Gostaríamos de agradecer nosso consultor, Dr. Eve Marder. Esta pesquisa foi apoiada pelo Instituto Nacional de Saúde concede NS17813 a Eva Marder e NS059255 para AE T.

Antes de começar este protocolo o estômago da lagosta deve ser retirado do animal e preparado como descrito no artigo companheiro Jove (Bierman &amp; Tobin, 2009). A maioria das etapas descritas abaixo devem ser realizados sob um microscópio de dissecação. Parte I-Isolar os gânglios comissural <ol><li> Orientar o prato de modo que a extremidade anterior (pelo lábio) está longe de você e da extremidade posterior (pelo piloro) é para você. </li><li> Garantir que o estômago é preso para que ele fique o mais plano possível no prato. <ol><li> Pin para o esôfago em cada lado do lábio. </li><li> Se necessário, reorganize os outros pinos colocados durante a dissecção macroscópica. </li></ol></li><li> Remova a hipoderme. <ol><li> Agarre a hipoderme perto do gm2/gm3 músculos, retire-os músculos e puxe na direção anterior. </li><li> Separar os hipoderme da gordura subjacente cortando as ligações. Maior utilização de cuidados ao redor da artéria central, que inclui uma parte fundamental do sistema nervoso stomatogastric. Deixe a hipoderme intacta dentro de cerca de um raio de 0,5 cm do gânglio stomatogastric, que fica entre os músculos GM1b. </li><li> Cortadas hipoderme separados. </li></ol></li><li> Separar o cérebro do tecido subjacente. <ol><li> Identificar os dois grandes nervos comissural esse projeto bilateral da parte anterior do cérebro para os gânglios comissurais na porção anterior do estômago. </li><li> Deixando estes nervos intactos, levante cuidadosamente o cérebro com uma pinça e corte para separar o cérebro do resto do tecido. Tenha cuidado como você fazer isso, como o nervo stomatogastric fica embaixo do cérebro e deve ser mantida intacta. </li></ol></li><li> Separar os gânglios comissural de ao redor do tecido. <ol><li> Siga um dos nervos comissural a um dos gânglios comissural. </li><li> Identificar o íon e filho - estes nervos devem permanecer intactos. O gânglio comissural terá muitos nervos que saem. Cortar a maioria deles (exceto o íon e filho), mas deixe um nervo ligado extras para manter o gânglio comissural no lugar quando você terminar a dissecção. </li><li> Repita com o gânglio outros comissural. </li></ol></li><li> Limpe o íon e filho de lateral para medial. Deixe o nervo labial intacta para ancorar o sistema nervoso para o estômago. O gânglio de esôfago fica eram os s ion se cruzam. Você pode limpar abaixo dela, agarrando o nervo ventricular inferior e puxando para cima a cortar por baixo. </li></ol> Parte II-Isolar o gânglio Stomatogastric (STG) e os nervos restantes. <ol start="7"><li> Separar os STG de ao redor do tecido. <ol><li> O STG fica dentro de uma artéria que corre entre os músculos GM1b e para baixo para os músculos gm2a, b. Olhe para a extremidade cortada da artéria perto os músculos gm2a, b. </li><li> Puxe a extremidade cortada da artéria para cima e anteriormente, para expor a DVN. Continue puxando para cima até chegar ao nível da hipoderme restantes. Uma vez separadas, a artéria pode ser cortado para o nível em que toca a hipoderme restantes. </li><li> Cortar o retalho do músculo que estava situada na tribuna da lagosta. </li><li> Levante a hipoderme acima da artéria, observando que a artéria faz um túnel dentro da gordura acima do STG. Coloque uma lâmina de sua tesoura dentro da artéria e cortar o músculo acima dela. Verifique se você pode ver o DVN como você cortar para ter certeza de que você está com segurança acima da STG. Continue o corte anterior, seguindo a artéria. Como você corte acima do STG, você será capaz de ver o stn emanando anteriormente da STG e para baixo mergulho para dentro do músculo. Continue o corte anteriormente ao separar totalmente o músculo acima do STG. </li><li> Limpar qualquer tecido longe da STN. </li></ol></li><li> Separar os s AGN do tecido circundante. (Mesmo se você não planeja gravar a partir da AGN, você vai precisar de uma porção do nervo intacto para fixar para baixo o gânglio para gravações intracelular.) <ol><li> Para ver melhor o s AGN, remover o restante hipoderme e uma fina camada do músculo subjacente. </li><li> Imediatamente posterior à STG, identificar um túnel escuro na gordura e músculos de cada lado da DVN. </li><li> Vara uma lâmina de tesoura dentro do túnel e cortar o tecido acima do nervo. Continue cortando lateralmente, seguindo o nervo, para isolá-lo. (Dois milímetros é suficiente para a pinagem do gânglio, mas expor pelo menos 5 mm da AGN, se você pretende gravar a partir dele.) </li><li> Destacar a AGN do músculo que inerva cortando as ligações. </li><li> Repita no outro lado para isolar a AGN outros. </li><li> Uma vez que ambos s AGN foram dissecados, cortar todos os músculos e tecido conectivo do STG para o estômago. </li></ol></li><li> Isolar o s. mvn Eles são um par bilateral de pequenos nervos que se ramificam do lvn e atravessando lateralmente embaixo da gordura. <ol><li> Usar tesoura ou duas pinças para cortar a gordura de um lado da DVN, perto de onde se ramifica em duas lvn s. Aqui estão longe do s mvn e não risco cortá-las. </li><li> Levante a camada de gordura, e identificar os debaixo mvn pequeno. Esses nervos executar medial para lateral posterior apenas ao nível dos músculos GM1b. </li><li> Remover a camada de gordura acima do mvn, deixando o nervo intacto para agora assim que não vai se tornar tangled. Você vai cortar a sua extremidade lateral de fora do