Financiado pela NSF (0.854.551).

1. Pré-experimental Preparação <ol><li> Preparar as seguintes soluções (ver Tabela 1 para as composições): (a) 1 NFR litro (Ringer-rã normal), (b) 100 ml de solução salina a 10%, (c) 100 ml CMF (Ca 2 + / Mg 2 + solução livre ), (d) 1 ml ITS (insulina-transferrina-selênio, Sigma I1884), (e) 100 ml de meio L-15 de cultura, (f) 10 mL de HCG (gonadotropina coriónica humana Sigma CG-10), (g) 100 ml de K +-solução interna, (h) 100 ml de K +-solução interna para a anfotericina B. A força osmótica de uma solução deve ser verificado para assegurar que é de aproximadamente 260 mOsM utilizando um osmómetro de pressão de vapor (por exemplo, modelo Wescor # 5100C) . </li><li> Filtrar esterilizar soluções (b) e (c). Soluções (a) (b) e (c) pode ser armazenado até três meses, a 4 ° C. Preparar a solução de (d) por adição de 1 ml de H2O desionizada ao frasco que contém o pó liofilizado por meio de uma agulha de seringa de calibre 25 e seringa de filtro. Esta solução final cum ser armazenado a 4 ° C durante 30 dias. </li><li> Preparar a solução de (e) numa câmara de fluxo laminar através da combinação de todos os componentes num recipiente ou frasco. Filtrar esterilizar a solução final e transferir alíquotas de 10 ml para tubos de centrífuga estéreis. Armazenar a -20 ° C. </li><li> Prepare o (solução (f)) HCG pela adição de 10 ml de H 2 0 desionizada ao frasco que contém o pó liofilizado por meio de uma agulha de seringa de calibre 25 e seringa de filtro. Esta solução final pode ser armazenado a 4 ° C durante 30 dias. </li><li> Fabricar, pelo menos, duas ferramentas de microdissecção colando um pino Minutien (26002-10, Science Tools finas) à extremidade de uma pipeta de Pasteur de vidro com cola de cianoacrilato. Permitir que a extremidade afiada do pino para estender para além da extremidade da pipeta de aproximadamente 0,5 cm. </li><li> Dois dias antes de preparar culturas, induzir procriação com o seguinte procedimento. Identificar um par de reprodução-pronto de Xenopus, observando uma cloaca, proeminente avermelhada na pigmentação feminino e escuro no su planarrface das patas dianteiras do sexo masculino. Um de um sapo tempo, cada rede e mantê-lo lado ventral para baixo em uma pia com a rede de modo a que não pode fugir. Injectar 1 mL de uma solução de (f) por meio da um líquido e por via subcutânea em dos sacos linfáticos dorsal. </li><li> Coloque o par reprodutor em conjunto num tanque de galão de água coberta 10. Para assegurar que os animais não pisar os ovos fertilizados e recém-estabelecidas, instalar um piso blindado com um tamanho de malha de cerca de ½ &quot;montados aproximadamente 1-2 polegadas acima do fundo do tanque (Figura 1A). </li><li> Deixe rãs não perturbadas por 12-48 horas até que os animais estão em amplexo e ovos fertilizados são observados no andar de baixo da tela (Figura 1B). </li><li> Retire os animais do tanque, mas deixar os ovos em repouso por pelo menos 24 horas por mais tempo. Este par de rãs pode ser re-produzido após seis semanas. </li><li> Soltar os embriões