O ensaio de matriz de colágeno foi desenvolvido e aperfeiçoado pelo trabalho de muitos diferentes grupos de pesquisa durante os últimos dois a três décadas. As abordagens descritas aqui para explantes e dopaminérgicos estriatais se beneficiar imensamente com esses estudos. Além disso, os autores gostariam de agradecer Asheeta Prasad por sua ajuda na criação de culturas de explante estriado. O trabalho no laboratório foi financiada pela Organização Human Programa de fronteira da Ciência (Prêmio de Desenvolvimento de Carreira), a Organização Neerlandesa de Pesquisa em Saúde e Desenvolvimento (ZonMW-VIDI e ZonMW-TOP), a União Europanian (mdDA-NeuroDev, FP7/2007-2011 , Grant 222999) (a RJP), e da Organização Holandesa para Pesquisa Científica (TopTalent; para ERES).

1. Preparação de Colágeno da cauda de rato <ol><li> Collect 6-10 caudas de ratos adultos (é possível armazenar as caudas a -20 ° C até à sua utilização). </li><li> Embeber as caudas em etanol a 95% durante a noite à temperatura ambiente (RT). </li></ol> Dissecção de caudas de rato (em capuz de cultura de tecido): (Manter as ferramentas em etanol a 70% quando não usá-los e se certificar de que todas as soluções, ferramentas e utensílios de vidro usados ​​em todo este procedimento são estéreis) <ol start="3"><li> Para a coleta de tendões da cauda, ​​corte a ponta da cauda. Mantenha extremidade grande da cauda com um par de pinças. Use um par de pinças para segurar, dobrar e quebrar o rabo perto de pinça outros. Separar e os tendões será deixado para trás (Figura 1A, B). </li><li> Colete os tendões em estéril H 2 O e depois de coletar todos os tendões movê-los para um novo com petridish estéril H 2 O. </li><li> Tome 2-3 tendões e retalhar-los usando 2 pares de paraceps em uma nova petridish com estéril H 2 O. Remover não-tecido do tendão, como veias (tecido do tendão é brilhante e reflexiva; Figura 1C). Após a remoção de todo o tecido não-tendão, cada cauda produz aproximadamente 100-150 mg de tendões. </li><li> Transfira os tendões a 300 ml de ácido acético a 3% estéril e agita-se lentamente durante a noite a 4 ° C. </li><li> Adicionar uma de 200 ml adicionais de ácido 3% estéril acético e agita-se lentamente durante a noite a 4 ° C. </li><li> Centrifugar a uma solução de ácido acético contendo os tendões a ~ xg 2700 durante 120 min a 4 ° C a pelota não dissolvidas tendões e não-tecidos do tendão. </li><li> Durante a centrifugação preparar tubo de diálise: <ul><li> cortar pedaços de 40 cm </li><li> ferver em 500 mM de EDTA durante 5 min </li><li> legal em estéril H 2 O </li></ul></li><li> Amarrá um nó em uma extremidade do tubo de diálise e encher o tubo com o sobrenadante da solução tendão centrifugada em gelo no capuz de cultura de tecido usando um pipetar automáticoE e uma pipeta 25 ml. Evitar a produção de bolhas. Nó a outra extremidade do tubo eo tubo loja em folha de alumínio estéril em gelo até que todos os pedaços de tubo são preenchidos (Figura 1D). </li><li> Preparar 10 litros de estéril 0,1 X MEM, pH 4,0, no capuz de cultura de tecidos. Adicionar flutuador para peças de tubagem e adicioná-los à solução pré-arrefecida MEM 0,1 X num copo de 10 l. Dializar durante a noite a 4 ° C enquanto lentamente sob agitação. Este diálise remove o excesso de ácido, mantendo o pH baixo. </li><li> Substitua por uma nova pré-arrefecido 10 l 0,1 X MEM e agitar a noite a 4 ° C. </li><li> Repetir o passo anterior, substituindo com o novo pré-arrefecida de 10 l 0,1 X MEM e agita-se durante a noite a 4 ° C. </li><li> Solução alíquota de colagénio em tubos de 50 ml estéreis. Loja alíquotas a 4 ° C. Mantenha 6-12 meses, apenas lidar com o estoque de colágeno na capa cultura de tecidos. </li><li> É importante para testar se o colagénio purificado precisa de ser diluído (em 0,1 x MEM) para se obter resultados óptimos na MATR colagénioensaio ix. Portanto, gerar uma série de diluições de colagénio (colagénio não diluído, 1:1, 1:2 e 1:5 diluído) e realizar ensaios de matriz de colagénio, tal como descrito abaixo para determinar a diluição de colagénio óptima. </li></ol> 2. Dissecção do mesencéfalo dopaminérgico 7,8 <ol><li> Dissecar E14.5 embriões de camundongos do útero da mãe e se manter em meio L15 no gelo até ser necessário. </li></ol> Todos os passos de dissecção subsequentes são realizados em meio L15 em gelo. <ol start="2"><li> Dissecar o cérebro. </li><li> Retire o telencéfalo cortando ao longo da parte medial de cada vesícula telencefálico (use as vesículas telencefálicos para explantes estriado). </li><li> Remover a bainha das meninges. </li><li> Faça um corte dorsoventral apenas rostral da flexura mesencefálica. </li><li> Faça outro corte dorsoventral apenas caudal da flexura mesencefálica. </li><li> Fazer um corte rostrocaudal ao longo da linha média dorsal usando um microdissection faca para expor o tecido do mesencéfalo ventral subjacente. Tenha cuidado para não bater o tecido mesencéfalo ventral, já que contém os neurônios dopaminérgicos (Figura 2). </li><li> Fazer dois cortes rostrocaudal lateral e paralelamente à linha média ventral para remover o tecido do mesencéfalo dorsal. </li><li> Divida o tecido mesencéfalo ventral restante em explantes usando uma faca microdissecção. </li><li> Armazenar os explantes em L15 meio contendo FBS a 5% em gelo até à utilização. </li></ol> 3. Dissecção do estriado Todos os passos são realizados em dissecação L15 médio em gelo. <ol><li> Use vesículas telencefálicos dissecados durante o esvaziamento do mesencéfalo. </li><li> Remover o tálamo cortando entre o tálamo e corpo estriado utilizando fórceps. </li><li> Faça um corte médio-lateral rostral para o estriado para remover estruturas rostrais como o bulbo olfativo. Repetir este passo para o tecido localizado caudalmente ao corpo estriado. </li><li> Posiçãoa fatia remanescente para se obter uma vista coronal do estriado (Figura 2). </li><li> O corpo estriado pode ser reconhecida como uma parte ligeiramente menos densa (mais transparente) de tecido. Dissecar para fora do corpo estriado e corte em explantes utilizando uma faca microdissecação. Evite o tecido um pouco mais escura perto da linha média, que contém neurônios que migram da eminência lateral e medial ganglionar. </li><li> Armazenar os explantes em L15 meio contendo FBS a 5% em gelo até à utilização. </li></ol> 4. Assembléia de colágeno Ensaios Matrix