<p class="jove_content"&gt; Agradecemos ao Dr. Yuval Gottlieb-Dror para a ilustração eletroporação. Este trabalho foi financiado por doações para DSD do Instituto Nacional de Psicobiologia em Israel e para o programa de cooperação Research Niedersachsen-Israel e por subvenções a AK da Fundação Israel Ciência, o Ministério da Saúde de Israel, e O Centro do Património Excellence-Legacy parceria Ciências Biomédicas.</p&gt;

1. Eletroporação hindbrain 1.1 manuseio Egg <ol><li> Colocar os ovos horizontalmente numa incubadora humidificada (37-38,5 ° C). Os embriões são electroporados após 65-70 horas de incubação, quando atingem 16-17 (HH) fase (25-30 somites). </li><li> Retire os ovos da incubadora, eles permanecem na posição horizontal. </li></ol> 1.2 Preparações <ol><li> Puxar capilares de vidro (0,5 mm de diâmetro). </li><li> Ligação dobrados em forma de L Genetrodes eléctrodos de ouro (diâmetro 3 mm) com uma placa de suporte para o gerador de impulsos. Parâmetros eletroporação incluem 25 volts, 5 números de pulso, 45 ms de comprimento de pulso, 300 intervalos de pulso ms. </li><li> Prepara-se uma pipeta de boca, agulha de seringa de 10 ml, as tiras de parafilme, uma escova macia, dissecando tesoura afiada e 25 ml de solução de PBS estéril. Esta quantidade é suficiente para lidar com uma bandeja de ovos (18 ovos). </li><li> Prepara-se uma mistura adequada de plasmídeos de ADN (5 mg / mL de cada) eadicionar cerca de corante Verde Rápido 0,025% para visualizar a injecção de ADN. Normalmente, um volume de 4 ul é suficiente para a injecção de um tabuleiro. </li></ol> 1.3 de janelas, Injeção e Eletroporação <ol><li> Limpe as cascas de ovos com etanol 70%. </li><li> Lidar com um ovo de cada vez para minimizar o tempo de exposição ao ar embrionário. </li><li> Fazer um buraco no pólo ovo com as tesouras de pontas e remover 3-4 ml de albumina com a seringa. Não remova a maior quantidade de albumina, uma vez que reduz a viabilidade. </li><li> Abra uma pequena janela oval (2,5 x 2 cm de tamanho) na parte central superior da casca do ovo usando a tesoura de dissecação. </li><li> Gotejamento algumas gotas de PBS em cima do embrião para evitar a secura durante todo o processo. </li><li> Coloque uma pequena quantidade da mistura de ADN no capilar de vidro usando uma pipeta de boca. </li><li> Coloque o embrião com a extremidade posterior em direção a você. Sem injeção de tinta é necessária nesta fase, o embrião é claramente visível. Evitar um aumento de injeção de tintasobrevivência embrionária. Penetrar o hindbrain caudal mantendo o capilar em ~ 45 °. Não retire nenhuma membrana ao redor do embrião. Injetar solução de DNA com cuidado para a luz hindbrain e expire sem formar bolhas de ar. Solução de ADN espalha anteriormente. </li><li> Imediatamente após a injeção de DNA, coloque os eletrodos paralelos no AP precisa e DV posição hindbrain você apontar para o alvo. Empurrar os eléctrodos ligeiramente ventralmente sem tocar no coração, em que pode reduzir a viabilidade. Pulsar o electroporator. Os eléctrodos deverão ser cobertas com bolhas para indicar condutividade elétrica (Figura 1A). </li><li> Cuidadosamente retire os eletrodos e pingar algumas gotas de PBS para esfriar o embrião e para remover as bolhas. Selar o ovo abrir corretamente com uma tira de parafilme para evitar o ressecamento durante a incubação. Lavar eléctrodos em PBS com o pincel macio. </li><li> Coloque ovos na incubadora para o período de tempo desejado. A sobrevivência de embriões de aproximadamente 90% depois de 2-3 diaselectroporação, e reduz-se para 50% de 12 dias após a electroporação. </li></ol> 2. Análise de Embriões 2.1 Preparação e imunofluorescência plano de montagem <ol><li> Preparações plano de montagem da parte posterior do cérebro podem ser realizadas até