a partir do fundo do tanque, e transferi-los para quatro ou cinco cult 60x15 milímetrosure pratos contendo solução salina a 10% (solução b). Classificar os embriões por estágio de acordo com o esquema de Niewkoop e Faber (ref. 20). Embriões úteis serão as que estão em fase de 22-24. Figura 2A mostra um embrião em fase de cerca de 22, enquanto a Figura 2B mostra um embrião que está demasiado longe ao longo do desenvolvimento para ser útil (cerca de fase 28). É importante escolher os embriões que são saudáveis: aqueles que são suaves na aparência com manchas castanho claro e branco são ideais. Embriões que têm grandes manchas pretas ou brancas são geralmente insalubre e inutilizável. </li></ol> 2. Microdissecação de embriões de Xenopus <ol><li> Dentro de um fluxo laminar etiqueta capuz, e encher até meio aproximadamente três 60x15 milímetros placas de cultura estéreis com solução salina a 10%, e um com CMF. </li><li> Usando uma solução estéril, de vidro Pasteur pipeta de transferência de fase 5-10 22-24 embriões em uma das placas contendo solução salina a 10%. Com o auxílio de um zoom estéreo dissecaring microscópio dentro do capô (0,6-5x com 10 x oculares), remova a camada de geléia e membrana vitelina de cada embrião usando dois pares de estéreis # 5 fórceps (11251-30, ferramentas Ciência Belas). (Veja Figuras 3A e 3B). </li><li> Lavar os embriões nus, passando-os, um de cada vez, através dos restantes dois pratos de solução salina a 10% e, finalmente, para o prato contendo CMF. Usar uma pipeta nova estéril para cada transferência e minimizar o volume de solução transferida de prato para prato. </li><li> Por sua vez, cada embrião segurar suave mas firmemente, com um par de pinças e, utilizando-se a ferramenta de microdissecção formado no passo 1.5, remover o tubo neural e myotomes associados que estão localizados na parte mais dorsal do animal. Fazer isso através de três fatias, uma em cada extremidade da localização do tubo neural e um terceiro apenas ventral a ele (Figura 4A). Mover cada tubo neural dissecado / myotome a uma parte limpa do prato para longe da granul gemaes e outros detritos (Figura 4B). O eixo dorsal-ventral, e os locais do tubo neural e myotomes são indicados na Figura 4. </li><li> Após cerca de 15 minutos nesta solução (CMF) fórceps usar para levantar a pele pigmentada livre do tecido dissecado e descartar. Após um adicional de 30-60 minutos, as células formarão uma &quot;pilha de areia&quot;, como eles estão dissociados um do outro (Figura 4C). </li></ol> 3. Preparação do nervo músculo-Co-culturas <ol><li> Descongelar um tubo de 10 ml de meio de cultura e adicionar assepticamente 70 uL ITS e 35 ng / ml de BDNF (Sigma B3795). </li><li> Rótulo e preencher estéreis 35 pratos de cultura (milímetros FD35-100 Precision Instruments Mundo) aproximadamente a meio L-15 com meio de cultura (solução (e)), um para cada embrião dissecados. </li><li> Fabricar uma pipeta de chapeamento, segurando cada ponta de uma pipeta de Pasteur de vidro, mantendo a porção afunilada mais de uma chama. Puxe as pontas para