Os autores agradecem membros de laboratório anteriores que contribuíram para o refinamento deste protocolo e os NIH para o apoio ao longo dos anos (e DA19808 DA15014 a OM). Anna Abt 1 é um companheiro da &quot;Formação Interdisciplinar de Pesquisa e translacional em neuroAIDS&quot; (T32-MH078795); assim, este trabalho foi apoiado em parte pelos Institutos Nacionais da Saúde sob Ruth Kirschstein National Research Service Award L. 5T32MH079785.

1. Dissection glia (~ 2 semanas antes de neurônios plating) <ol><li> Para se preparar para as ferramentas de dissecção lugar estéril em etanol 70%, adicione 4 mL de médio dissecção fria para 60 pratos mm (um prato por cérebro), e coloque 2,5% de tripsina e DNase no gelo a derreter (ver detalhes sobre instrumentos cirúrgicos na tabela VI ). </li><li> Use uma tesoura grande para decapitar 2-4 dias de idade os filhotes e as cabeças coloque num prato de 100 milímetros estéril. </li><li> Use uma tesoura média para fazer um corte na linha média através da pele e puxar a pele para expor o crânio. Use uma tesoura curva para fazer um corte na linha média e dois cortes laterais através do crânio, sem danificar o tecido cerebral por baixo. Remova a parte superior do crânio para expor o cérebro. </li><li> Extrair o cérebro e colocá-lo em seu prato milímetros própria 60 contendo 4 ml de meio de dissecação a frio (ver tabela I para composição). Coloque o prato sob um estereomicroscópio (Leica ZOOM 2000, zoom de controle: 7-30) e utilize uma pinça No.5 para separar os hemisférios, remova o mesencéfalo, e descasque off as meninges. </li><li> Recolher todos os córtices cerebral dissecado em um prato fresco 60 milímetros, com média dissecação fria, mince e do tecido com uma tesoura curva. </li><li> Transferência do tecido picada a um tubo mL estéreis 15. Obter um volume de 4,5 mL, com média dissecação fria e adicionar 500 mL de tripsina 2,5%. Enrole o tubo em Parafilm e flutuar em banho-maria a 37 ° C por 15 minutos, misturando por inversão a cada 5 minutos. (Nota: aqui descrevemos o protocolo que geralmente seguem ao usar até 5 cérebros, se os filhotes são mais usados, volumes podem precisar de ser ajustado adequadamente) </li><li> Transfira o tecido para um tubo novo mL 15, minimizando o volume de meio contendo tripsina dissecção transitadas. Obter um volume de 4.8mL com meio de dissecção frescos e adicionar 200 mL DNase para alcançar uma concentração final de 60 ug / mL (solução estoque: 3 mg de DNase e 5 mg MgSO4 em 2 mL de meio de dissecção). Triturar gentilmente ~ 20 vezes com uma pipeta estéril 5 mLte, em seguida, adicione 10 mL de meio de revestimento de glia (ver tabela II para a composição). </li><li> Permitir que grandes pedaços de tecido que se contentar com 4-5 minutos, em seguida, transferir os 80% superior da suspensão de células para um tubo de 50 ml. Repita a trituração do tecido com um adicional de 2-3 mL de médio plating glia (e usando uma pequena pipeta, se necessário); mais uma vez, permitir que o tecido para resolver, e adicionar os 80% superior à coleção anterior. Trazer o volume total de 20 mL com meio glia chapeamento e centrifugar por 15 minutos a 280 g (ou 1300 rpm em uma centrífuga IEC Centra CL2). </li><li> Aspirar o sobrenadante e ressuspender o pellet celular em meio de revestimento de glia (cerca de 5-7 mL usando dependendo do número de filhotes usados); em seguida, adicione meio de revestimento adicional, a fim de ter 10 mL para cada cérebro dissecado. Misture bem e placa 10 mL de suspensão de células em cada frasco de 75 cm 2 (ou seja, um cérebro por frasco). </li><li> Mova frascos de cultura a um conjunto de incubadora a 37 ° C (5% a 10 CO2; proporcional à bicarbona de sódiote conteúdo do meio). Alterar meio de cultura após 24-48 horas, depois a cada 3-4 dias. </li></ol> 2. Glia secundário (48 horas antes de neurônios plating) <ol><li> Prepare thermanox lamínulas (se os neurônios serão semeadas em pratos) pela adição de 3 gotas de cera derretida paraplast em torno da circunferência de cada thermanox lavada e autoclavada. Células serão plaqueadas sobre este lado da thermanox. Nosso thermanox são feitas à mão insere reutilizáveis ​​~ 60 milímetros de corte de plástico (ver tabela VI de reagentes) com dois furos ~ 50 milímetros perfurado completamente, embora a cultura disponíveis comercialmente bem inserções podem ser usados ​​no lugar. </li><li> Lavar a glia primária com PBS duas vezes, agitando vigorosamente cada frasco para remover as células solto. Cada frasco com glia principal confluentes irá fornecer cerca de 10 milhões astroglia, por isso usam regularmente 2-4 frascos por experimento. </li><li> Separar as células com 0,05% tryspin-EDTA (1 mL10X solução estoque de 0,5% de tripsina-EDTA diluída em 9 mL de PBS). Combine as células de 2 frascos em um tubo de 50 mL cônico e adicionar uma quantidade igual de médio plating glia (ver tabela II de reagentes). Centrifugar por 15 minutos a 280 g. Aspirar o sobrenadante e dissolver o sedimento em glia alguns mililitros de meio de chapeamento glia. </li><li> Contar as células usando uma hematocytometer e diluir a suspensão de células necessárias (~ 1x10 6 células / mL); placa as células tanto em pratos de 60 milímetros (se os neurônios serão semeadas em lamínulas), ou em lamínulas thermanox (se os neurônios serão semeadas em 60 pratos mm). As células devem ser banhado em 500.000 células por prato ou thermanox usando médio plating glia. No caso de thermanox lamínulas, 600 mL placa de células de uma forma gota a gota, em seguida, após 2 horas thermanox flip e células submergir em meio plating glia. </li><li> Se preparar 60 pratos mm de glia secundário para lamínulas neuronal, a mudança de médio a N2.1 na noite anterior à dissecção neuronal. </li></ol> 3. Dissection neuronale Cultura <ol><li> Prepare lamínulas de vidro (15 mm de diâmetro) pela adição de 3 gotas de cera derretida paraplast em torno da circunferência de cada lamela estéril, a fim de separar fisicamente as camadas de células e prevenir a raspagem de células. Células serão plaqueadas sobre este lado da