Ce travail est soutenu par NIH NS065183 et des fonds de démarrage de l&#39;École de médecine de Rutgers Robert Wood Johnson.

Une. Préparation Rat Astrocyte Culture pour les milieux conditionnés et astrocyte-neurone co-cultures. <ol><li> Préparer dissection tampon (BSS, voir le tableau 1 pour la recette) et conserver à 4 ° C jusqu&#39;à utilisation. </li><li> Anesthésier les ratons nouveau-nés (P0-P2) avec de l&#39;isoflurane dans un bécher de 500 ml. </li><li> Lorsque les chiots sont immobiles, pulvériser avec 70% d&#39;éthanol et décapiter. </li><li> Retirez le cerveau. <ol><li> Tenez fermement la tête avec une paire de Dumont # 5 forceps, et utiliser des ciseaux fins de faire une incision médiane à travers la peau et le crâne. </li><li> Exposer le cerveau en réfléchissant le crâne sur les côtés, et retirer le cerveau dans un plat contenant BSS froid. </li></ol></li><li> Séparer les hémisphères cérébraux du diencéphale et le tronc cérébral sous un microscope à dissection. Retirez soigneusement toutes les méninges en stabilisant le tissu avec une paire de pinces et en tirant doucement les méninges avec une autre paire de pinces. </li><li> Recueillir tous les hémisphères in une boîte de 60 mm. L&#39;utilisation de petits ciseaux, tissu mince aussi finement que possible. Puis transférer le tissu haché dans un tube conique de 50 ml. </li><li> Ajouter 1,5 ml de 1% DNase I et 1,5 ml de 2,5% de trypsine et 12 ml de BSS (volume total de 15 ml) de cerveau. Incuber dans un bain d&#39;eau agité pendant 15 min à 37 ° C. Pour assurer un bon mélange, le tube est tourbillonné à la main toutes les 5 min. </li><li> Transférer le surnageant dans une passoire 70 um de cellule sur un nouveau 50 ml tube conique. Ajouter 3 ml de FBS à ce surnageant. </li><li> Pour les morceaux restants, ajouter 13,5 ml de BSS et 1,5 ml 2,5% de trypsine et incuber au bain-marie en agitant pendant encore 15 minutes. </li><li> Transférer le surnageant restant à travers le tamis de la cellule et de combiner avec le surnageant de l&#39;étape 1.8. </li><li> Centrifuger à 1000 rpm pendant 5 min pour sédimenter les cellules. </li><li> Remettre en suspension les pastilles dans 5 ml de milieu gliales. Le surnageant peut être de nouveau centrifugé pour sédimenter les cellules restantes. </li><li> Compter les cellules en utilisant un hématimètre. </li><li> Plaque cells à une densité de 1 x 10 7 par cm 2 flacon 150. </li><li> Changer de support aux médias gliales frais le jour après l&#39;étalement. Ensuite, nourrir les cellules deux fois par semaine avec les médias gliales. </li><li> Lorsque les cellules ont atteint&gt; 80% de confluence (environ 10 jours après placage), geler les cellules dans 90% de sérum de cheval et 10% de DMSO et maintenir les stocks dans l&#39;azote liquide. Les cellules sont congelées à 2 x 10 6 par flacon. Environ 5 flacons peuvent être congelés dans chaque flacon. </li><li> Environ 10-14 jours avant la mise en place de la culture de l&#39;hippocampe, les cellules gliales sont étalées dans les stocks surgelés. Nous plaquons habituellement cinq plaques de 6 puits, ce qui est suffisant pour soutenir la croissance de 90 lamelles de neurones de l&#39;hippocampe (trois lamelles couvre 15 mm rondes par puits). Nous plaquons également une huit à dix plats de 60 mm supplémentaires de cellules gliales, qui est utilisé pour le conditionnement de médias N2.1 pour la transfection. La veille de culture neuronale, les cellules doivent être nourris avec les médias NB27. Les astrocytes devraient idéalement être&gt; 90% de confluence à eest le point. Nous constatons que la congélation réduit considérablement le nombre de cellules microgliales dans des cultures astrocytaires. Etant donné que l&#39;augmentation neurotoxicité est observée