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

REMARQUE: Les animaux ont été maintenus et manipulés en conformité avec les directives institutionnelles et toutes les procédures d&#39;animaux ont été approuvés par la protection des animaux de l&#39;Imperial College et le Home Office et de l&#39;Université Harvard institutionnel de protection des animaux et l&#39;utilisation Comité (IACUC) examen éthique du corps (de AWERB) et, en conformité avec les règlements fédéraux et étatiques. 1. réactif et le matériel Setup <ol><li> Thaw, aliquote et stocker 1 mg / ml solution à -80 ºC laminine. Dissoudre 2 pi dans 1 ml de DMEM / F12, résultant en une concentration de revêtement de 1-2 mg / cm 2. REMARQUE: Thaw laminine lentement sur la glace et se dissoudre dans le froid DMEM / F12 et immédiatement ajouter aux plaques / lamelles. </li><li> Diluer 75 ml de 0,01% Poly-L-ornithine dans 425 ml de PBS et conserver à 4 ºC. REMARQUE: La durée de conservation de la solution de travail est à un mois. </li><li> Dissoudre 25% (poids / volume) de BSA dans du PBS, pH 7,4, et conserver à 4 ° C jusqu&#39;à un year. </li><li> Préparer 50% de FBS (dans HBSS) Moyens de désactivation. </li><li> Préparer un milieu complet par addition de 50 U / ml de pénicilline et de la streptomycine, 1x supplément N2, 5% (vol / vol) de FBS, 0,36% de D - (+) - glucose (poids / volume), 0,25% de SAB (poids / volume) de DMEM / F12 et conserver à 4 ºC. REMARQUE: La durée de conservation est de 10 jours. </li><li> La place des lamelles à 70% (vol / vol) d&#39;éthanol bouillant pendant au moins 30 minutes pour éliminer toute trace de graisse et autoclave. </li><li> La place des lamelles dans des plaques à 24 puits et ajouter 500 ul de solution de poly-L-ornithine à chaque puits. Incuber 1 h sous le capot de culture de tissus à la température ambiante puis, laver 3 fois avec 500 pi d&#39;eau. Ajouter la solution de laminine 500 ul à chaque plaque et incuber O / N dans le humidifié incubateur de culture tissulaire à 37 ° C. REMARQUE: si aucune lavages sont nécessaires après le traitement de la laminine, en lavant les poly-L-ornithine moins de trois fois entraîne la mort des cellules 24 h après la mise en culture. </li><li> Essuyez les conseils de pipettes en verre plus de gaz naturel ou torche fboiteux. NOTE: Après cette étape, le diamètre de la pointe devrait être d&#39;environ la moitié de sa taille normale. </li><li> Couper les bouchons de 1,5 ml microtubes, en utilisant un autoclave ciseaux et autoclave. </li></ol> 2. Dissection du mésencéphale embryonnaire ventral <ol><li> Narcotiser chronométré enceintes (E12.5) barrages par inhalation de CO 2 puis euthanasier par dislocation cervicale. </li><li> Pulvériser l&#39;abdomen avec 70% d&#39;éthanol et de faire une incision abdominale jusqu&#39;à ce que les sacs de l&#39;utérus sont tous exposés. Aide d&#39;une pince coupe l&#39;attachement vaginale et supprimer utérus. Placez l&#39;utérus glacée HBSS, dans une boîte de Pétri de 100 mm. </li><li> Retirer embryons de l&#39;utérus et le sac amniotique, en utilisant une pince (figure 1C). La place embryons dans HBSS glacée fraîche. NOTE: Le rectangle dans la figure 1D indique l&#39;emplacement du mésencéphale ventral embryonnaire. </li><li> Placez les embryons sous un microscope de dissection (10x de grossissement) et disséquer le mésencéphalearc en coupant le cerveau à l&#39;isthme et la région frontière mésencéphalique-diencéphalique, en utilisant la bioscissor et pince (figure 1E, se il vous plaît se référer à la figure 1A pour les lignes de l&#39;incision). </li><li> Après avoir sorti l&#39;ensemble du mésencéphale, faire une coupe dans le mésencéphale médiodorsal (figure 1F). </li><li> Retirer méninges, en utilisant deux pinces (figure 1H). REMARQUE: ÉTAPE CRITIQUE: Cette étape est essentielle pour un rendement optimal et la survie neuronale. Étant donné que les conditions de culture sont optimales pour les cellules du cerveau, la plupart des cellules du tissu environnant meurent après l&#39;étalement et le signal apoptotique à partir de ce tissu peut endommager l&#39;intégrité des cultures. </li><li> Aplatir le tissu mésencéphale sur la boîte de Pétri pour observer la forme de papillon et couper environ la moitié de chaque aile, à l&#39;aide d&#39;une lame de rasoir stérilisé (Figure 1 H, I). REMARQUE: la figure 1J représente le mésencéphale ventral isolé. </li><li> Transférer lemorceau de mésencéphale