lamela. </li><li> Revestir as placas ou lamelas com 0,5 mg / mL polilisina (ou poli-L-lisina ou poli-D-lisina dissolvido em tampão borato; ver tabela VI) por 2 horas ou durante a noite. Lavar com água estéril 2-3 vezes e deixe secar completamente. Nota: lamínulas são colocados numa placa de Petri hidrofóbicos para evitar o derramamento / disseminação das soluções durante o revestimento com revestimento de poli-lisina ou célula, que pode facilmente ocorrer se as lamelas são colocadas em placas de cultura de tecidos. </li><li> Prepare-se para a dissecção neuronal como para a dissecção glia (passo 1.1). Euthanize um rato de 17 dias de gravidez, pulverizar a pele com álcool 70%, e corte medialmente através da pele para expor o útero. Remover todos os fetos e colocá-los em umprato milímetros estéril 100. </li><li> Rapidamente dissecar livre e decapitar cada feto, colocando cada cabeça em seu prato milímetros própria 60 com média dissecação fria (4 mL). Nós normalmente dissecar fetos 4-5 por experiência como aproximadamente 20 milhões de neurônios são derivados de cada feto E17. Se os embriões são necessários mais, certifique-se que todo o procedimento (a partir do passo 3,4-3,12) não duram mais de 9-10 minutos. </li><li> Usando um microscópio estereoscópico e No.1/No.5 forceps isolar o córtex cerebral, puxando abrir a pele e crânio, extrair o cérebro, separando os hemisférios, e remoção do mesencéfalo e meninges. </li><li> Recolher todos os córtices dissecada em um prato fresco 60 milímetros, com média dissecação fria, mince e do tecido com tesoura curva em pedaços ~ 1 mm. </li><li> Transferência do tecido picada a um tubo mL estéreis 15. Obter um volume de 4,5 mL, com média dissecação fria e adicionar 500 mL de tripsina 2,5%. Enrole o tubo em Parafilm e flutuar em um banho de água 36 ° C por 15 minutes, misturando por inversão a cada 5 minutos. </li><li> Transfira o tecido para um tubo novo mL 15, minimizando o volume de meio contendo tripsina dissecção transitadas. Trazer para 4,8 mL com meio dissecção frescos e adicionar 200 mL DNase para alcançar uma concentração final de 60 ug / mL. Triturar cerca de 10 vezes com uma pipeta estéril de 5 ml ou 2; repita esta etapa, se necessário usando um fogo-polido (frio) pipeta Pasteur para dissociar a maior parte do tecido. Então permitir que os pedaços de tecido (em geral, muito poucos, se houver) para resolver por 4-5 minutos. </li><li> Remover a suspensão de uma única célula superior, deixando para trás pedaços de tecido resolvido, e colocar essa solução em um novo tubo com igual volume de meio neurônio chapeamento (ver tabela III). Misture bem, mas com cuidado, em seguida, contar as células; placa em 15 lamínulas mm (35.000 células em 200 mL cada) ou 60 pratos mm (1x10 6 células em cada 3 mL), conforme necessário. Transferência de células para a incubadora (36,5 ° C, 5-10% CO2). </li><li> Depois de 3-4 horas combine da camada de neurônio ea glia secundário como se segue. Para os neurônios em 60 pratos mm, lavar as células com DMEM quente, mudar a médio e 4 ml de meio livre de soro (N2, veja a tabela IV), e adicionar um thermanox com glia para cada placa (células gliais deve estar voltado para os neurônios). Para lamínulas neuronal, transferência 3-5 lamínulas para cada prato 60 milímetros de glia secundário, com os neurônios voltado para baixo. </li><li> 24 horas após o plaqueamento os neurônios, adicionar 10 mM de citosina-β-D-arabinofuranoside para cada prato, a fim de evitar a proliferação da glia. Preparamos quatro soluções estoque mM, diluir 1:10 com o meio N2, e adicionar 25 uL / ​​mL da solução diluída para cada prato. </li><li> As células são geralmente utilizados para experimentos após 7-10 dias em cultura, embora o tempo exato depende estágio de diferenciação desejado, pois eles têm sido cultivados por até 4 semanas sem uma redução significativa na sobrevida. Após a primeira semana, recomenda-se que a metade do volume de meios de ser substituído a cada semana. </li></ol> Poucas horas depois de neurônios ter anexado ao substrato, eles começam a estender lamelliopodia. Processos de continuar a alongar (Fig 2A) e polarizar longo de vários dias em cultura, e os axônios e dendritos estendida são claramente visíveis (Fig 2B; MAP2 coloração). Neurônios maduros como os mandris dendríticas tornam-se mais elaboradas e desenvolvem contatos sinápticos, cerca de 2-3 semanas após o plaqueamento muitas espinhas dendríticas são visíveis (Fig 2C) 11. <img alt="Figura 1" src="/files/ftp_upload/3257/3257fig1.jpg" /> Figura 1. Fluxograma do procedimento para os neurônios de rato cultura cortical junto com uma camada de alimentação glial (células gliais secundário). Em primeiro lugar, 11 dias antes da dissecção neuronal, uma cultura de glia principal é preparado a partir de ratos recém-nascidos (P2-4). Quando as células são confluentes (cerca de nove dias após o plaqueamento da glia primário e dois dias antes da dissecção neuronal), os astrócitos são mechanically separados a partir de células precursoras de oligodendrócitos e microglia e banhado tanto em pratos ou thermanox (glia secundário). Neurônios são então obtidos a partir do córtex de embriões E17 e banhado quer em lamelas ou em pratos (revestidas com poli-lisina). Quatro horas após o plaqueamento, a camada neuronal é adicionado à glia secundário. <img alt="Figura 2" src="/files/ftp_upload/3257/3257fig2.jpg" /> Figura 2 Exemplos de culturas em várias fases (por exemplo, 5 horas, 12 DIV, 21 DIV).. Números são imagens representativas de neurônios corticais em lamínulas em momentos diferentes. Depois de neurônios anexar ao substrato neurônios começam a estender lamelliopodia. Imagem de contraste de fase mostra uma neurites poucos estendida cinco horas após o plaqueamento (A). A imagem no meio (B, de Cook et al. 2010, com permissão) mostra 12 neurônios corticais DIV fixa e histoquímica com o marcador neuronal MAP2 dendríticas. Coloração Hoechst foi usado como um counterstaining. Como neurônios maduros espinhas dendríticas também se tornam visíveis (C). Barras de escala: A, B = 25 mm; C = 10 mM.