lorsque la microglie est présent dans les cultures astrocytaires, on utilise toujours la culture astroglial préparé à partir de stocks congelés de co-culture avec les neurones. </li></ol> 2. Neuronale Culture <ol><li> Nettoyer les lamelles de verre de premier rinçage dans milliQ 2x de l&#39;eau. Elles sont ensuite trempées dans de l&#39;acide nitrique concentré pendant 24 heures, et on les rince avec de l&#39;eau milliQ pendant au moins cinq fois au cours d&#39;un total de 2 heures. Lamelles sont séchées et stérilisées par cuisson dans un four à 225 ° C pendant 6 heures. </li><li> Transfert des lamelles couvre 60 mm plats après la stérilisation. Placer quatre points de paraffine stérile à proximité du bord extérieur de chaque lamelle afin de maintenir séparés des neurones au cours de la co-culture des cellules gliales. </li><li> Enduire les lamelles de 1 mg / ml de poly-L-lysine dans un tampon borate 0,1 M pendant une nuit à 37 ° C incubateur. </li><li> Le lendemain, rincez deux fois en lamelles m stérileilliQ eau pendant au moins 15 minutes à chaque fois. Ils sont ensuite incubés pendant la nuit dans les médias de placage à 37 ° C incubateur. </li><li> Euthanasier rat enceinte (E18) par l&#39;anesthésie isoflurane suivie par un pneumothorax, et euthanasier les rats embryonnaires par décapitation. Retirer le cerveau et disséquer les hémisphères cérébraux, comme décrit ci-dessus. </li><li> Disséquer l&#39;hippocampe de l&#39;hémisphère cérébral. Placez tous les hippocampes dans un tube conique de 15 ml, et digérer avec 0,25% de trypsine (0,5 ml 2,5% de trypsine, 4,5 ml BSS) à 37 ℃ pendant 15 min. </li><li> Hippocampes digérés sont rincées dans BSS 3x pendant 5 minutes chacun. Ils sont ensuite triturés avec une pipette Pasteur en verre, et la densité cellulaire est déterminée en utilisant un hématimètre. Les neurones sont ensuite ensemencées à une densité de 2 x 10 5 cellules par boîte de 60 mm. </li><li> Environ 2-4 heures après le placage, le transfert des lamelles couvre avec les neurones attachés aux plats contenant des cellules astrocytes, avec les neurones vers le bas vers la glie. Au DIV3, ajouter la cytosine arabinoside à lales cellules à une concentration finale de 5 uM pour arrêter la prolifération des cellules gliales. </li></ol> 3. Calcium transfection <ol><li> Médias condition de N2.1 sur la glie le jour avant la transfection. </li><li> Le jour de la transfection, les représentants des médias de N2.1 conditionné hors de la glie et transférer dans un nouveau plat. Transfert des lamelles couvre avec les neurones dans les médias de N2.1 conditionné. Laissez s&#39;équilibrer dans l&#39;incubateur pour 10-30 min. </li><li> Pour une série de tubes stériles, combiner 1-4 ug d&#39;ADN, 12,5 ul de 2 M de CaCl 2, et H 2 O stérile pour un volume total de 100 pl. À un second ensemble de tubes, ajouter 100 ul de HBS 2x. </li><li> Ajouter le volume ⅛ de HBS 2x (12,5 pi) à la fois dans le tube contenant le mélange de CaCl2 / d&#39;ADN, vortex pendant quelques secondes, à chaque fois. Laisser les tubes reposer pendant 15 min. </li><li> Après 15 min d&#39;incubation, ajouter goutte à goutte le mélange de transfection aux cellules. Incuber pendant 1-1,5 heures. Une couche de précipités de sable-comme doit être visiblesous le microscope en utilisant un objectif 10X. </li><li> Après incubation, rincer lamelles fois avec du tampon de lavage chaud HBS. </li><li> Retour à lamelles plats originaux avec des cellules gliales, ajouter de l&#39;acide kynurénique à une concentration finale de 0,5 mM. </li><li> Neurones transfectées peuvent être visualisés en direct ou traitées pour immunocytochimie dès le lendemain. Les neurones peuvent survivre jusqu&#39;à trois semaines après la transfection. </li></ol>