E6.5 para visualizar facilmente axônios sob um microscópio ou um estéreo-microscópio. </li><li> Dissecar o embrião cuidadosamente separando-a de membranas que envolvem e vasos sanguíneos. Colocar o embrião num pequeno silicone - revestido placa de Petri contendo PBS. Conecte o embrião para o prato com pinos de tungstênio enfrentam dorsal. Realize uma fenda dorsal na parte posterior do cérebro telhado placa usando sharp capilares de vidro. </li><li> Usando pinças afiadas e micro-tesouras segurar a parte superior da cabeça e puxe o tecido hindbrain com muito cuidado a partir de rostral para caudal. Após separação da rombencéfalo remover restantes tecidos ventrais para alcançar uma preparação limpa. </li><li> Incubar a hindbrain com 4Solução% paraformaldeído (PFA) por 1-2 horas em temperatura ambiente (RT). Lava-se a parte posterior do cérebro com PBT (PBS/0.1% de Triton X-100) para remover a PFA. </li><li> Para imunofluorescência em hindbrains plana montados adicionar 500 ul de solução de bloqueio (5% de soro de cabra em PBT e incubar durante 2 horas à temperatura ambiente com um anticorpo Incubar r ON a 4 ° C. A lavagem com PBT (15 minutos x 3). Incubar 2 nd com anticorpo durante 2 horas à temperatura ambiente. Lavar com PBT (15 minutos x 3). </li><li> Colocar o rombencéfalo virada dorsalmente numa lâmina de vidro. Adicione meios de montagem fluorescentes na parte posterior do cérebro. Use capilares de vidro ou agulhas de tungstênio para achatar a hindbrain no slide. </li><li> Adicionar pequena quantidade de massa lubrificante, em cada canto para impedir a compressão do tecido por a lamela de cobertura. Cuidadosamente coloque uma cobertura de vidro na parte superior da parte posterior do cérebro e evitar bolhas de ar. Para adesão adicionar unha polonês para cobrir as bordas de vidro. </li></ol> 2.2 Cryo-seções e Imunofluorescência <ol><li> Cryo-seções do tronco cerebral can ser realizada em qualquer fase de visualizar trajectórias axonais. No entanto, depois de E6.5 este deve ser o método preferido, devido à dificuldade em visualizar os axônios no embrião fixa-montado, que se torna demasiado espessa. Além disso, a manifestação de sinapses requer o seccionamento do tecido e uma vista de alta ampliação com um microscópio. </li><li> Dissecar o embrião e remover todos os tecidos circundantes. Lavar com PBS. Corte o embrião na parte caudal da medula usando micro-tesouras e pinças afiadas. Fazer uma ampla incisão entre o cerebelo eo mesencéfalo e remover todo o tronco cerebral. O tecido deve conter a medula, ponte e cerebelo. </li><li> Fixar o tronco cerebral em PFA a 4% durante a noite (ON) a 4 ° C, lavar com PBS (10 minutos x 3), e incubar com 30% de sacarose para LIGADO, a 4 ° C até afundamento. </li><li> Incorporar o rombencéfalo num molde de crio com outubro (temperatura óptima de corte) de composto, e colocá-lo a -20 ° C, até o congelamento, conforme descrito noutro local 11. </li><li&gt; Coloque o bloco outubro no criostato no eixo transversal desejado. Largura da seção é de 12 mM. </li><li> Seque as lâminas. Adicionar 100 ul de solução de bloqueamento em cada lâmina, durante 2 horas à temperatura ambiente. Adicionar 100 ul de anticorpos (diluído em solução de bloqueio à concentração desejada). O período de incubação para a 1 ª e 2 ª anticorpos está ligado a 4 ° C ou 2 horas à temperatura ambiente, respectivamente. Para cada tempo de incubação, adicionar suavemente parafilme tira na parte superior da corrediça para evitar a secura. Lavar com PBS (10 minutos x 3) entre os anticorpos. Se for necessário, adicionar 1:1000 Dapi (4&#39;-6-diamidino-2-fenilindole), diluiu-se em PBS durante 20 min à TA. Lavar com PBS. </li><li> Montar o slide com meios de montagem fluorescentes. Deixe secar por 1 hora antes da análise. </li></ol>