<ol>
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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuronal+P53+activity+by+CXCR+4.">Regulation of neuronal P53 activity by CXCR 4.</a> Mol. Cell. Neurosci. 30, 58-66 (2005).</li><li>Patel, J. P. <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+neuronal+CXCR4+by+the+micro-opioid+agonist+DAMGO.">Modulation of neuronal CXCR4 by the micro-opioid agonist DAMGO.</a> J. Neurovirol. 12, 492-500 (2006).</li><li>Shimizu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+transcription+factor+E2F1+in+CXCR4-mediated+neurotoxicity+and+HIV+neuropathology.">Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology.</a> Neurobiol. Dis. 25, 17-26 (2007).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+receptor+CXCR4+regulates+cell-cycle+proteins+in+neurons.">The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons.</a> J. Neurovirol. 9, 300-314 (2003).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+CXCL12+promotes+survival+of+postmitotic+neurons+by+regulating+Rb+protein.">The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein.</a> Cell. Death. Differ. 15, 1663-1672 (2008).</li><li>Khan, M. 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Este protocolo fornece um método para cultura primária de neurônios corticais de ratos na presença de células da glia, permitindo que os neurônios para ser facilmente isolado para análise experimental. O apoio ao desenvolvimento glia de um fenótipo neuronal saudáveis, ao mesmo tempo, modulando respostas neuronal para tratamentos experimentais de forma fisiologicamente relevantes. Além disso, por passaging da glia primária antes de cultura com os neurônios desta população de células torna-se seletivamente ...