estômago no final da dissecção. </li><li> Repita o processo do outro lado para isolar o mvn outros. </li></ol></li><li> Isolar o s lvn e nervos posterior. <ol><li> Partida onde o ramo lvn s lateralmente fora da DVN, siga um lvn cortando a gordura acima dela. Dependendo da preparação, por vezes, toda a camada de gordura acima do nervo pode ser puxado para fora. </li><li> Continue revelando a lvn até o ponto onde os ramos psn off. </li></ol></li><li> Isolar o pyn. <ol><li> Localize o pyn, é um nervo que viajam posteriormente sobre o piloro. </li><li> Remover a fina membrana que cobre a região. </li><li> Isolar o pyn cortando uma pequena secção do piloro, anexado ao pyn. </li><li> Levante cuidadosamente a secção do piloro e cortar o tecido de cada lado do pyn até chegar ao ramo que atribui ao PDN. </li></ol></li><li> Isolar o pdn. <ol><li> Localize o pdn, é um nervo viajando posteriormente para o músculo dilatador pilórica. </li><li> Corte uma pequena parte do músculo dilatador pilórica, anexado ao PDN. </li><li> Levante cuidadosamente a secção do músculo pilórico dilatador, cortar o tecido de cada lado do pdn até chegar ao ramo que atribui à pyn. </li></ol></li><li> Isolar o lvn ventral, que liga o pyn e pdn ao lvn. <ol><li> No ponto de ramificação triangular onde o pyn e pdn conectar à lvn ventral, isolar qualquer pequena parte do nervo que se ramifica a lvn ventral, como o vpln. Este será usado como um identificador para puxar o lvn ventral. </li><li> Levante a alça e cortar o tecido de cada lado do lvn ventral, trabalhando na direção anterior até chegar ao ponto onde os ramos psn off. </li></ol></li><li> Repita os passos 10-13 para isolar os nervos lvn e posterior do outro lado. </li><li> Retire o sistema nervoso stomatogastric do estômago. <ol><li> Para remover completamente a lvn do estômago, puxe a psn e cortar todos os tecidos e nervos ligados ao lvn lvn e ventral, exceto para o pdn e pyn. Repita o processo do outro lado. </li><li> Corte a extremidade lateral de cada mvn. Levante a extremidade lateral e corte quaisquer anexos estranhos entre o nervo eo tecido subjacente estômago. </li><li> Na extremidade anterior, cortar quaisquer anexos estranhos para os gânglios comissural, e cortar o nervo vestibular em cada lado. </li><li> Visualmente a varredura do sistema nervoso stomatogastric sistema para identificar e depois cortar quaisquer anexos adicionais ao tecido do estômago. </li><li> Agarre-se a extremidade anterior de ambos os nervos comissural, acima dos gânglios comissural, e descasque o sistema nervoso stomatogastric fora do estômago e movê-lo para o lado do prato. Ao fazer isso, cortar quaisquer anexos restantes para libertar o sistema nervoso. </li></ol></li><li> Preparar o prato Sylgard pequeno. <ol><li> Passe um pouco de tecido do estômago completamente na superfície do Sylgard no prato pequeno. Isto irá diminuir a hidrofobicidade do Sylgard, de modo que a solução salina irá distribuir uniformemente em todo o prato. </li><li> Enxaguar e encher o prato Sylgard com soro fisiológico gelado. </li></ol></li><li> Transferir o sistema nervoso para o prato preparado por Sylgard agarrando as extremidades anterior de ambos os nervos comissural, levantando o sistema nervoso fora do grande prato, e estabelece-lo no pequeno prato. </li><li> Pin do STNS no prato pequeno, utilizando uma combinação de pinos Minuten e pinos mais finos corte de fio de tungstênio. <ol><li> Garantir que a preparação é o lado dorsal, verificando que o nervo ventricular inferior está apontando longe do Sylgard. </li><li> Remover o cérebro e os nervos pin comissural. </li><li> Puxe o sistema nervoso apertado e plana, puxando cada um dos s psn posteriormente e lateralmente, e fixando-os para baixo. </li><li> Retire o músculo dilatador pilórica do s pdn, e fixar o s. pdn </li><li> Remova osegmento pilórica do s pyn, e fixar o s. pyn </li><li> Identificar qualquer nervos remanescentes isolados, como os nervos labial, o s mvn e AGN s. </li></ol></li><li> Desheath o STG. <ol><li> Garantir a STG fica encostado ao Sylgard e mantida tensa por lateral AGN s, a stn anterior e posterior DVN. </li><li> Providenciar a iluminação para permitir a visualização dos axônios dentro da DVN. </li><li> Usando um pequeno alfinete ou agulha de tungstênio fino, faça um pequeno furo raso na bainha ao redor da DVN, apenas posterior à STG. </li><li> Puxe a agulha através da DVN, dorsal para os axônios, para criar uma lágrima horizontal na bainha do nervo. </li><li> Agarre a retalho de bainha do nervo posterior à STG anteriormente e puxe para remover a bainha do lado dorsal do STG. </li></ol></li></ol><table border="1"><tbody><tr><td colspan="2"> Abreviaturas: </td></tr><tr><td> STNS </td><td> Sistema nervoso Stomatogastric </td></tr><tr><td> STG </td><td> Gânglio Stomatogastric </td></tr><tr><td> CoG </td><td> Gânglio comissural </td></tr><tr><td> stn </td><td> nervo stomatogastric </td></tr><tr><td> mvn </td><td> ventricular nervo medial </td></tr><tr><td> íon </td><td> esofágico inferior do nervo </td></tr><tr><td> filho </td><td> nervo esofágico superiores </td></tr><tr><td> AGN </td><td> nervo gástrica anterior </td></tr><tr><td> DVN </td><td> nervo dorsal ventricular </td></tr><tr><td> lvn </td><td> nervo ventricular laterais </td></tr><tr><td> psn </td><td> nervo posterior do estômago </td></tr><tr><td> vpln </td><td> nervo posterior ventral lateral </td></tr><tr><td> pyn </td><td> nervo pilórica </td></tr><tr><td> pdn </td><td> nervo dilatador pilórica </td></tr></tbody></table>