ventral dans glacée HBSS dans un tube conique de 15 ml et procéder à la prochaine embryon. REMARQUE: les étapes 2.1 à 2.8 ne devrait pas prendre plus d&#39;une heure. Garder les embryons au-delà de ce laps de temps sur la glace diminue de manière significative la viabilité des cellules. </li></ol> 3. La dissociation des cellules ventrale Mésencéphale <ol><li> Transférer les morceaux de mésencéphale ventral sous une hotte à flux laminaire. Retirez le HBSS et ajouter 1 ml préchauffé (37 ºC) 0,05% de trypsine-EDTA dans le tube conique (volumes assez pour jusqu&#39;à 12 morceaux de mésencéphale ventral). Incuber le tissu à 37 ° C pendant 5 à 10 min. </li><li> Retirer la trypsine-EDTA sous le capot et ajouter du milieu de désactivation de 1 ml (le sérum désactivera la trypsine) pour le tissu. </li><li> Éliminer le milieu de désactivation et laver le tissu dans 1 ml de milieu complet deux fois. REMARQUE: Evitez de retirer tout le milieu du tube et pipetage le tissu, pour éviter de jeter les cellules dissociées. </li><li> Ajouter 1 ml de milieu complet et triturate le tissu avec une pipette en verre poli-le-feu. Eviter la formation de bulles et de continuer jusqu&#39;à ce trituration suspension cellulaire unique est atteint. Note: Habituellement, 8-10 traverse la pointe restreint sont obligatoires pour la dissociation complète. </li><li> Centrifuger les cellules à 400 g pendant 5 min à température ambiante et retirez le support. </li><li> Remettre en suspension le culot dans 1 ml de milieu complet en pipettant doucement de haut en bas jusqu&#39;à quatre fois. </li></ol> 4. Les cellules Placage ventrale Mésencéphale <ol><li> Mélanger 10 ul de la suspension cellulaire avec une solution de bleu Trypan 90 pi et de compter le nombre de cellules avec un hémocytomètre. NOTE: La viabilité des cellules peut également être vérifiée à ce stade. </li><li> Placez stérilisés bouchon d&#39;un tube à centrifuger de 100 mm boîtes de Pétri et de transférer les poly-L-ornithine / laminine lamelles, en utilisant une pince, un à la fois au-dessus des bouchons. REMARQUE: Ne pas laver les lamelles et éviter le séchage de la laminine, car cela pourrait provoquer uneven cultures et diminution de la capacité de survie des cellules. </li><li> Ajuster le volume à 1 500 cellules par ul de 1 par addition de milieu complet et ajouter 100 ul (total de 150 000 cellules) de la suspension de cellules au-dessus de chaque lamelle (le volume est optimale pour couvrir la surface de la lamelle couvre-objet sans déversement). Fermez les boîtes de Pétri et incuber pendant 1 h dans un incubateur de culture de tissu humidifié (37 ° C, 5% de CO 2). REMARQUE: étape critique: Cette étape est nécessaire pour améliorer la fixation et la viabilité des cellules et pour augmenter le nombre de lamelles par embryon. Alternativement, les cellules peuvent être transférés directement dans les puits (contenant lamelles couvre), mais le nombre de cellules devrait être porté à 350 000 par puits (au lieu de 150 000). En d&#39;autres termes, en utilisant cette méthode, 8-10 lamelles peuvent être acquis, tandis que le transfert direct donne environ 3-4 lamelles en raison des cellules, qui se fixent sur les plaques (à côté ou au-dessous des lamelles). La moyenne viabilité des cultures, en utilisant cette méthode était d&#39;environ 90%. </li></ol> 5. Culture croissance et l&#39;entretien <ol><li> Après 1 h d&#39;incubation, transférer soigneusement les lamelles y compris le milieu dans des plaques à 24 puits contenant 400 ul préchauffé (à 37 ° C) du milieu complet (volume total: 500 ul). Incuber les cellules O / N à 37 ºC. </li><li> De 24 h après l&#39;incubation des puits, ajouter doucement 500 pi de milieu complet dans chaque puits. REMARQUE: Les premières heures est l&#39;étape la plus critique pour la survie des neurones dopaminergiques et le nombre de cellules survivantes ne change pas de façon significative au-delà de ce point. </li><li> Ne ajoutez pas supplémentaire pour les cultures pour les deux premières semaines ou jusqu&#39;à ce que le milieu devient jaune. Si la culture depuis plus de deux semaines ou des changements de couleur moyennes au jaune, l&#39;échange de la moitié des médias (500 pi) avec frais milieux complets préchauffées (généralement toutes les deux semaines). NOTE: les neurones dopaminergiques au sein de lacultures seraient survivre pendant plus de six semaines dans ces conditions. </li></ol>