<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>16 mM</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>22 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.22 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>Osmolarity</td>
			<td>310&plusmn;10 mOsm</td>
		</tr>
	</tbody>
</table>
Table 1. Dissection Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>50 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
Tabe 2. Glia Plating Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>
Table 3. Neuron Plating Medium.
<table>
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>50 mg/100mL</td>
		</tr>
	</tbody>
</table>
Table 4. N2 Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Sodium Borate</td>
			<td>12.5 mM</td>
		</tr>
	</tbody>
</table>
Table 5. Borate Buffer (for poly-lysine).
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
		</tr>
		<tr>
			<td>High glucose Dulbecco's Modified Eagle 
			Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat-inactivated</td>
			<td>Hyclone</td>
			<td>26400-044</td>
		</tr>
		<tr>
			<td>Horse Serum, Heat-inactivated</td>
			<td>Hyclone</td>
			<td>H1138</td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL)</td>
			<td>Invitrogen</td>
			<td>15750-060</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Grade VI 
			(Ovalbumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A2512</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Invitrogen</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas 
			(DNase)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5025</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Fisher</td>
			<td>12-646-106</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
		</tr>
		<tr>
			<td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C6645</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica ZOOM 2000</td>
		</tr>
		<tr>
			<td>Cover glasses, Circles, 15 mm, Thickness 
			0.13-0.17 mm</td>
			<td>Carolina</td>
			<td>633031</td>
		</tr>
		<tr>
			<td>Thermanox sheets</td>
			<td>Grace BioLabs</td>
			<td>HS4550</td>
		</tr>
		<tr>
			<td>Large forceps</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>70-4000</td>
		</tr>
		<tr>
			<td>Fine-tipped No.5 forceps</td>
			<td>Fine Science 
			Tools</td>
			<td>91150-20</td>
		</tr>
		<tr>
			<td>Pattern No.1 forceps</td>
			<td>Biomedical 
			Research</td>
			<td>10-1400</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors, straight, sharp-blunt</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>28-1435</td>
		</tr>
		<tr>
			<td>Micro Dissecting scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-2070</td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-1395</td>
		</tr>
	</tbody>
</table>