Preparação do colágeno (todos os passos sobre o gelo, use a ponteira resfriada) <ol><li> Misturar 860 uL de colagénio diluída com 100 ul de MEM de 10x e 40 ul de bicarbonato de sódio 1M (NaHCO3) e manter em gelo. Se neste momento o colágeno aquece vai solidificar. </li><li> Adicionar uma gota de 20 ul (cerca de 5 mm de diâmetro) de colagénio preparada para uma lamela em um poço de um prato 4-bem Nunce deixar repousar num incubador de CO2 a 37 ° C e 5% de CO2 durante 30 min (ambiente concentração de O 2 é suficiente). Durante esta incubação o colagénio será gelatinizar. </li></ol> Instalação de co-cultura em gel de colágeno <ol start="3"><li> Após o colagénio tem gelatinizado, utilizar uma pipeta com uma ponta 200 uL de transferir um explante dopaminérgico ou estriado para o colagénio. </li><li> Mover os explantes em estreita proximidade entre si, utilizando uma agulha. Afastá-los a uma distância de aproximadamente o diâmetro de um explante (~ de 200-300 uM) (Figura 2). </li><li> Remover o excesso de médio e adicionar 20 uL de colagénio preparada em cima dos explantes. Isso muitas vezes fazer com que os explantes para se mover. Reposicionar os explantes utilizando uma agulha, tal como descrito em 4.4. </li><li> Deixe a colagénio solidificar durante 15 min a RT seguido por 30 min a 37 ° C e 5% de CO 2. </li><li> Depois que o colágeno tem definido, adicionar 400 ul de emédio xplant e crescer durante 2-3 dias num incubador de CO2 a 37 ° C e 5% de CO 2. </li></ol> 5. Análise por imuno-histoquímica e Quantificação Imunohistoquímica <ol><li> Para corrigir explantes, suavemente adicionar 400 uL de paraformaldeído 8% (PFA) em PBS a 400 uL de meio (diluindo o PFA a 4%) e deixar repousar durante 1 hora à RT. </li><li> Lavar 15 min 3x em PBS à temperatura ambiente. </li><li> Incubar em tampão de bloqueio (BB; PBS + 1% de FBS + Triton 0,1% X-100) durante 2 horas. </li><li> Incubar durante uma noite com o anticorpo primário em APA a 4 ° C. Para axônios dopaminérgicos usar anti-tirosina hidroxilase anticorpos (1:1000) 7, para os axônios estriatais anti-DARPP32 anticorpos (1:500) e 9 para visualizar todos os axônios anti-βIII tubulina (1:3000) 7. </li><li> Lavar horas uma 5x em PBS à temperatura ambiente. </li><li> Incubar com anticorpo secundário conjugado com o fluoróforo adequado (1:500) em APA durante a noite a 4° C. A partir deste passo em minimizar a exposição dos explantes à luz (por exemplo, os cobrir com folha de alumínio). </li><li> Lavar durante a noite em PBS a 4 ° C, seguida por várias lavagens durante o dia seguinte. </li><li> Monte explantes em microscópio lâminas usando Prolongar reagente Antifade meio de montagem. Adicionar uma gota de ~ 10 ul numa lâmina de microscópio de vidro. Pegue a lamela e gentilmente coloque-o sobre a queda de meio de montagem com o lado explante voltado para baixo. Evite prender o ar sob a lamela de por muito suavemente baixá-lo sobre a queda de meio de montagem. </li></ol> Quantificação por meio do cálculo P / D rácio <ol start="9"><li> Aquisição de imagens digitais dos explantes utilizando um microscópio de epifluorescência (Figura 3). </li><li> Usando essas imagens, dividir cada explante em quadrantes para gerar um quadrante proximal (isto é, parte do explante mais próximo do explante adjacente) e um quadrante distai (parte do explante mais distante da explicação adjacentent) (Figura 2, 4). </li><li> Medir o comprimento de 20 mais longos neurites que emergem do explante tanto nos quadrantes proximais e distais. </li><li> Use o comprimento médio das neurites mais longas 20 para calcular a relação P / D para cada explante individual. AP / D relação&gt; 1 indica atração axônio, enquanto uma proporção &lt;1 indica repulsão axônio. </li><li> Em alguns casos, o crescimento de neurites densa impede a avaliação da duração de neurites individual. Nestas situações, a quantificação pode ser realizada através da medição da distância entre a borda do explante e da frente principal do crescimento axonal nos quadrantes proximais e distais. </li></ol> 6. Imagens representativas Após cultura de sucesso de explantes dopaminérgicos e estriatal um grande número de axónios são visíveis por microscopia de campo brilhante ou como visualizado por anti-tubulina βIII imunocitoquímica (não mostrado). Um subconjunto destes axónios será dopaminérgico ou s<html> axónios triatal como imunocitoquímica utilizando visualizada (Figura 3). Dividindo os explantes em quadrantes, tal como descrito e determinando P / D rácios, um efeito axónio putativo atraente ou repulsivo pode ser quantificada (Figura 3E). <img alt="A Figura 1" src="/files/ftp_upload/3691/3691fig1.jpg" /> Figura 1. Fotos ilustram a preparação de colagénio a partir de tendões da cauda de rato. A) separando a cauda de rato expõe os tendões. B) Os tendões são visíveis como corda-como feixes. C) Os tendões são fáceis de distinguir de veias por sua aparência brilhante branco. D) Depois de se dissolver os tendões em ácido acético, a solução é transferida para tubo de diálise. Para manter a frio colagénio dissolvido, os tubos são colocados em folha de alumínio estéril em gelo. e 2 &quot;src =&quot; files/ftp_upload/3691/3691fig2.jpg / &quot;/&gt; Figura 2. Esquemático indicando as diferentes etapas do processo. Regiões do cérebro adequadas são dissecados e cortados para gerar explantes. Um único e mesencéfalo explante estriatal estão posicionados na proximidade de uma matriz de colagénio e deixada crescer durante 48-72 horas a 37 ° C. Axónios são visualizados por imuno-histoquímica de fluorescência e uma relação de P / D é calculado para quantificar as respostas quimiotrópicos. <img alt="A Figura 3" src="/files/ftp_upload/3691/3691fig3.jpg" /> Figura 3. Os resultados representativos que mostram o crescimento axonal em uma cultura matriz de colagénio. A) explante Midbrain corados com anticorpo anti-tirosina hidroxilase revelando o crescimento axonal. A linha pontilhada indica explante adjacente. B) ampliação de um neurites mostram individuais e cones de crescimento (setas). C) explante estriatal corados com anti-DARPP32. D) Ampliação de C mostrando neurites individuais. Exemplo E) doP / D quantificação proporção. Explantes são divididos em quadrantes iguais. O quadrante proximal está voltada para o explante adjacente enquanto o quadrante distai está virada para fora dele. Barras de escala indicam 100 mm.

