Agradecemos Dr. Shirin Bonni para proporcionar o construto pSil-GFP, Dr. Alper Uzun para a ilustração da Figura 1, e com a instalação bioimaging Leduc para microscopia confocal. EMM é suportado pelo Prémio Carreira para a Ciência Médica da Burroughs Wellcome Fund, uma NARSAD Prêmio jovens investigadores, e NCRR NIH COBRE P20 RR018728-01. SBL é suportado pelo NIH NCRR COBRE P20 RR018728-01, e tem recebido apoio do PHS NRSA 5T32MH019118-20.

1. Preparar Soluções Cultura e Mídia (e não em vídeo) <ol><li> Prepare 1 litro de solução de Hank completa do sal equilibrada (HBSS) contendo HBSS 1x, 2,5 mM de HEPES (pH 7,4), 30 mM de D-glucose, 1 mM de CaCl2, 1 mM de MgSO4 e 4 mM de NaHCO3. Adicione a água bidestilada (DDH 2 O). Filtrar esterilizar com um filtro de 0,2 mícrons e armazenar a 4 ° C. </li><li> Preparar meio de cultura usando fatia 35 mL de Basal Eagle Médium meios de comunicação, 12,9 mL de HBSS completos, 20 mM de D-glucose (1,35 mL de solução 1 M), 1 mM de Glutamax (0,25 mL de uma solução 200 mM), 0,5 ml de penicilina -estreptomicina estoque 100x. Filtrar esterilizar com um filtro de 0,2 mícrons, em seguida, adicionar calor soro de cavalo inactivado a uma concentração final de 5%. </li><li> Preparar solução de trabalho laminina, fazendo um 1 mg / mL de solução stock laminina com água destilada estéril desionizada. Preparar alíquotas de 100 uL em tubos de 0,5 mL eppendorf e congelar a -80 ° C. </li><li> Prepare Poly-L-lisina de trabalho paralução pela adição de 5 mL de estéril de H2O a 5 mg de poli-L-lisina para fazer uma 1 mg / mL de solução stock. Preparar alíquotas de 1 ml e congeladas a -20 ° C. </li><li> Preparar solução de revestimento por diluição de 1 mL de poli-L-lisina e 100 uL de laminina a um volume final de 12 mL com água estéril. Faça desta nova solução de cada vez. </li></ol> 2. Preparando Insere Slice organotípicas (e não em vídeo) <ol><li> Prepare-se dois períodos de seis poços com uma inserção de cultura por bem, usando uma pinça estéril. Adicionar 2 mL de estéril ddH2O debaixo das inserções de cultura. </li><li> Adicionar 1 mL da solução de revestimento no topo da membrana tendo o cuidado de não perfurar a membrana. Incubar durante a noite numa incubadora humidificada a 37 ° C e 5% de CO 2. </li><li> Remova a mídia de revestimento e lave com membrana estéreis H 2 0 três vezes. Deixe insere secos antes do uso. Adicionar 1,8 mL de meio de cultura e fatia lugar em 37 ° C incubadora. Enrole as placas não utilizados com Parafilme armazenar a 4 ° C durante até 4 semanas. </li></ol> 3. Preparando-se para eletroporação (e não em vídeo) <ol><li> Prepare construções RNAi para eletroporação. Dupla fita inserções hairpin RNA foram clonados num vector pSilencer. O contém o plasmídeo: 1) um promotor que dirige a U6 duplo filamento geração de RNA, e 2) uma cassete de expressão da GFP conduzida pelo promotor CMV 8,9. Este plasmídeo foi descrito previamente por Konishi e colaboradores 9 e outros 7,8. Os plasmídeos são purificados utilizando um kit Qiagen maxi-prep, e utilizado a uma concentração de 1 mg / mL. </li><li> A fim de visualizar o DNA enquanto injectando-o, preparar uma solução 0,5% de corante verde rápido e usá-lo às 1:20 com o DNA a ser injectado (geralmente 20 uL de ADN com 1 uL de verde rápido). Sobra DNA-rápido mistura corante verde pode ser armazenada a -20 ° C durante até uma semana. </li><li> Área de dissecção Limpo, dissecting ferramentas e vasos vibratome com etanol 70%. Arrefeça HBSS completas de modo que é arrefecida em gelo. Frio navio vibratome por embalagem de gelo ao seu redor dentro do vibratome com um pouco de água para o resfriamento rápido. Preparar 3% de agarose de baixo ponto de fusão usando HBSS completos. Microondas durante 1 min. Evitar o excesso de ebulição. Manter em um banho-maria a 42 ° C até à utilização. </li><li> Eletroporação parâmetros são definidos da seguinte forma. Para um uso de embriões E15 35 V, 5 pulsos, comprimento de 100 ms, 900 ms de intervalo entre os pulsos. Para os animais mais velhos, para assegurar a electroporação, utilizar uma tensão mais elevada até 50 V ou aumentar o número de pulsos até 8 pulsos. Para evitar danificar o tecido em animais jovens, usar pulsos ou menos, ou até 2 leguminosas e inferior de tensão até 25 V. Estes parâmetros podem ser variados e determinada empiricamente, dependendo da idade do animal. </li></ol> 4. Dissecção e eletroporação (em vídeo) <ol><li> Após eutanásia de uma mulher grávida, dissecar embriões para fora geladas HBSS completos. Keep cada embrião nos seus individuais sacos da placenta. </li><li> Dissecar embrião fora e cortar a cabeça após a primeira vértebra. Tenha em geladas HBSS completos. </li><li> Para injecção, colocar a cabeça sobre um pedaço de Parafilm no topo de uma placa de Petri. Usando feitos Hamilton seringa (ver Tabela I) injectar cerca de 6-8 ul de DNA: mistura rápida do corante verde através do ventrículo terceiro, a fim de encher, em ambos os ventrículos laterais nas vesículas corticais. Alternativamente, a injecção pode ser feito directamente em cada ventrículo lateral. </li><li> Para a eletroporação in vivo ex, usar eletrodos BTX-pinça de platina. Coloque o eléctrodo positivo para o lado do córtex deseja electroporate topo isto é, da cabeça para o córtex dorsal. </li><li> Após a electroporação, as cabeças incubar em gelo durante pelo menos 5 minutos antes de dissecação. </li><li> Dissecar cérebros em HBSSby gelada de uma pequena incisão na parte lateral da cabeça e descamação da pele fora dos lados da cabeça. Em seguida, compinça fina descascar delicadamente fora da pia do cérebro. Remover o cérebro intacto do crânio, tendo o cuidado para não danificar o córtex. </li></ol> 5. Incorporação e Seccionamento de córtices eletroporados (em vídeo) <ol><li> Transferir a 3% de agarose de baixo ponto de fusão dentro de um molde grande colocada em gelo. A parte inferior do molde irá começar a solidificar rapidamente o que irá impedir cérebros de afundar para baixo para o fundo do molde. Suavemente, transferir cérebros com fórceps finos, um por um tampão após a remoção do excesso com uma Kimwipe ou papel de filtro. Usar uma ponta de pipeta 10 uL de redemoinho os cérebros no interior do molde para assegurar a interface entre o máximo de agarose e tecido cerebral. </li><li> Orientar os cérebros para assegurar que todos os cérebros são na mesma orientação e em aproximadamente o mesmo nível no agarose. Deixe a