além de cerca de 10 cm e, em seguida, se separam ao fim de produzir uma ponta de cerca de 0,2 mm. </li><li> Utilizar a pipeta de chapeamento para sugar a &quot;pilha de areia&quot; a partir de um embrião de minimizar a quantidade de solução desenhada. Expulsar as células na parte inferior de um prato de cultura. Placa de células em várias linhas. Observar banhados células indiferenciadas (Figura 5A e 5B). </li><li> Deixe os pratos de células plaqueadas não perturbadas por pelo menos quinze minutos para permitir que o tempo de células para anexar ao prato. </li></ol> 4. Patch clamping nervo-músculo Sinapses <ol><li> Após 12-24 h em cultura, as células banhados a assumir características morfológicas distintas: as células musculares se tornam fusiforme e neurônios espinhais ampliar os processos longos (Figura 6). Contato sináptico funcional entre varicosidades neurite e células musculares pode ser confirmado com simultâneas emparelhados registros eletrofisiológicos. &quot;M&quot;, &quot;S&quot; e &quot;V&quot; referem-se, respectivamente, à célula muscular, neuronalsoma e varicosidades pré-sináptica. </li><li> Preparar dois eléctrodos de patch para gravação: uma para o varicosidade pré-sináptica e um para as células musculares. </li><li> Para o eléctrodo de pré-sináptica, preparar uma solução de anfotericina contendo o seguinte: Adicionam-se 100 ul de DMSO para um tubo de microcentrífuga de 1,5 ml, contendo 5 mg de anfotericina B (Sigma A4888). Vortex esta solução durante 10 seg. Em seguida, adicionam-se 10 ul desta solução para um tubo de microcentrífuga de segundo que contém 625 ul de K +-interno solução de anfotericina B (solução (h)). Vortex como acima. </li><li> Encher o eléctrodo de pré-sináptica com o K + contendo anfotericina-interno solução preparada no passo 4.3 e o eléctrodo de pós-sináptica com K +-interno solução (solução de (g)). </li><li> Substituir a mídia na placa de cultura com NFR e realocá-lo em um microscópio invertido equipado com óptica de contraste de fase. </li><li> Posicione os dois eléctrodos apenas acima de suas respectivas metas. Obter o co de células inteirasnfiguration com o eletrodo de célula muscular antes da configuração de patch perfurada com o eletrodo varicosidades. </li><li> Para confirmar a funcionalidade da sinapse, despolarizar a varicosidade com um amplificador de patch clamp (Axopatch 200B, por exemplo, Molecular Devices), sob o controlo de software (por exemplo, por pCLAMP 9, Molecular Devices) e observar as correntes simultâneas pré-sinápticos e pós-sinápticos. Figura 7 mostra as correntes vistos em resposta aos passos de despolarização de tensão dadas ao varicosidade pré-sináptica em incrementos de 10 mV a partir de -30 mV a +40 mV. As correntes para dentro visto na célula pré-sináptica são transportados por Na + e Ca 2 + e as correntes exteriores por K +. Correntes gravados na célula pós-sináptica são respostas a neurotransmissor libertado do varicosidades e são realizados principalmente por meio de canais de Na + dos receptores nicotínicos da acetilcolina. </li></ol>