<ol>
	<li>van den Heuvel, D. M., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+connected+in+the+dopamine+system.">Getting connected in the dopamine system.</a> Prog. Neurobiol. 85, 75-93 (2008).</li><li>Lobo, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+striatonigral+and+striatopallidal+medium+spiny+neurons+past,+present,+and+future.">Molecular profiling of striatonigral and striatopallidal medium spiny neurons past, present, and future.</a> Int. Rev. Neurobiol. 89, 1-35 (2009).</li><li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298, 1959-1964 (2002).</li><li>Chilton, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+mechanisms+of+axon+guidance.">Molecules mechanisms of axon guidance.</a> Dev. Biol. 292, 13-24 (2006).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J., Jessel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+guidance+of+developing+axons+in+the+mammalian+central+nervous+system.">Chemotropic guidance of developing axons in the mammalian central nervous system.</a> Nature. 336, 775-778 (1988).</li><li>Kolk, S. M., Gunput, R. A., Tran, T. S., van den Heuvel, D. M., Prasad, A. A., Hellemons, A. J., Adolfs, Y., Ginty, D. D., Kolodkin, A. L., Burbach, J. P., Smidt, M. P., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+3F+is+a+bifunctional+guidance+cue+for+dopaminergic+axons+and+controls+their+fasciculation,+channeling,+rostral+growth,+and+intracortical+targeting.">Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channeling, rostral growth, and intracortical targeting.</a> J. Neurosci. 29, 12542-12557 (2009).</li><li>Fenstermaker, A. G., Prasad, A. A., Bechara, A., Adolfs, Y., Tissir, F., Goffinet, A., Zou, Y., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/planar+cell+polarity+signaling+controls+the+anterior-posterior+organization+of+monoaminergic+axons+in+the+brainstem.">Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem.</a> J. Neurosci. 30, 16053-16064 (2010).</li><li>Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip2+controls+the+differentiation+of+medium+spiny+neurons+and+the+establishment+of+the+cellular+architecture+of+the+striatum.">Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.</a> J. Neurosci. 28, 622-632 (2008).</li><li>De Wit, J., Toonen, R. F., Verhage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+local+retention+of+secretory+vesicle+cargo+in+cortical+neurons.">Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.</a> J. Neurosci. 29, 23-37 (2009).</li><li>G&#228;hwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, 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=Organotypic+slice+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends Neurosci. 20, 471-477 (1997).</li></ol>