bilaminar co-culture of primary rat cortical neurons and glia

Este vídeo irá guiá-lo através do processo de neurônios corticais de ratos cultura na presença de uma camada glial alimentador, um sistema conhecido como um bilaminar ou co-cultura modelo. Este sistema é adequado para uma variedade de necessidades experimental exigindo um vidro de substrato de crescimento ou de plástico e também pode ser usado para a cultura de outros tipos de neurônios. Neurônios de rato cortical obtidos a partir da fase tardia embrionário (E17) são semeadas em lamínulas ou pratos de cultura de tecidos de frente para uma camada de alimentador de glia cultivadas em pratos de plástico ou lamelas (conhecido como Thermanox), respectivamente. A escolha entre as duas configurações depende da técnica específica experimental usado, o que pode exigir, ou não, que os neurônios são cultivados em vidro (por exemplo, imagens de cálcio em relação Western blot). A camada glial alimentador, uma cultura astroglia enriquecido secundária de glia mista, é separado preparado a partir do córtex de filhotes de ratos recém-nascidos (P2-4) antes da neuronaldissecção. Uma grande vantagem deste sistema de cultura em relação a uma cultura de neurônios é apenas o suporte de crescimento neuronal, sobrevivência e diferenciação proporcionada por factores tróficos secretados a partir da camada alimentador glial, que se assemelha mais precisão o ambiente do cérebro in vivo. Além disso, a co-cultura pode ser usado para estudar as interações neuronal glial-1. Ao mesmo tempo, a contaminação glia na camada neuronal é impedido por diferentes meios (cultura de baixa densidade, além dos inibidores da mitose, a falta de soro e uso de meio de cultura otimizado), levando a uma camada praticamente pura neuronal, comparável a outros métodos estabelecidos um -3. Neurônios podem ser facilmente separados da camada glial a qualquer momento durante a cultura e utilizadas para diferentes aplicações experimentais que vão de eletrofisiologia 4, biologia celular e molecular 5-8, bioquímica 5, de imagem e microfoneroscopy 4,6,7,9,10. Os neurônios primários estender axônios e dendritos para formar sinapses funcionais 11, um processo que não é observado em linhagens de células neuronais, embora algumas linhagens celulares se estendem processos. Um protocolo detalhado de neurônios do hipocampo de ratos cultura usando este sistema de co-cultura tem sido descrito anteriormente 4,12,13. Aqui detalhamos um protocolo modificado adequado para neurônios corticais. Como cerca de 20x10 6 células são recuperados de cada embrião de rato, este método é particularmente útil para experimentos que requerem um grande número de neurônios (mas não preocupado com uma população altamente homogênea neuronal). A preparação de neurônios e células gliais precisa ser planejada de maneira tempo específico. Vamos fornecer o protocolo passo a passo para os neurônios corticais de ratos cultura, bem como a cultura de células gliais para apoiar os neurônios.

Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Aqui nós fornecemos um protocolo para a cultura de neurônios corticais de ratos na presença de uma camada de alimentação glial. Os neurônios cultivados estabelecer polaridade e criar sinapses, e pode ser separada da glia para uso em diversas aplicações, tais como eletrofisiologia, imageamento de cálcio, ensaios de sobrevivência celular, imunocitoquímica e RNA / DNA / isolamento de proteínas.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e 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 ...

bilaminar-co-culture-of-primary-rat-cortical-neurons-and-glia

Research

JoVE Journal

Neuroscience

19.5K visualizações.  Drexel University College of Medicine. Aqui nós fornecemos um protocolo para a cultura de neurônios corticais de ratos na presença de uma camada de alimentação glial. Os neurônios cultivados estabelecer polaridade e criar sinapses, e pode ser separada da glia para uso em diversas aplicações, tais como eletrofisiologia, imageamento de cálcio, ensaios de sobrevivência celular, imunocitoquímica e RNA / DNA / isolamento de proteínas.

Vídeo: Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

No útero Eletroporação seguido por Cultura Neuronal primário para estudar a função do gene no subconjunto dos neurônios corticais

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neurociência

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

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

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Determinação de Membrana Mitocondrial Espécies de Oxigênio Reativas Potencial e em neurônios de rato vivo Cortical

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Análise de imagens de Neuron Glia para Interação em Plataforma Cultura microfluídicos (MCP) baseado Axon Neuronal e Sistema Glia co-cultura

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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.

Imagiologia espinhas dendríticas dos neurônios do hipocampo de rato primária usando 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. ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantificação de filamentosas actina (F-actina) puncta no Rat neurônios corticais

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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.

Geração de neuroesferas do hipocampal preliminar misturado e dos neurônios corticais isolados de E14-E16 embrião do rato de Sprague Dawley

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 ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migração, Quimio-Atração e Ensaios de Cocultura para Células Endoteliais derivadas de células-tronco humanas e neurônios gabaergicos

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Isolamento e Cultura dos Neurônios Primários e Glia da Bexiga Urinária de Ratos Adultos

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Cocultura de Neurônios de Gânglio Axotomizado de Rato Retinal com Glia Ensheathing Olfactory, como um Modelo In Vitro de Regeneração Axonal Adulta

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...