agarose solidificar durante cerca de 5 min. Use substância adesiva (cola maluca) para anexar os blocos de agarose para que os bulbos olfatórios são em pé. Uma vez que os blocos estão ligados, imediatamente adicionar iHBSS ce frio e aparar o agarose para certificar-se fatias individuais são obtidos para cada cérebro. </li><li> Para cortar os blocos, definir a velocidade do vibratome a uma baixa velocidade (cerca de metade do máximo) e definir a freqüência de vibração lâmina na configuração mais alta. Gerar 250-mm de espessura cortes coronais. Recuperar fatias usando uma espátula dobrada bem e transferi-las para poços de tecidos com um pincel fino ou fórceps. </li><li> Em capuz de cultura de tecidos, transferir fatias em pastilhas revestidas. Adicionar 500 ul de meio de cultura de fatia para cada inserção para fazer a transferência fácil. Até 5 fatias pode ser colocado por pastilha. Remover o excesso de mídia do topo das fatias e incubar a 37 ° C em incubadora humidificada. </li></ol> 6. Cultura e Análise de fatias organotípicas (em vídeo) <ol><li> A fim de manter fatias saudáveis, meios frescos deve ser adicionado pelo menos um dia todos os outros por baixo da membrana através da substituição de metade dos meios de cada vez. </li><li> A fim de analisar fatias após a ddia esired em cultura, fixar as fatias na membrana. Lavar com 1x tampão salina de fosfato (PBS) a 37 ° C três vezes durante 10 min de cada vez. Em seguida, fixar com paraformaldeído a 4% (PFA) durante a noite a 4 ° C ou durante 1 hora à temperatura ambiente. </li><li> Fatias podem ser analisados ​​com diferentes marcadores celulares ou coradas com Hoechst sozinho para visualizar as células eletroporados e não-electroporado. Permeabilizar e bloquear fatias para 2 h à temperatura ambiente com soro de cabra a 10%, Triton 0,1% em PBS 1x com agitação suave. </li><li> Stain com a Hoechst durante 1 h à temperatura ambiente, lavar 3 vezes com PBS 1x 10 min de cada vez com agitação suave. </li><li> Para montar as fatias cortar a membrana com um bisturi, e usar um fórceps finos para transferir a membrana contendo fatias para um vidro fosco desliza em uma câmara de água. Até 5 fatias pode ser colocado por lâmina de vidro. Retire o excesso de água e adicione uma gota de solução Flourmount a cada fatia do cérebro. Suavemente colocar uma lamela sobre as fatias de cérebro e remover umay bolhas de ar. Analise fatias utilizando um microscópio confocal. </li></ol> 7. Inclusão em Parafina alternativa de fatias organotípicas (e não em vídeo) <ol><li> Fatias organotípicas também podem ser incorporados para parafina para uma análise mais fina morfológica, para este fim, a membrana contendo as fatias organotípicas pode ser fixado em 4% PFA como descrito acima. </li><li> As fatias fixos são incorporados em 1% de agarose (pré-aquecido a 37 ° C), e solidificado em gelo durante 30 min. Os blocos de agarose pode então ser pós-fixadas em 4% PFA a 4 ° C durante 30 min. </li><li> O bloco de agarose contendo a fatia organotípico será então embebidos em parafina e processados ​​para imunofluorescência como descrito anteriormente 10. </li></ol> 8. Os resultados representativos Uma representação esquemática do electroporação de córtex de murino e de cultura de fatias organotípicas é mostrado na Figura 1. Este método é uma estratégia útil para rapid avaliação da função de genes envolvidos no desenvolvimento neuronal 11. Dependendo da quantidade de ADN electroporado eo estágio embrionário em electroporação, a eficiência de transfecção irá variar. Fatias começará expressando GFP, pelo menos, 8 horas após a electroporação e células serão submetidos a sequência normal dos acontecimentos neurogénicos (proliferação, migração e diferenciação neuronal precoce) em cultura. A Figura 2 mostra uma fatia do cérebro electroporado que está expressando um controlo pSilencer-GFP vetor e pode-se observar progenitores neuronais, neurônios que migram e neurônios diferenciados da fatia. Fatias organotípicas irá manter a sua morfologia, enquanto que são mantidas em uma interface de meios de comunicação de ar-boa sobre as membranas e pode ser usado até pelo menos 5 dias em cultura. <img alt="A Figura 1" src="/files/ftp_upload/3621/3621fig1.jpg" /> Figura 1. Ilustração de eletroporação in vivo e ex fatia organotípicode cultura de ensaio. E14.5 embriões são dissecados, e individualmente injectados com DNA misturado com corante verde rápido a fim de visualizar o local de injecção. O ADN pode ser injectado em ambos os ventrículos laterais, conforme ilustrado na figura ou no ventrículo terceiro, a fim de encher os ventrículos laterais. Após a injecção, os cérebros são electroporados com um electroporator onda quadrada, colocando o eléctrodo positivo sobre o lado desejado do cérebro. Os cérebros são incorporados em 3% de agarose de baixo ponto de fusão e seccionados usando um vibratome. 250 fatias de cérebro iM são colocados em 0,4 inserções iM e cultivadas até uma semana. GFP pode ser observado após 8 horas pós transfecção. <img alt="A Figura 2" src="/files/ftp_upload/3621/3621fig2.jpg" /> Figura 2. Análise de fatias de cérebro eletroporados. Fatias de cérebro electroporado foram coradas para Hoescht. Córtex dorsal mostra eletroporados progenitores neuronais na zona ventricular (VZ). Neurônios na cortical placa (cp) são delimitadas por a zona marginal (mz). As setas brancas mostram os neurônios que migram. Neste caso, os cérebros foram injectados em E15.5 e electroporado com um vector de controlo pSilencer GFP. As secções representam explantes corticais 4 dias após a electroporação. Barra de escala de 100 um. Solução de problemas: <ol><li> Baixa eficiência de transfecção: Ajustar a concentração de ADN utilizado para pelo menos 1 ug / uL. Sempre use DNA muito limpa a partir de uma preparação maxi se necessário utilizar uma endo-livre Quiagen kit para purificar o DNA. </li><li> As células transfectadas de uma área do cérebro diferente do que o desejado um: Assegure-se que os eléctrodos estão correctamente posicionada e com a posição do eléctrodo positivo para o lado do cérebro a ser electroporado. </li><li> Fatias organotípicas perder morfologia: Troca de mídia a cada dia e garantir que os cortes não estão flutuando na mídia </li><li> Fatias de sair da agarose como eles estão a ser cortada na vibratome: Certifique-se que uma boa interface é feita ao incorporar os cérebros no baixo ponto de fusão de agarose. </li></ol>