<ol>
	<li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generators+and+the+control+of+rhythmic+movements.">Central pattern generators and the control of rhythmic movements.</a> Curr. Biol. 11, R986-R996 (2001).</li><li>Nusbaum, M. P., Beenhakker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-systems+approach+to+motor+pattern+generation.">A small-systems approach to motor pattern generation.</a> Nature. 417, 343-350 (2002).</li><li>Clemens, S., Massabuau, J. C., Legeay, A., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+interacting+central+pattern+generators+in+lobster+stomatogastric+ganglion:+influence+of+feeding+and+partial+pressure+of+oxygen.">In vivo modulation of interacting central pattern generators in lobster stomatogastric ganglion: influence of feeding and partial pressure of oxygen.</a> J. Neurosci. 18, 2788-2799 (1998).</li><li>Bierman, H. S., Tobin, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gross+dissection+of+the+stomach+of+the+lobster,+Homarus+americanus.">Gross dissection of the stomach of the lobster, Homarus americanus.</a> J Vis Exp. 27, (2009).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268, 161-220 (1974).</li><li>Herrick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+American+lobster.">Natural history of the American lobster.</a> Bull. U.S. Bur. Fish. 29, 219-252 (1909).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Bucher, D., Taylor, A. L., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generating+neurons+simultaneously+express+fast+and+slow+rhythmic+activities+in+the+stomatogastric+ganglion.">Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion.</a> J. Neurophysiol. 95, 3617-3632 (2006).</li><li>Mizrahi, A., Dickinson, P. S., Kloppenburg, P., F&#233;nelon, V., Baro, D. J., Harris-Warrick, R. M., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+channel+distribution+in+a+central+pattern+generator+neuron+by+neuromodulatory+inputs+revealed+by+decentralization+in+organ+culture.">Long-term maintenance of channel distribution in a central pattern generator neuron by neuromodulatory inputs revealed by decentralization in organ culture.</a> J. Neurosci. 21, 7331-7339 (2001).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+channel+expression+in+identified+single+and+electrically+coupled+neurons+in+different+animals.">Variable channel expression in identified single and electrically coupled neurons in different animals.</a> Nat. Neurosci. 9, 356-362 (2006).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+profiling+of+identified+neurons+reveals+cell-specific+constraints+on+highly+variable+levels+of+gene+expression.">Quantitative expression profiling of identified neurons reveals cell-specific constraints on highly variable levels of gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 13187-13191 (2007).</li></ol>