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No conflict of interest declared.

Les neurones dopaminergiques dans le mésencéphale sont la principale source de la dopamine dans le système nerveux central. Ils sont divisés en trois groupes, substantia nigra pars compacta (SNPC), Surface tegmentale ventrale (VTA) et sur ​​le terrain retrorubral (RRF) 10, 11. Les neurones dans SNPC et VTA donnent lieu à des grandes voies dopaminergiques, mésocorticales, mésolimbiques et nigro-striée, impliquées dans les fonctions telles que le contrôle de l&#39;émotion, la motivation et le com...

La perte de neurones dopaminergiques de la substantia nigra pars compacta conduit à des symptômes moteurs cardinal de la maladie de Parkinson (MP), le deuxième trouble neurodégénératif le plus répandu. La cause sous-jacente de la disparition de cette population neuronale mésencéphalique ne est pas connu. Pour étudier les voies biochimiques responsables du développement et de moduler les propriétés neurophysiologiques et la survie des neurones MESDA, plusieurs culture cellulaire et des systèmes de modèles animaux ont été utilisés. Les lignées cellulaires immortalisées, y compris la dopaminergique chez le rat lignée cellulaire 1RB 3 AN 27 (N27), le dopaminergique humain lignée cellulaire de neuroblastome SH-SY5Y, la lignée cellulaire hybride dopaminergique de la souris MN9D et mésencéphalique humaine LUHMES cellules ont été utilisées pour des études mécanistiques biochimiques et limitées 1 -5. Pour l&#39;étude de la perte spécifique de neurones MESDA, plusieurs neurotoxine-fondées et les modèles génétiques ont été développés 6-8. Cultures mésencéphale ventrales primaires, Fournir un outil indispensable pour étudier les propriétés neuronales et synaptiques des neurones dopaminergiques et les voies impliquées dans la pathogenèse de cette maladie fréquente. Ici, nous présentons un protocole détaillé pour l&#39;isolement des neurones dopaminergiques mésencéphaliques, qui contient des modifications résultant en la capacité de survie plus élevé et un rendement accru de lamelles par embryon. L&#39;utilisation de la souris E12.5 mésencéphale pré-mature (E14.5 chez le rat) améliore la survie. À cet âge neurones ne ont pas encore développé axones, ce qui laisse des cellules intactes lors de la dissection et minimise la contrainte augmentant ainsi considérablement la viabilité. En outre, dissection minutieuse du mésencéphale ventral, tel que décrit dans l&#39;article 2 de ce protocole, améliore encore la capacité de survie. Pour augmenter le nombre de lamelles par embryon, une méthode de placage de remplacement est présenté dans la section 4 de ce protocole. Cela conduit à un rendement allant jusqu&#39;à 10 lamelles par embryon par rapport aux lamelles de moins de 4conditions de placage classiques réduisant ainsi la quantité d&#39;animaux par expérience. Neurones en culture exposition excroissance des axones et des dendrites, forment des connexions synaptiques et révèlent la présence de marqueurs neuronaux et synaptiques faisant ces cultures appropriée pour l&#39;imagerie de cellules vivantes, immunocytochimique et des études électrophysiologiques. En outre, l&#39;utilisation de cultures de neurones facilite la manipulation génétique et pharmacologique. Excroissance de neurites de deux jours in vitro permet d&#39;études sur le développement. En outre, la survie à long terme des cultures (jusqu&#39;à six semaines) les rend appropriés pour l&#39;étude de la lente, de la dégénérescence progressive de ces neurones.

<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.

Immunohistochimie contre la tyrosine hydroxylase (Th) montre que, entre 0,5 à 1% des cellules en culture sont dopaminergique. Projections neuronales apparaissent dans deux heures après placage et par le premier jour, les axones et les dendrites se distinguent (Figure 2), en utilisant la tyrosine hydroxylase (TH) et la protéine 2 (carte2) anticorps associée aux microtubules (Figure 3). Les neurones de survivre pendant plus de six semaines, et montrent une vaste excroissance. Le ratio...

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.

Isolement, Culture et maintenance à long terme des mésencéphalique primaire dopaminergiques neurones à partir de cerveaux embryonnaires de rongeurs

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

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JoVE Journal

Neuroscience

13.8K vues.  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.

Vidéo: 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.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

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

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Neurosciences

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.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

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.

L&#39;isolement et la culture de neurones de l&#39;hippocampe de souris pendant la grossesse

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 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.

Isolement et culture de cellules endothéliales de la embryonnaire du cerveau antérieur

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.

Culture primaire de souris dopaminergiques neurones

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.

Isolement et culture de dissocié neurones sensoriels De embryons de poulet

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.

Une méthode simplifiée pour les ultra-basse densité, à long terme primaire hippocampique Neuron Culture

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.

L&#39;isolement et la culture des neurones de la moelle épinière chez les souris néonatales

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.

Génération et entretien à long terme sans nerve<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 ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolement et Extraction de l’ARN des neurones, les Macrophages et les cellules microgliales du cerveau de larves de poisson zèbre

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...