<ol>
	<li>Augustine, G. J., Eckert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divalent+cations+differentially+support+transmitter+release+at+the+squid+giant+synapse.">Divalent cations differentially support transmitter release at the squid giant synapse.</a> J. Physiol. 346, 257-271 (1984).</li><li>Spitzer, N. C., Lamborghini, 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=The+development+of+the+action+potential+mechanism+of+amphibian+neurons+isolated+in+culture.">The development of the action potential mechanism of amphibian neurons isolated in culture.</a> Proc. Natl. Acad. Sci. U.S.A. 73, 1641 (1976).</li><li>Weldon, P. R., Cohen, M. W. <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+synaptic+ultrastructure+at+neuromuscular+contacts+in+an+amphibian+cell+culture+system.">Development of synaptic ultrastructure at neuromuscular contacts in an amphibian cell culture system.</a> J. Neurocytol. 8, 239-259 (1979).</li><li>Tabti, N., Poo, M. -. M., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 137-153 (1991).</li><li>Yazejian, B., DiGregorio, D. A., Vergara, J. L., Poage, R. E., Meriney, S. D., Grinnell, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurements+of+presynaptic+calcium+and+calcium-activated+potassium+currents+regulating+neurotransmitter+release+at+cultured+Xenopus+nerve-muscle+synapses.">Direct measurements of presynaptic calcium and calcium-activated potassium currents regulating neurotransmitter release at cultured Xenopus nerve-muscle synapses.</a> J. Neurosci. 17, 2990 (1997).</li><li>DiGregorio, D. A., Peskoff, A., Vergara, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+action+potential-induced+presynaptic+calcium+domains+at+a+cultured+neuromuscular+junction.">Measurement of action potential-induced presynaptic calcium domains at a cultured neuromuscular junction.</a> J. Neurosci. 19, 7846 (1999).</li><li>Yazejian, B., Sun, X. P., Grinnell, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+presynaptic+Ca2++dynamics+during+neurotransmitter+release+with+Ca2+-activated+K++channels.">Tracking presynaptic Ca2+ dynamics during neurotransmitter release with Ca2+-activated K+ channels.</a> Nat. Neurosci. 3, 566 (2000).</li><li>Sun, X. P., Chen, B. M., Sand, O., Kidokoro, Y., Grinnell, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-induced+Ca2++entry+preferentially+evokes+release+of+large+quanta+in+the+developing+Xenopus+neuromuscular+junction.">Depolarization-induced Ca2+ entry preferentially evokes release of large quanta in the developing Xenopus neuromuscular junction.</a> J. Neurophysiol. 104 (5), 2730-2740 (2010).</li><li>Xie, S. P., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+events+in+the+formation+of+neuromuscular+synapse:+rapid+induction+of+acetylcholine+release+from+embryonic+neurons.">Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neurons.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 7069 (1986).</li><li>Li, P. P., Chen, C., Lee, C. W., Madhavan, R., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+filopodial+asymmetry+induced+by+synaptic+target.">Axonal filopodial asymmetry induced by synaptic target.</a> Mol. Biol Cell. 22 (14), 2480-2490 (2011).</li><li>Feng, Z., Ko, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+promote+synaptogenesis+at+the+neuromuscular+junction+via+transforming+growth+factor-beta1.">Schwann cells promote synaptogenesis at the neuromuscular junction via transforming growth factor-beta1.</a> J. Neurosci. 28 (39), 9599-9609 (2008).</li><li>Song, H. J., Ming, G. L., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP-induced+switching+in+turning+direction+of+nerve+growth+cones.">cAMP-induced switching in turning direction of nerve growth cones.</a> Nature. 17 (6639), 275-279 (1997).</li><li>Morimoto, T., Wang, X. H., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+synaptotagmin+modulates+short-term+synaptic+plasticity+at+developing+neuromuscular+junctions.">Overexpression of synaptotagmin modulates short-term synaptic plasticity at developing neuromuscular junctions.</a> Neuroscience. 82 (4), 969-978 (1998).</li><li>Lu, B., Czernik, A. J., Popov, S., Wang, T., Poo, M. M., Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+synapsin+I+correlates+with+maturation+of+the+neuromuscular+synapse.">Expression of synapsin I correlates with maturation of the neuromuscular synapse.</a> Neuroscience. 74 (4), 1087-1097 (1996).</li><li>Schaeffer, E., Alder, J., Greengard, P., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+IIa+accelerates+functional+development+of+neuromuscular+synapses.">Synapsin IIa accelerates functional development of neuromuscular synapses.</a> Proc. Natl. Acad. Sci. U.S.A. 26 (9), 3882-3886 (1994).</li><li>Liou, J. C., Tsai, F. Z., Ho, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiation+of+quantal+secretion+by+insulin-like+growth+factor-1+at+developing+motoneurons+in+Xenopus+cell+culture.">Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture.</a> J. Physiol. 553 (Pt. 3), 719-7128 (2003).</li><li>Peng, H. B., Yang, J. F., Dai, Z., Lee, C. W., Hung, H. W., Feng, Z. H., Ko, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+neurotrophins+and+schwann+cell-derived+signals+on+neuronal+survival/growth+and+synaptogenesis.">Differential effects of neurotrophins and schwann cell-derived signals on neuronal survival/growth and synaptogenesis.</a> J. Neurosci. 23 (12), 5050-5060 (2003).</li><li>Dan, Y., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hebbian+depression+of+isolated+neuromuscular+synapses+in+vitro.">Hebbian depression of isolated neuromuscular synapses in vitro.</a> Science. 12 (5063), 1570-1573 (1992).</li><li>Xiao, Q., Xu, L., Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+regulation+of+neurotransmitter+specification+and+embryonic+neuronal+calcium+spike+activity.">Target-dependent regulation of neurotransmitter specification and embryonic neuronal calcium spike activity.</a> J. Neurosci. 30 (16), 5792-5801 (2010).</li><li>Niewkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin). , (1967).</li></ol>