<ol>
	<li>Angevine, J. B., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+study+of+cell+migration+during+histogenesis+of+cerebral+cortex+in+the+mouse.">Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.</a> Nature. 192, 766-766 (1961).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-392 (2004).</li><li>Barnes, A. P., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+axon-dendrite+polarity+in+developing+neurons.">Establishment of axon-dendrite polarity in developing neurons.</a> Annu. Rev. Neurosci. 32, 347-347 (2009).</li><li>Haydar, T. F., Bambrick, L. L., Krueger, B. K., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+for+analysis+of+proliferation,+cell+death,+and+migration+in+the+embryonic+neocortex.">Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex.</a> Brain Res. Brain Res. Protoc. 4, 425-425 (1999).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. 2002, pl9-pl9 (2002).</li><li>Guerrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-BAR+domain+of+srGAP2+induces+membrane+protrusions+required+for+neuronal+migration+and+morphogenesis.">The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.</a> Cell. 138, 990-990 (2009).</li><li>Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+regulation+of+axonal+morphogenesis+by+the+Cdh1-APC+target+SnoN.">Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.</a> Neuron. 50, 389-389 (2006).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PLoS Genet. 4, e1000111-e1000111 (2008).</li><li>Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303, 1026-1026 (2004).</li><li>Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+paraffin-embedding+of+mouse+tissues+for+GFP+visualization.">Fixation and paraffin-embedding of mouse tissues for GFP visualization.</a> Cold Spring Harb Protoc. 2009, 5298-5298 (2009).</li><li>Hand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-45 (2005).</li><li>Taniguchi, Y., Young-Pearse, T., Sawa, A., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Electroporation+as+a+Tool+for+Genetic+Manipulation+in+Vivo+to+Study+Psychiatric+Disorders:+From+Genes+to+Circuits+and+Behaviors.">In Utero Electroporation as a Tool for Genetic Manipulation in Vivo to Study Psychiatric Disorders: From Genes to Circuits and Behaviors.</a> Neuroscientist. 18, 169-179 (2012).</li></ol>