A anatomia do estômago lagosta e STNS tem sido bem descrita anteriormente 5,6, e as variações deste procedimento dissecção têm sido utilizados em numerosos estudos. Recomendamos familiarizar-se com a anatomia do estômago 5-7 e STNS 5,8 antes dissecções início. Essa familiaridade vai diminuir o risco de danos acidentais aos nervos e tecidos. Para manter a saúde da preparação, é fundamental para manter a preparação em uma temperatura baixa. Isso é facilmente alcançado através da troca de solução salina a cada 30 minutos com 4 ˚ C salina mantidos em uma geladeira. Quando imersos em solução salina fria, a preparação STNS isolada é viável para muitas horas. Gravações em gammarus relacionadas Homarus demonstraram atividade rítmica estável a partir do STG todo cinco dias in vitro 9; robustez semelhante foi visto em H. americanus (pers. obs.). Entrada modulatórios para o STG dos gânglios comissural é necessário para preservar os padrões motores em curso. Portanto, para manter uma atividade normal rítmica estes gânglios e do íon, filho, e os nervos stn deve ser mantida intacta. A dissecção descrevemos preserva vários nervos motores (que contém os axônios do neurônio motor): a s AGN (contendo moinho gástrico (GM) axônios), o mvn s (contendo Lateral gástrica (LG) e Inferior Cardíaca (IC) axônios), a s pyn (contendo Pilórica neurônio (PY) axônios), a s pdn (contendo Dilatador Pilórica (PD) axônios), e os s lvn (contendo Pilórica Lateral (LP), Gastric Medial (MG), os axônios de GLP, bem como os da GM, PD e PY). Dissecando adicionais (ou diferentes) os nervos, outras os axônios do neurônio motor pode ser registrado. Cada tipo individual de axônio do neurônio motor pode ser registrado individualmente por dissecação e gravação do nervo que inervam o músculo para que este tipo de projectos neurônio. Esta preparação pode ser usado para uma variedade de estudos focados em função do sistema nervoso. A atividade dos neurônios pode ser registada utilizando intracelular, gravações eletrodo afiada de somata ou axônios, ou por registro de impulsos elétricos dos nervos extracelularmente. Muitos canais iônicos dos neurônios STG ter sido parcialmente clonado e mRNA pode ser medido a partir somata manualmente puxado 10,11.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Tool</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Cut into 1-2mm pieces to use as fine pins.</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Tool</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Tool</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Tool</td>
<td>FST</td>
<td>15370-52</td>
<td>These are the top of the line. There are more economical spring scissors available from the same company.</td>
</tr>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Tool</td>
<td>Fischer</td>
<td>08-757-12</td>
<td>Fill 2-4mm with Sylgard 182.</td>
</tr>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Tool</td>
<td>FST</td>
<td>26018-17</td>
<td>Optional. Desheathing can be done with scissors and fine forceps alone.</td>
</tr>
<tr>
<td>Super fine forceps #5SF</td>
<td>Tool</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Tool</td>
<td>FST</td>
<td>10130-05</td>
<td>Optional. Desheathing can be done with scissors or with pins cut from fine tungsten wire.</td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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homarus americanus stomatogastric nervous system dissection