<ol>
	<li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30, 6171-6177 (2010).</li><li>Zeitelhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+mammalian+neurons+via+nucleofection.">High-efficiency transfection of mammalian neurons via nucleofection.</a> Nat. Protoc. 2, 1692-1704 (2007).</li><li>Janas, J., Skowronski, J., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+delivery+of+RNAi+in+hippocampal+neurons.">Lentiviral delivery of RNAi in hippocampal neurons.</a> Method Enzymol. 406, 593-605 (2006).</li><li>Graham, F. L., vander Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52, 456-467 (1973).</li><li>Craig, A. M., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+cultured+neurons.">Transfecting cultured neurons.</a> Culturing Nerve Cells. , (1998).</li><li>Alavian, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13, 1224-1233 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+synaptic+function+by+VAC14,+a+protein+that+regulates+the+phosphoinositides+PI(3,5)P(2)+and+PI(5)P.">Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P.</a> EMBO J. 31, 3442-3456 (2012).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Curr. Protoc. Neurosci. Chapter 3, Unit 3 11 (2001).</li><li>Goslin, K. A. H., Banker, G., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Hippocampal+Neurons+in+Low-Density+Culture.">Rat Hippocampal Neurons in Low-Density Culture.</a> Culturing Nerve Cells. , (1998).</li><li>Kohrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+convenient,+and+effective+method+to+transiently+transfect+primary+hippocampal+neurons.">Fast, convenient, and effective method to transiently transfect primary hippocampal neurons.</a> J. Neurosci. Res. 58, 831-835 (1999).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Goetze, B., Grunewald, B., Baldassa, S., Kiebler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+formation+of+a+DNA/calcium+phosphate+coprecipitate:+application+for+transfection+of+mature+hippocampal+neurons.">Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.</a> J. Neurobiol. 60, 517-525 (2004).</li></ol>

Aucun conflit d&#39;intéressé déclaré.

Il ya plusieurs paramètres clés qui doivent être soigneusement contrôlée pour transfections toujours réussies 10,11. Le paramètre le plus critique pour la transfection au phosphate de calcium est la valeur de pH de 2 x HBS, qui dans nos mains varie habituellement entre 7.10 à 7.15. Nous vous conseillons d&#39;effectuer trois lots de stocks avec des valeurs de pH par pas de 0,05 pour tenir compte de la différence entre les pH-mètres. Autrement, le kit de transfection mammifère Clontech fournit 2x HB...

Neurones primaires sont l&#39;un des types cellulaires les plus difficiles à transfecter car ils sont post-mitotiques et sont très sensibles aux changements des micro-environnement. Il existe quatre types de méthodes pour l&#39;expression de gènes exogènes et ARN court en épingle à cheveux (shRNA) dans ces cellules une couramment utilisés. Chacun a ses propres avantages et inconvénients. Par exemple, l&#39;électroporation est généralement effectuée sur les neurones fraîchement isolés 2, les cellules doivent être transférés dans des cuvettes pour la transfection. infection par le virus peut généralement atteindre un rendement très élevé 3, mais il est plus de main-d&#39;œuvre et risqué pour les opérateurs. Beaucoup de réactifs de transfection médiée par un lipide sont disponibles dans le commerce, avec des degrés variables de succès dans les neurones et les différents niveaux de cytotoxicité. la transfection au phosphate de calcium représente une méthode pratique et économique pour l&#39;introduction de gènes étrangers dans les neurones. La méthode a été utilisée pour la première à introduire de l&#39;ADN de l&#39;adénovirus dans cel mammifèresls par Graham et Van Der Eb (1973) 4. La transfection a été réalisée en mélangeant du chlorure de calcium avec de l&#39;ADN recombinant dans un tampon de phosphate. Ceci permet la formation de phosphate de l&#39;ADN / de calcium précipite, qui, en cas de chute progressivement sur ​​une monocouche de cellules, d&#39;adhérer à la surface de la cellule, sont repris par endocytose et finalement pénétrer dans le noyau 5. Ce processus conduirait à l&#39;expression de gènes étrangers introduits dans la cellule cible. Efficacité typiques de calcium gamme de transfection de phosphate entre 0,5-5% 6-8. Cependant, avec une optimisation soigneuse et constante de l&#39;exécution du protocole expérimental, il est possible d&#39;atteindre une efficacité de transfection de près de 50%. Nous décrivons ici notre protocole détaillé pour la transfection au phosphate de calcium de neurones hippocampiques primaires, qui sont co-cultivées avec des cellules astrogliales dans un format en sandwich 9.

