dopaminergic neurons

Dopaminergen Neuronen weniger als 1% der gesamten Anzahl von Neuronen im Gehirn. Diese geringe Menge an Neuronen regelt wichtige Hirnfunktionen wie Motorsteuerung, Motivation und Arbeitsgedächtnis. Nigrostriatalen dopaminergen Neuronen selektiv degenerieren bei Parkinson-Krankheit (PD). Diese fortschreitende Verlust von Nervenzellen ist eindeutig mit den Motoren Symptome der Pathologie (Bradykinesie, Ruhetremor und Muskelstarre) assoziiert. Die wichtigsten Vertreter der dopaminergen Neuronen verantwortlich Degeneration ist noch unbekannt. Allerdings scheinen diese Neuronen extrem anfällig in unterschiedlichen Bedingungen zu sein. Primärkulturen stellen eine der wichtigsten Modelle Eigenschaften und Merkmale von dopaminergen Neuronen zu untersuchen. Diese Kulturen können auf verschiedene Stressmittel, die PD Pathologie imitieren und zu neuroprotektiven Verbindungen, um zu stoppen oder zu verlangsamen neuronale Degeneration vorgelegt werden. Die zahlreichen transgenen Mausmodellen der PD, die Gener habenergaben sich im letzten Jahrzehnt weiter erhöht das Interesse der Forscher für dopaminerge Neuronenkulturen. Hier konzentriert sich das Video-Protokoll auf den empfindlichen Dissektion der embryonalen Gehirn der Maus. Präzise Exzision der ventralen Mittelhirn ist entscheidend für die neuronalen Kulturen ausreichend reich an dopaminergen Zellen zu erhalten, um nachfolgende Studien zu ermöglichen. Dieses Protokoll kann mit embryonalen transgenen Mäusen realisiert werden und ist für die Immunfluoreszenzfärbung, quantitative PCR, second messenger Quantifizierung oder zum neuronalen Tod / Überleben Beurteilung.

Eine illustrierte Flussdiagramm der Mittelhirn-Kultur Schritte ist in Abbildung 1 dargestellt. Kurz gesagt, nach dem Sammeln von E13.5 Embryonen von einer schwangeren Schweizer Maus, Ventralmesenzephalons von der gesamten Embryo seziert. Die isolierten Gehirnfragmente werden nacheinander auf die enzymatische Verdauung und mechanische Dissoziation eingereicht. Dissoziierten Zellen werden durch Zentrifugation pelletiert, in Kulturmedium resuspendiert und in vorbeschichteten 12 oder 24-Well-Platten ausplat...

Watch this Scientific Journal Video about Primary Culture of Mouse Dopaminergic Neurons at JoVE.com

Primary Culture of Mouse Dopaminergic Neurons

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.

Primärkultur von Maus dopaminergen Neuronen

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

primary-culture-of-mouse-dopaminergic-neurons

Research

JoVE Journal

Neuroscience

37.6K Aufrufe.  Institut de Génomique Fonctionnelle, Montpellier. 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.

Serie: Primary Culture of Mouse Dopaminergic Neurons

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 und Primärkultur von embryonalen Maus-Hippocampus und kortikalen Neuronen

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

Forschung

Neurowissenschaften

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

The Culture of Primary Motor und sensorischen Neuronen in definierten Medien auf Elektrogesponnene Poly-L-Lactid Nanofiber Gerüste

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminäre Co-Kultur von primären Ratten-kortikalen Neuronen und Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Dissection und Kultur von Maus-dopaminergen und Striatale Explantate in dreidimensionalen Kollagen-Matrix-Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calciumphosphat-Transfektion von primären Hippocampus-Neuronen

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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.

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.

Isolation, Kultur und langfristige Wartung von Primär mesencephalen dopaminergen Neuronen aus embryonalen Nagetier Brains

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

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellierung von neuronalen Tod und Degeneration in Maus primären zerebelläre Granulat Neuronen

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Live-Bilder von GLUT4 Protein Menschenhandel Maus primäre hypothalamische Neurone mit Dekonvolution Mikroskopie

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

Primärzellkultur von gereinigten Gurified GABAergic oder Glutamatergic Neuronen, die durch Fluoreszon-Sortierung gegründet wurden

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Primäre Kultur der Neuronen isoliert von Embryonal Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...