Não há conflitos de interesse declarados.

Principais passos para o sucesso co-culturas de neurónios motores e as células musculares são a utilização de forma adequada encenado embriões produzidos a partir da reprodução induzida de rãs Xenopus, e a dissecção cuidadosa asséptico dos neurónios espinais indiferenciadas e mioblastos. Ovos fertilizados deve ser deixado em repouso até alcançarem aproximadamente estágio 10 ou assim como movê-los mais cedo, muitas vezes interrompe o seu desenvolvimento. Embriões saudáveis ​​são identificados por uma aparência suave e uma coloração marrom e branco manchado. Fase 22-24 embriões são os mais úteis porque este é o momento durante o desenvolvimento apenas após o encerramento do tubo neural e antes que os miócitos foram diferenciadas de forma significativa. Além disso, as células de embriões mais velhos deixam de dissociar bem em CMF. Devem ser tomados cuidados durante a remoção da membrana vitelina em que adere fortemente ao embrião. A membrana deve ser rasgada com pinças cortantes, para que o embrião emerge intacta. Outra importante precaution é cuidadosamente colocar as células na parte inferior da placa de cultura (em vez de deixá-los resolver lá). Este método é preferível, pois isso aumenta a probabilidade de que as células aderem à placa de cultura. Neurotransmissão funcional entre nervo e músculo pode ser determinada com a gravação, pós-sináptica e muitas vezes pode ser determinado pela observação da contração muscular espontânea após inervação. Células não-musculares inervados não se contorcer na cultura. Gravação pós-sináptica só é útil para gravar correntes de placa terminal em miniatura ou potenciais, mas as medidas de liberação evocado exige estimulação pré-sináptica, e correlação de correntes de pré e pós-sináptica requer o dobro de patch-clamp. Para além do método de gravação de patch-emparelhado aqui descrito, esta preparação oferece a oportunidade de introduzir uma pipeta terceiro na soma neuronal 5 Isto permite a geração de um potencial de acção that pode propagar para o varicosidades pré-sináptico e levar à liberação do neurotransmissor. Além disso, os agentes putativos que se pensa mediar ou modular a transmissão sináptica pode ser introduzido a um ou outro lado da sinapse: via pipeta de células musculares ou por meio de difusão a partir de uma pipeta de terceiro colocado na soma.

<table border="1"><tbody><tr><td> Equipamentos / Suprimentos </td><td> Vendedor </td><td> Catálogo / Número modelo </td><td></td></tr><tr><td> A insulina-transferrina-selênio </td><td> Sigma </td><td> I1884 </td><td></td></tr><tr><td> Gonadotrofina Coriônica Humana </td><td> Sigma </td><td> CG-10 </td><td></td></tr><tr><td> Vapor Pressure Osmometer </td><td> Wescor </td><td> 5100C </td><td></td></tr><tr><td> Pinos Minutien </td><td> Belas Science Tools </td><td> 26002-10 </td><td></td></tr><tr><td> A anfotericina B </td><td> Sigma </td><td> A4888 </td><td></td></tr><tr><td> Fórceps </td><td> Belas Science Tools </td><td> 11251-30 </td><td></td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Nome da solução </td><td> Receita </td></tr><tr><td> (A) NFR (Ringer-rã normal (em mM) </td><td> 116 NaCl, KCl 2, 1,8 de CaCl2, 5 de Na +-HEPES, pH 7.35. </td></tr><tr><td> (B) 10% de solução salina </td><td> 10 ml NFR 90 ml de H2O desionizada </td></tr><tr><td> (C) CMF (Ca 2 + / Mg 2 + solução gratuita) </td><td> 125 NaCl, KCl 2, 1,2 de EDTA, 5 de Na + HEPES, pH 7.35. </td></tr><tr><td> (E) Media L-15 Cultura </td><td> 50 ml de L-15 mídia (Sigma L1518) + 50 ml NFR. </td></tr><tr><td> (G) K +-solução interna (em mM) </td><td> 116 KCl, 1 de NaCl, 1 de MgCl2, 10 EGTA, 5 HEPES, pH 7,3 </td></tr><tr><td> (H) K +-interno solução de anfotericina B (em mM) </td><td> 52 K 2 SO 4, 38 KCl, 1 EGTA, 5 HEPES, pH 7,3 </td></tr></tbody></table></td></tr></tbody></table> Table 1.