<table border="0" cellpadding="0" cellspacing="0" width="879">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">Dumont #5 forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Fine scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td>straight / sharp 8.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Vannas-Tubinger spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15008-08</td>
			<td>angled / sharp / 8.5 cm / 5 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Standard pattern forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11000-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Student surgical scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">91401-14</td>
			<td>blunt / 14.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td>angled to side / 9 cm / 10 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Isoflurane</td>
			<td style="width:207px;">Webster Veterinary</td>
			<td style="width:119px;">14043-225-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Poly-L-lysine</td>
			<td>Sigma</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Sigma</td>
			<td>M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">100 mM Sodium pyruvate</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Glucose</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x HBSS</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">1 M HEPES, pH 7.3</td>
			<td>Gibco/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Gibco/Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Media</td>
			<td>Gibco/Invitrogen</td>
			<td>21130-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement (50x)</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">2.5% Trypsin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">DNase I</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Cytosine arabinoside</td>
			<td style="width:207px;">Calbiochem/EMD Millipore</td>
			<td align="right" style="width:119px;">251010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Kynurenic acid</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium phosphate transfection of primary hippocampal neurons

précipitation au phosphate de calcium est une méthode pratique et économique pour la transfection de cellules en culture. Avec l&#39;optimisation, il est possible d&#39;utiliser cette méthode sur des cellules difficiles à transfecter comme les neurones primaires. Ici, nous décrivons notre protocole détaillé pour le phosphate de calcium transfection de neurones de l&#39;hippocampe co-cultivés avec des cellules astrocytes.

Lorsque les différents paramètres de transfection sont optimisés et contrôlés soigneusement de expérience à l&#39;autre, il est possible d&#39;obtenir des efficacités de transfection allant jusqu&#39;à 50%. Figure 1 montre un domaine de neurones qui sont transfectées avec la GFP sur DIV4. Le champ contient un total de 28 neurones, parmi lesquels 16 ont été transfectées. Cela représente un rendement de plus de 50%. Un échantillon des autres champs de la même lamelle montre le rendement gl...

Watch this Scientific Journal Video about Calcium Phosphate Transfection of Primary Hippocampal Neurons at JoVE.com

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Phosphate de calcium transfection de primaire neurones de l&#39;hippocampe

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

calcium-phosphate-transfection-of-primary-hippocampal-neurons

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JoVE Journal

Neuroscience

25.1K vues.  Rutgers University.  précipitation au phosphate de calcium est une méthode pratique et économique pour la transfection de cellules en culture. Avec l'optimisation, il est possible d'utiliser cette méthode sur des cellules difficiles à transfecter comme les neurones primaires. Ici, nous décrivons notre protocole détaillé pour le phosphate de calcium transfection de neurones de l'hippocampe co-cultivés avec des cellules astrocytes. 

Vidéo: Calcium Phosphate Transfection of Primary Hippocampal 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.

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 ...

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Neurosciences

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 ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

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 ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Faible densité primaire hippocampique Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Mesurer l’endocytose de la vésicule synaptique dans des neurones Hippocampal cultivés

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Génération de neurosphères à partir de neurones hippocampes et corticales primaires mixtes isolées de l'embryon de rat Sprague Dawley E14-E16

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Utilisation de neurones hippocampiques cultivés primaires pour étudier l’assemblage des segments initiaux axonaux

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Culture primaire de faible densité facile et reproductible utilisant un stock congelé de neurones embryonnaires de l’hippocampe

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imagerie calcique d’ensembles neuronaux dans des réseaux de neurones sensoriels primaires dans des ganglions radiculaires dorsaux intacts

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...