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O ensaio de matriz de colagénio aqui descrita foi usada e melhorado por muitos laboratórios diferentes nas últimas décadas para investigar uma variedade de moléculas de direccionamento axonal e sistemas neuronais (por exemplo, ver as referências 5-8). Estes estudos demonstraram que este ensaio é uma ferramenta poderosa para estudar os efeitos de regulação e de moléculas de direccionamento axonal secretadas por diferentes tecidos (Intermediário) alvo. No entanto, deve notar-se que ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Soro fetal de vitelo </td><td> BioWhittaker </td><td> 14-801F </td><td></td></tr><tr><td> Glutamina (200mm) </td><td> PAA </td><td> M11-004 </td><td></td></tr><tr><td> Hepes </td><td> VWR International </td><td> 441476L </td><td></td></tr><tr><td> β-mercaptoetanol </td><td> Merck </td><td> 444203 </td><td></td></tr><tr><td> Mínimo de mídia Essenciais (MEM) </td><td> Gibco </td><td> 61100-087 </td><td></td></tr><tr><td> Neurobasal </td><td> Gibco </td><td> 21103-049 </td><td></td></tr><tr><td> B27 </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> L-15 de Leibovitz Médio </td><td> Gibco </td><td&gt; 11415-049 </td><td></td></tr><tr><td> Penicilina-Estreptomicina </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Prolongar Reagente Antifade Ouro </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> Tubo de diálise </td><td> Espectro Labs </td><td> 132660 </td><td></td></tr><tr><td> Hidroxilase anti-tirosina Coelho </td><td> Pel-Freez </td><td> P40101-0 </td><td></td></tr><tr><td> De coelho anti-Darpp32 (H-62) </td><td> Santa-Cruz </td><td> Sc-11365 </td><td></td></tr><tr><td> Rato tubulina anti-βIII </td><td> Sigma </td><td> T8660 </td><td></td></tr><tr><td> Alexa Fluor rotulado anticorpos secundários </td><td> Invitrogen </td><td></td><td></td></tr></tbody></table>

dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

Mesencéfalo a dopamina (mdDA) projeto neurônios através do feixe medial do prosencéfalo para diversas áreas do telencéfalo, incluindo o 1 striatum. Reciprocamente, médio neurónios espinais no estriado que dão origem à via (directo) Estriatonigral inervam a substantia nigra 2. O desenvolvimento desses setores axônio é dependente das ações combinatória de uma infinidade de crescimento do axônio e pistas de orientação, incluindo as moléculas que são liberadas por neurites ou por regiões (Intermediário)-alvo 3,4. Estes factores solúveis podem ser estudada in vitro por mdDA cultura e / ou explantes do estriado em uma matriz de colagénio que proporciona um substrato tridimensional para os axónios que imitam o ambiente extracelular. Além disso, a matriz de colagénio permite a formação de gradientes relativamente estáveis ​​de proteínas libertadas por explantes outros ou células colocadas na proximidade (por exemplo, ver as referências 5 e 6). Aqui descrevemos métodos para os finsificação de colágeno da cauda de rato, microdissecção de explantes e dopaminérgicos no estriado, a sua cultura em géis de colágeno e análise imuno-histoquímica e quantitativa posterior. Primeiro, o cérebro de embriões de camundongos E14.5 são isoladas e dopaminérgicos estriatais e explantes são microdissectados. Estes explantes são, em seguida, (co) cultivados em géis de colagénio em lamelas durante 48 a 72 horas in vitro. Posteriormente, as projeções axonais são visualizados por meio de marcadores neuronais (eg tirosina hidroxilase, DARPP32, ou tubulina βIII) e crescimento do axônio e respostas axônio atrativas ou repulsivas são quantificados. Esta preparação neuronal é uma ferramenta útil para estudos in vitro dos mecanismos celulares e moleculares do crescimento do axônio mesostriatal e estriatal e orientação durante o desenvolvimento. Utilizando este ensaio, também é possível avaliar outros (Intermediário) alvos para axônios dopaminérgicos e estriado ou para testar específicos pistas moleculares.

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Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Explantes a partir do sistema de dopamina mesencéfalo e corpo estriado são utilizados num ensaio de matriz de colagénio para o<em&gt; In vitro</em&gt; Análise do desenvolvimento via mesostriatal e estriatal. Neste ensaio crescimento axonal e orientação podem ser manipulados e quantificado. Ele também pode ser modificado para avaliar outras regiões ou pistas moleculares.

Dissecção e Cultura do mouse e dopaminérgico do estriado explantes em três dimensões Ensaios matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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25.6K visualizações.  University Medical Center Utrecht. Explantes a partir do sistema de dopamina mesencéfalo e corpo estriado são utilizados num ensaio de matriz de colagénio para o In vitro Análise do desenvolvimento via mesostriatal e estriatal. Neste ensaio crescimento axonal e orientação podem ser manipulados e quantificado. Ele também pode ser modificado para avaliar outras regiões ou pistas moleculares.

Vídeo: Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Dissecção e Cultura do Gânglio pintainho Statoacoustic e Explantes da Medula Espinhal em géis de colágeno para Ensaios crescimento de neuritos

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

A Unidade Funcional Motor na placa de cultura: Co-cultivo de explantes da Medula Espinhal e as células musculares

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Avaliando fenótipos Neurodegenerativas em<em&gt; Drosophila</em&gt; Neurônios dopaminérgicos subindo Ensaios e toda Imunocoloração Cérebro

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Isolamento e Quantificação da neurotoxina botulínica a partir de matrizes complexas usando os BoTest Matrix Ensaios

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primária de neurônios dopaminérgicos mouse

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Longo prazo Time Lapse Imagem do mouse cocleares explantes

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Protocolo para tridimensional confocal morfométrica Análise de astrócitos

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

Conexões de Fibra da Área Suplementar de Motor Revisitada: Metodologia de Dissecção de Fibras, DTI e Documentação Tridimensional

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirurgia estereotáxica para manipulação genética em células estriadas de cérebro de rato Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...