Não temos nada a divulgar.

Estes métodos envolvem a eletroporação ex vivo de plasmídeos codificando a fita dupla de RNA grampos 8 e cultura de fatias organotípicas 4 oferece diversas vantagens. Primeiro, estes métodos permitem uma avaliação rápida de RNAi derivados de fenótipos. A inclusão de uma GFP codificação expressão cassete no vector pSilencer mesmo que contém o promotor U6 que impulsiona o gancho de cabelo de ARN de cadeia dupla permite uma identificação rápida e caracterização de células...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Hamilton seringa </td><td> HAMILTON </td><td> 80008 </td><td> 31 gauge, 0,5 centímetros de comprimento, PT-4 (nível de chanfradura ponto), o volume de 10 ul </td></tr><tr><td> Tweezertrodes Platinum </td><td> BTX </td><td> 45-0489 </td><td> Tamanho 5 milímetros </td></tr><tr><td> ECM830 electroporator </td><td> BTX </td><td> 45-0002 </td><td></td></tr><tr><td> BTX Footswitch </td><td> BTX </td><td> 45-0208 </td><td> Para utilização com ECM830 electroporator </td></tr><tr><td> Vibratório micrótomo de lâmina </td><td> LEICA </td><td> VT1000 S </td><td></td></tr><tr><td> 6 - prato bem para usar com inserções </td><td> FALCON </td><td> 353502 </td><td> Contém entalhes para encaixar inserções </td></tr><tr><td> Cultura inserções de tecido </td><td> FALCON </td><td> 353090 </td><td> 0,4 micrômetro </td&gt; </tr><tr><td> Verde rápido </td><td> SIGMA </td><td> F7252 </td><td></td></tr><tr><td> Baixo ponto de fusão Agarose </td><td> FISHER </td><td> BP165-25 </td><td> DNA grau </td></tr><tr><td> A laminina </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P5899 </td><td></td></tr><tr><td> Basal Eagle Médium </td><td> Sigma Aldrich </td><td> B-1522 </td><td></td></tr><tr><td> HBSS 10x sem Ca e Mg </td><td> GIBCO </td><td> 14180-046 </td><td></td></tr><tr><td> HEPES livre de ácido </td><td> Sigma-Aldrich </td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