Com o objetivo de entender como os sistemas nervosos produzem atividade e respondem ao ambiente, os neurocientistas vez para modelar sistemas que exibem a atividade de interesse e são acessíveis e passíveis de métodos experimentais. O sistema nervoso stomatogastric (STNS) da lagosta americana (<i&gt; Homarus americanus</i&gt;; Também sabem foi a lagosta Atlântico ou Maine) foi estabelecida como um sistema modelo para estudar o ritmo de geração de redes e neuromodulação das redes. O STNS consiste de 3 gânglios anterior (2 gânglios comissural e um gânglio esofágico), contendo neurônios moduladores que o projeto central para o gânglio stomatogastric (STG). O STG contém aproximadamente 30 neurônios que compõem o padrão de duas redes de centrais de geração, as redes pilórica e gástricas que estão subjacentes comportamentos alimentares em crustáceos<sup&gt; 1,2</sup&gt;. Embora seja possível estudar este sistema<i&gt; In vivo</i<sup&gt; 3</sup&gt;, O STNS continua a produzir a sua atividade rítmica quando isolados<i&gt; In vitro</i&gt;. Isolamento físico dos STNS em um prato permite fácil acesso ao somata nos gânglios para intracelular gravações eletrofisiológicas e para os nervos do STNS para gravações extracelular. Isolar o STNS é um processo em duas partes. A primeira parte, dissecando o estômago do animal, é descrito em um artigo de vídeo que acompanha<sup&gt; 4</sup&gt;. Neste artigo de vídeo, técnicas de dissecção fina são usadas para isolar o STNS do estômago. Este procedimento resulta em uma preparação do sistema nervoso que está disponível para as gravações eletrofisiológicas.

Watch this Scientific Journal Video about Homarus Americanus Stomatogastric Nervous System Dissection at JoVE.com

Homarus Americanus Stomatogastric Nervous System Dissection

Descrevemos a dissecção fina do sistema nervoso stomatogastric do estômago da lagosta americana (<i&gt; Homarus americanus</i&gt;).

Homarus Americanus Stomatogastric Dissecção do Sistema Nervoso

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

homarus-americanus-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Homarus Americanus Stomatogastric Nervous System Dissection

11.1K visualizações.  Brandeis. Descrevemos a dissecção fina do sistema nervoso stomatogastric do estômago da lagosta americana ( Homarus americanus).

Vídeo: Homarus Americanus Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissecção de 6,5 embriões de camundongos dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

Isolamento de células mononucleares do Sistema Nervoso Central de ratos com EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval Dissection MNJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissecção da Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

Câncer Borealis Dissection Sistema Nervoso Stomatogastric

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Dissecação bruta do Estômago da lagosta, Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissecção de Órgãos do Zebrafish Adulto

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissecção e coloração de Drosophila Larval Ovários

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissecção do rim Zebrafish Adulto

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...