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+precerebellar+nuclei+in+the+rat:+II.+The+intramural+olivary+migratory+stream+and+the+neurogenetic+organization+of+the+inferior+olive.">Development of the precerebellar nuclei in the rat: II. The intramural olivary migratory stream and the neurogenetic organization of the inferior olive.</a> J. Comp. Neurol. 257, 490-512 (1987).</li><li>Rose, M. F., Ahmad, K. A., Thaller, C., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+neurons+of+the+proprioceptive,+interoceptive,+and+arousal+hindbrain+networks+share+a+developmental+requirement+for+Math1.">Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 22462-22467 (2009).</li><li>Storm, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bHLH+transcription+factor+Olig3+marks+the+dorsal+neuroepithelium+of+the+hindbrain+and+is+essential+for+the+development+of+brainstem+nuclei.">The bHLH transcription factor Olig3 marks the dorsal neuroepithelium of the hindbrain and is essential for the development of brainstem nuclei.</a> Development. 136, 295-305 (2009).</li><li>Lumsden, 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+cellular+basis+of+segmentation+in+the+developing+hindbrain.">The cellular basis of segmentation in the developing hindbrain.</a> Trends Neurosci. 13, 329-335 (1990).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+precerebellar+neuron+development+by+Olig3+bHLH+transcription+factor.">Control of precerebellar neuron development by Olig3 bHLH transcription factor.</a> J. Neurosci. 28, 10124-10133 (2008).</li><li>Muller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bHLH+factor+Olig3+coordinates+the+specification+of+dorsal+neurons+in+the+spinal+cord.">The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord.</a> Genes Dev. 19, 733-743 (2005).</li><li>Avraham, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+dorsal+root+ganglion+axons+serve+as+choice+points+for+the+ipsilateral+turning+of+dI3+axons.">Motor and dorsal root ganglion axons serve as choice points for the ipsilateral turning of dI3 axons.</a> J. Neurosci. 30, 15546-15557 (2010).</li><li>Avraham, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4, 21 (2009).</li><li>Avraham, O., Zisman, S., Hadas, Y., Vald, L., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+axonal+pathways+of+genetically+defined+groups+of+neurons+in+the+chick+neural+tube+utilizing+in+ovo+electroporation.">Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation.</a> J. Vis. Exp. (39), e1792 (2010).</li><li>Kohl, A., Hadas, Y., Klar, A., Sela-Donenfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Patterns+and+Targets+of+dA1+Interneurons+in+the+Chick+Hindbrain.">Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain.</a> J. Neurosci. 32, 5757-5771 (2012).</li><li>Vogel, J., Mobius, C., Kuschinsky, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+delineation+of+ischemic+tissue+in+rat+brain+cryosections+by+high-contrast+staining.">Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining.</a> Stroke. 30, 1134-1141 (1999).</li><li>Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Lumpkin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr. Patterns. 3, 389-395 (2003).</li><li>Lu, Y., Lin, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transgenic+strategies+in+the+developing+chicken+spinal+cord.">PiggyBac transgenic strategies in the developing chicken spinal cord.</a> Nucleic Acids Res. 37, e141 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-like+elements+in+the+pink+bollworm,+Pectinophora+gossypiella.">piggyBac-like elements in the pink bollworm, Pectinophora gossypiella.</a> Insect Mol. Biol. 19, 177-184 (2010).</li><li>Alsina, B., Vu, T., Cohen-Cory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+synapse+formation+in+arborizing+optic+axons+in+vivo:+dynamics+and+modulation+by+BDNF.">Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.</a> Nat. Neurosci. 4, 1093-1101 (2001).</li><li>Leal-Ortiz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piccolo+modulation+of+Synapsin1a+dynamics+regulates+synaptic+vesicle+exocytosis.">Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis.</a> J. Cell Biol. 181, 831-846 (2008).</li><li>Gardzinski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+synaptotagmin+I+C2A+calcium-binding+domain+in+synaptic+vesicle+clustering+during+synapse+formation.">The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation.</a> J. Physiol. 581, 75-90 (2007).</li><li>Nowack, A., Yao, J., Custer, K. L., Bajjalieh, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV2+regulates+neurotransmitter+release+via+multiple+mechanisms.">SV2 regulates neurotransmitter release via multiple mechanisms.</a> Am. J. Physiol. Cell Physiol. 299, C960-C967 (2010).</li><li>Itasaki, N., Sharpe, J., Morrison, A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+Hox+expression+in+the+vertebrate+hindbrain:+influence+of+paraxial+mesoderm+and+rhombomere+transposition.">Reprogramming Hox expression in the vertebrate hindbrain: influence of paraxial mesoderm and rhombomere transposition.</a> Neuron. 16, 487-500 (1996).</li><li>Clarke, J. D., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+repetition+of+neuronal+phenotype+sets+in+the+chick+embryo+hindbrain.">Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain.</a> Development. 118, 151-162 (1993).</li><li>Diaz, C., Glover, J. C., Puelles, L., Bjaalie, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hodological+and+cytoarchitectonic+organization+in+the+vestibular+complex+of+the+11-day+chicken+embryo.">The relationship between hodological and cytoarchitectonic organization in the vestibular complex of the 11-day chicken embryo.</a> J. Comp. Neurol. 457, 87-105 (2003).</li><li>Marin, F., Puelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+fate+of+rhombomeres+in+quail/chick+chimeras:+a+segmental+analysis+of+hindbrain+nuclei.">Morphological fate of rhombomeres in quail/chick chimeras: a segmental analysis of hindbrain nuclei.</a> Eur. J. Neurosci. 7, 1714-1738 (1995).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li></ol>