O córtex cerebral dirige funções cognitivas superiores. Esta estrutura de seis camadas é gerado no interior um do primeiro, a maneira de fora para último, em que os neurónios primeiros nascidos permanecem mais próximos do ventrículo enquanto que os neurónios últimos nascidos migrar passado os neurónios primeiros nascidos no sentido da superfície do cérebro 1. Além disso a migração neuronal 2, um processo chave para a função cortical normal é a regulação da morfogénese neuronal 3. Enquanto morfogênese neuronal pode ser estudada in vitro em culturas primárias, há muito a ser aprendido a partir de como esses processos são regulados em ambientes de tecido. Nós descrevemos técnicas para analisar a migração neuronal e / ou morfogénese em fatias organotípicas do córtex cerebral 4,6. Um vetor pSilencer modificado é usado que contém um promotor U6 que impulsiona as duplas de RNA hairpin encalhados e um cassete de expressão separado que codifica a proteína GFP dirigido bya promotor de CMV 7-9. Nossa abordagem permite a rápida avaliação de defeitos no crescimento de neuritos em knockdown específica de genes candidatos e tem sido utilizado com sucesso em uma tela para os reguladores de crescimento neurite 8. Como apenas um subconjunto de células irão expressar as construções RNAi, as fatias organotípicas permitir uma análise do mosaico dos fenótipos potenciais. Além disso, porque esta análise é feita de uma aproximação próxima da ambiente in vivo, ele fornece uma alternativa de baixo custo e rápida para a geração de animais transgénicos ou knockout para os genes de função desconhecida cortical. Finalmente, em comparação com a tecnologia in vivo electroporação, o sucesso de experiências de electroporação ex vivo não é dependente de desenvolvimento proficiente cirurgia habilidade e pode ser realizada com um menor tempo de formação e habilidade.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Para realizar uma avaliação rápida da função de genes no desenvolvimento de córtex cerebral, que descrevem métodos que envolvem a<em&gt; Ex vivo</em&gt; Electroporação de plasmídeos co-expressando RNA inibitória (RNAi) e GFP no córtex embrionário de murídeo. Este protocolo é favorável ao estudo de vários aspectos do neurodesenvolvimento, como neurogênese, migração neuronal e morfogênese neuronal incluindo dendrito e crescimento axonal.

Métodos de Estudo da morfogênese neuronal:<em&gt; Ex vivo</em&gt; Eletroporação RNAi no Cortex Cerebral Murina embrionárias

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

11.7K visualizações.  Brown University. Para realizar uma avaliação rápida da função de genes no desenvolvimento de córtex cerebral, que descrevem métodos que envolvem a Ex vivo Electroporação de plasmídeos co-expressando RNA inibitória (RNAi) e GFP no córtex embrionário de murídeo. Este protocolo é favorável ao estudo de vários aspectos do neurodesenvolvimento, como neurogênese, migração neuronal e morfogênese neuronal incluindo dendrito e crescimento axonal.

Vídeo: Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Eletroencefalografia simultânea, em tempo real de medição da concentração de lactato e Manipulação optogenética da atividade neuronal do córtex cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Eletroporação Abordagens para estudar a excitabilidade neuronal subpopulações e Conectividade uma única célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Induzindo a recombinação de Cre-lox no córtex Cerebral de rato através da eletroporação no Utero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Eletroporação e culturas Organotypic fatia do cérebro embrionário Mouse para imagens ao vivo de migração gabaérgica interneurônios

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...