simultaneous pre- and post-synaptic electrophysiological recording from xenopus nerve-muscle co-cultures

Muita informação sobre o acoplamento de pré-sinápticas correntes iônicas com a liberação do neurotransmissor foi obtida a partir de preparações de invertebrados, principalmente a lula gigante sinapse 1. No entanto, excepto para a preparação aqui descritos, as preparações de vertebrados existem poucos em que é possível fazer medições simultâneas de libertação de neurotransmissores e pré-sinápticos correntes iónicas. Motoneurónios embrionários de Xenopus e células musculares podem ser cultivadas juntas no meio de cultura simples à temperatura ambiente, eles vão formar sinapses funcionais dentro de 12-24 horas, e pode ser utilizado para estudar o desenvolvimento do nervo e células de músculo e as interações sinápticas durante vários dias (até supercrescimento ocorre). Algumas vantagens de estas co-culturas mais preparações vertebrados outros incluem a simplicidade da preparação, a capacidade de manter as culturas e de trabalho à temperatura ambiente, e a pronta acessibilidade das sinapses formadas duas-4. A preparação tem sido amplamente utilizada para estudar as propriedades biofísicas de canais de íons pré-sinápticas e à regulação da liberação do transmissor 5-8. Além disso, a preparação prestou-se a outras utilizações, incluindo o estudo de crescimento de neurites e sinaptogênese 9-12, os mecanismos moleculares da libertação do neurotransmissor 13-15, o papel de mensageiros difusíveis em neuromodulação 16,17, in vitro e plasticidade sináptica 18 - 19.