<p class="jove_content"&gt; Não há conflitos de interesse declarados.</p

Na eletroporação in ovo é uma ferramenta viável, confiável e eficaz para examinar especificação celular e orientação axonal durante garota desenvolvimento do sistema nervoso 20. Neste protocolo, descrevemos um modo de eletroporação na hindbrain garota em E2.75 usando elementos potenciador que permitem a rotulagem condicional de interneurons específicas. Esta estratégia é combinada com o sistema de transposição PiggyBac mediada para inserir genes estranhos no genoma do pintinh...

A parte posterior do cérebro representa um cubo do relé-chave do sistema nervoso por meio da comunicação entre os sistemas nervosos central e periférico via ascendente e descendente redes neuronais. Ele regula as funções básicas, incluindo respiração, consciência, audição e coordenação motora 1-3. Durante o desenvolvimento embrionário inicial, a parte posterior do cérebro dos vertebrados é transitoriamente subdividida ao longo do eixo ântero-posterior (AP) em rhombomeres repetitivas, em que os tipos de células neuronais distintas são formadas e gerar múltiplos centros de núcleos do tronco cerebral 4. A parte posterior do cérebro também é dividido ao longo do seu eixo dorsal-ventral (DV) em uma placa basal e alar, em que os progenitores neuronais discretas e diferenciar-se especificadas em locais distintos 3,5,6 DV. Como o início da AP e DV específicos padrões neuronais estão governando o estabelecimento de circuitos do tronco cerebral funcional é em grande parte desconhecido. Para adquirir conhecimentos sobre esta fundamentaisquestão, são necessárias ferramentas, a fim de rotular subconjuntos específicos de neurônios no cérebro posterior cedo e traçar suas trajetórias axonal e conectividade em estágios mais avançados. Nós já utilizou elementos de melhoria específica, e um sistema de expressão condicional Cre / baseado LoxP para acompanhar a trajetória axonal de interneurônios da coluna dorsal no embrião de galinha cedo 7-9. No manuscrito atual que têm como alvo o cérebro posterior e atualizado o paradigma experimental para a rotulagem final interneurons rombencéfalo embrionárias, axônios e seus alvos sinápticas, usando uma estratégia de eletroporação modificado eo PiggyBac - Transposição DNA mediada. A nossa nova estratégia permite a marcação de subtipos neuronais distintas em um dos lados da parte posterior do cérebro e a localização dos seus locais de projecções axonais e sinápticos em vários estádios de desenvolvimento embrionário, a partir de 2 até 12 dias após a electroporação. Com base neste método, que classificou o dorsal, mais subgrupo de interneurons rombencéfalo (dA1/Atoh1 + </sup&gt; células) e revelou dois padrões de projeção axonal ascendentes contralateral, cada um deriva de um local AP diferente e alonga em um funículo distinta. da1 axônios foram encontrados para projeto e sinapses formulário em núcleos auditivos, mesencéfalo e em múltiplas camadas do cerebelo 10. A combinação de garota eletroporação, rastreamento genético de neurônios e análise de locais de projeção em estágios muito avançados de desenvolvimento fornece uma plataforma única de estudar a formação de redes neuronais no cérebro e para elucidar os mecanismos moleculares que governam a formação de circuitos.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