A Figura 4B mostra a vista dorsal de um espinal medula isolado / myotome imediatamente após a sua remoção a partir de uma fase de 22 embriões de Xenopus 4C. Figura mostra que, após a solução de remoção da pele e incubação em +-Mg 2 + solução livre de Ca 2 (CMF, ( c)), as células de dissociar-se em um &quot;monte de areia&quot; e está pronto para revestimento. Imediatamente após o plaqueamento em meio de cultura de células apresentam pouca variabilidade morfológica (Figura 5B), mas assumem formas distintas, depois de vinte e quatro horas de cultura. As células musculares se fusiforme enquanto os neurônios permanecem esférico enquanto neurites estendendo que varicosites elaborados em sinapses com as células musculares (Figura 6). Gravação simultânea remendo emparelhado na varicosidade pré-sináptica e pós-sináptica da célula muscular (Figura 7) mostra as correntes de entrada e de saída pré-sinápticos associadas com a libertação de neurotransmissores e o resultant excitatórios correntes pós-sinápticos. <img src="/files/ftp_upload/50253/50253fig1.jpg" alt="Figura 1"/> Figura 1 A:.. Par Criação de rãs em balde de acasalamento sobre tela B: Rãs em amplexo com ovos fertilizados. <img src="/files/ftp_upload/50253/50253fig2.jpg" alt="Figura 2"/> Figura 2 A:. Fase B &quot;adequado&quot; 22 embrião:. Palco &quot;inadequado&quot; 28 embrião. Barra de escala representa 1 mm. <img src="/files/ftp_upload/50253/50253fig3.jpg" alt="Figura 3"/> Figura 3 A:. Stage 22 embrião antes da remoção da membrana vitelina e casaco geléia. A seta aponta para o bordo exterior do revestimento de gelatina. A membrana vitelina adere estreitamente à embryo e é praticamente invisível B:. embrião Bare. Barra de escala representa 1 mm. <img src="/files/ftp_upload/50253/50253fig4.jpg" alt="Figura 4"/> Figura 4 A: embrião Stage 22 com uma linha a tracejado indica a-ser dissecado porção B:.. Agudamente isolado porção dorsal de embriões, &quot;d&quot; e &quot;v&quot; referem-se aos aspectos dorsal e ventral dos embriões, enquanto que &quot;m&quot; e &quot;n&quot; indica as localizações aproximadas dos mytomes e tubo neural C:. &quot;pilha de areia&quot; de células após 60 min em CMF. Barra de escala representa 1 mm para uma e 0,5 mm para B e C. <img src="/files/ftp_upload/50253/50253fig5.jpg" alt="Figura 5"/> Figura 5 A:. Vista poder 5x de células em cultura, imediatamente após o plaqueamento. B: mesma cultura em 40x. Escalabarra representa 40 mm para A e 5 mm para B. <img src="/files/ftp_upload/50253/50253fig6.jpg" alt="Figura 6"/> Figura 6. Junção neuromuscular em cultura com identificação de soma neuronal (S), varicosidade pré-sináptico (V), e células do músculo pós-sináptico (M). Barra de escala: 5 m. <img src="/files/ftp_upload/50253/50253fig7.jpg" alt="Figura 7" fo:content-width="5in" fo:src="/files/ftp_upload/50253/50253fig7highres.jpg"/> Figura 7. Pré-sináptica (em cima) e pós-sináptico (inferior) correntes observados em resposta à aplicação de voltagem 10 mV passos incrementais apresentado ao varicosidade pré-sináptica de -30 mV a +40 mV. Degraus de tensão são indicados junto a alguns dos vestígios pré-sinápticos. O potencial de retenção para ambas as células foi de -70 mV.

Watch this Scientific Journal Video about Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures at JoVE.com

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Este vídeo demonstra os procedimentos usados ​​para crescer culturas primárias de embrião<em&gt; Xenopus</em&gt; Células nervosas e musculares ea utilidade desta preparação para fazer gravações simultâneas de fixação pré-e pós-sináptica patch.

Simultânea de pré e pós-sináptica gravação eletrofisiológico de<em&gt; Xenopus</em&gt; Nervo músculo-Co-culturas

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

simultaneous-pre-post-synaptic-electrophysiological-recording-from

Research

JoVE Journal

Neuroscience

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

10.8K visualizações.  David Geffen School of Medicine at UCLA. Este vídeo demonstra os procedimentos usados ​​para crescer culturas primárias de embrião Xenopus Células nervosas e musculares ea utilidade desta preparação para fazer gravações simultâneas de fixação pré-e pós-sináptica patch.

Vídeo: Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivação de Enriquecido Culturas oligodendroglial e Neuron / oligodendroglial Myelinating Co-culturas de tecidos pós-natais murino

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Neurociência

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

De gravação simultânea de sinais de cálcio dos neurônios identificados e comportamento alimentar de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neurais culturas de explante de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Prosencéfalo gravação eletrofisiológico em larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Gravação eletrofisiológica no cérebro de Intacta Zebrafish Adulto

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

Um Método para Sistemática de Eletroquímica e eletrofisiológica Avaliação do Neurais gravação Eletrodos

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Eletrofisiológica Gravação De<em&gt; Drosophila</em&gt; Labellar Prove sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Gravação simultânea eletrofisiológica e micro-injeções de agentes inibidores no cérebro do roedor

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Gravação simultânea de Eletrorretinografia e potenciais evocados visuais em ratos anestesiados

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...