A electroporação do tubo neural embrionário pintainho tem muitas vantagens, tais como ser rápido e eficiente para a expressão de genes estranhos em células neuronais. Neste artigo, procuramos fornecer um método que demonstra unicamente como electroporate DNA no hindbrain aviária em E2.75, a fim de marcar especificamente um subconjunto de células progenitoras neuronais, e como seguir suas projeções axonais e metas sinápticas em estágios muito avançados de desenvolvimento, até E14.5. Utilizamos ferramentas genéticas novas, incluindo elementos específicos do realçador, Cre / Lox - plasmídeos base e do sistema de transposição de DNA PiggyBac mediada para dirigir a expressão GFP em um subtipo de células rombencéfalo (o dorsal mais subgrupo de interneurônios, da1). Trajetórias e metas da da1 axônios axonal são seguidas em fases embrionárias precoces e tardias em várias regiões do tronco cerebral. Esta estratégia contribui com técnicas avançadas para as células de interesse alvo na parte posterior do cérebro embrionário e para tracing formação circuito em vários estágios de desenvolvimento.

Este protocolo foi usado recentemente para descobrir os padrões axonal e locais de projeção de da1 subgrupo de interneurônios na garota hindbrain 10. Para identificar especificamente esses axónios, um elemento potenciador (Atoh1), que tenha sido previamente caracterizadas como específico para neurónios espinais ED1 8,12,13, foi confirmado para ser expresso em células rombencéfalo da1 10. O elemento foi clonado a montante Cre recombinase e co-electroporados em E2.75 juntamente co...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Como as redes neuronais são estabelecidas no cérebro embrionário é uma questão fundamental no desenvolvimento da neurobiologia. Aqui nós combinamos uma técnica de eletroporação com novas ferramentas genéticas, como Cre / Lox-plasmídeos e sistema de transposição de DNA PiggyBac mediada no hindbrain aviária para rotular interneurons dorsal e rastrear suas projeções axonais e metas sinápticas em diferentes estágios de desenvolvimento.

Eletroporação do rombencéfalo rastrear Trajetórias axonal e Metas Synaptic no embrião de galinha

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K visualizações.  The Hebrew University of Jerusalem. Como as redes neuronais são estabelecidas no cérebro embrionário é uma questão fundamental no desenvolvimento da neurobiologia. Aqui nós combinamos uma técnica de eletroporação com novas ferramentas genéticas, como Cre / Lox-plasmídeos e sistema de transposição de DNA PiggyBac mediada no hindbrain aviária para rotular interneurons dorsal e rastrear suas projeções axonais e metas sinápticas em diferentes estágios de desenvolvimento.

Vídeo: Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Rotulagem e Células de imagem no rombencéfalo Zebrafish

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neurociência

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Produção de Extrato de embrião de galinha para o cultivo de células-tronco neurais murino Crest

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Uso de pHluorin para avaliar a dinâmica dos receptores de orientação de axônio na cultura celular e no embrião de filhotes

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

O Sistema Olfativo como um modelo para estudar os padrões de crescimento axonal e Morfologia<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultânea de dois fótons<em&gt; In Vivo</em&gt; Imagem de Synaptic Entradas e metas pós-sinápticos no córtex do rato retrosplï¿½ico

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

O uso do condicionamento de rastreamento Eyeblink clássico para avaliar disfunção Hippocampal em um modelo do rato de transtornos do espectro do álcool Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Uso de zinco sináptica histoquímica para revelar as diferentes regiões e lâminas no cérebro em desenvolvimento e adulto

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinação de Optogenetics com microRNAs Artificial para caracterizar os efeitos do Gene Knockdown na função pré-sináptica dentro circuitos neuronais intacta

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...