NIH K08 EB003996

1. Poli-L-láctico (PLLA) Spinning Solution <ol><li> Dissolver 0,4 g PLLA em 9 mL de clorofórmio por agitação em fogo baixo. </li><li> Adicionar 1 mL dimetilformamida para a solução, trazendo a concentração final da solução para PLLA 4% (w / v) em clorofórmio: DMF 9:1 (v / v). </li><li> Coloque a solução em um polipropileno ou seringa de vidro com uma ponta de metal blunt 23ga. </li></ol> 2. Spinning Preparação do Substrato 1 <ol><li> Faça uma solução de 8% (w / v) de PLGA 85:15 (poli-lático-co-glicólico ácido) em clorofórmio por agitação em fogo baixo. </li><li> Revestimento de vidro limpo lamínulas em PLGA cobrindo a superfície de cada lamínula com a solução de PLGA. Permitir que o PLGA para secar a uma película fina (aprox. 30 min). </li></ol> 3. Eletrofiação 2 <ol><li> Seguro PLGA tampa de vidro revestido desliza para colecionador com fita de carbono condutora. Para fibras alinhadas, o coletor é uma roda motorizada. Para fibras aleatórias, o coletor é uma placa estacionária. </li><li> Seringa lugar na ponta da bomba com 20 cm de roda de colecionador. Conjunto de bomba a cerca de 2 mL / hr e se estiver usando uma roda, conjunto do motor para 300-400 RPM. Se possível, aplique um viés -2 kV DC para o coletor e 15 kV a ponta de metal. Na ausência de acesso a uma fonte de alimentação bipolar, terra do coletor. </li><li> Fibras será jato da ponta da seringa e recolher na roda girando. Continue girando até que a densidade desejada de fibras é obtido. Passe a ponta de metal com uma toalha de papel afixado a uma haste não-condutor periodicamente para evitar entupimento na ponta. </li></ol> 4. Moating 1 <ol><li> Spinning seguinte, &#39;fosso&#39; as amostras electrospun pipetando uma linha de PLGA em todo o perímetro da lamínula. Permitir que o PLGA para secar. Isto irá manter o alinhamento das fibras durante a cultura. </li></ol> 5. Revestimento de proteína <ol><li> Fibras de vidro electrospun e controles precisam ser revestidas em proteínas antes de cultura de células. Cobrir os substratos em uma solução de proteína, pelo menos, uma hora e depois aspirar o excesso de solução e continuar aspirando até os substratos estão secos. Soluções de proteína possíveis incluem: PLL (poli-L-lisina) (100 mcg / mL), laminina (25 mcg / mL) ou fibronectina (50 mcg / mL). Se PLL é utilizado, os substratos devem ser lavados em água estéril e seco duas vezes após o revestimento inicial. </li></ol> 6. Obtenção de embriões de ratos 3 E15 * Todos os experimentos foram feitos de acordo com o Guia de NIH para Cuidado e Uso de Animais de Laboratório, aprovado pela Universidade de Michigan Comissão de Uso e Cuidados dos Animais. <ol><li> Preparação: Verifique se o animal está grávida. Descongelar 3 mL de tripsina, 3 mL FBS e uma garrafa quente do L15. Fogo polonês uma pipeta Pasteur. Desinfectar todos os instrumentos cirúrgicos em etanol 70% por 30 min. </li><li> Preparar e aquecer media chapeamento (ver Tabela 1). </li><li> Eutanásia: Realizar uma injeção IP de cetamina / xilazina. Esperar 10-15 minutos e verificar se há falta de córnea e pedal reflexos de retirada. Uma vez que os reflexos estão ausentes, execute uma injeção de pentobarbital IC (FatalPlus). </li><li> Tenda a pele do abdômen. Corte através da pele e camadas musculares para expor a cavidade abdominal. Localize o útero e retirá-la no colo do útero e ovários. </li><li> Remoção dos embriões do útero com uma tesoura e coloque-os imediatamente em L15 quente. (Todos os seguintes procedimentos sejam concluídos em um microscópio de dissecação e embriões e os cabos devem ser dissecados submerso em L15 quente em todos os momentos.) Decapite os embriões fechando forceps em torno do queixo e abaixo do osso occipital do crânio. </li></ol> 7. Dissecção 3 <ol><li> Coloque o embrião de um lado. Proteger a medula espinhal com um conjunto de pinças enquanto usa o outro conjunto para descascar afastado membros e órgãos abdominais. </li><li> Vire o embrião e mantê-lo firme com um conjunto de fórceps. Snip ao longo do comprimento do embrião, do pescoço à cauda, ​​até que o cabo inteiro é exposto. </li><li> Segure a extremidade do cabo com cuidado e puxe-o sobre si mesmo para a cauda para remover. O cabo vai puxar livre com membrana e gânglios da raiz dorsal (DRG) em anexo. </li><li> Se DRG explante ou cultura dissociada neurônio sensorial é a ser executada, o DRG snip fora o cabo e transferi-los para um prato de doce L15 quente. <ol><li> DRG para a cultura de explantes, pule para o passo 10,2 </li><li> Para a cultura neurônio sensorial dissociada, pule para o passo 9 </li><li> Para motor a cultura de neurônios, o DRG serão removidos com a membrana no passo 7,5 </li></ol></li><li> Remover a membrana da medula espinhal, segurando-o na extremidade superior da medula e peeling-lo para baixo, semelhante ao de remover uma meia longa. Repita o procedimento para todos os embriões e depois cortar as cordas em pequenos (2-3 mm) peças. </li></ol><pclass = &quot;jove_title&quot;&gt; 8. Motor Neuron (MN) Processamento 5,6,7 <ol><li> Despeje os cabos cortados e L15 em um cone, girando em 1000 RPM por 2-3 min para agregar as peças. </li><li> Deitar fora o sobrenadante e adicionar 3 mL de tripsina para os cabos peletizada. Incubar em banho-maria a 37 ° C por 20 min. </li><li> Adicionar 3 mL FBS a cônica e girar a 1000 RPM por 2-3 min para re-pellet. </li><li> Deitar fora o sobrenadante e casaco de um incêndio polido Pasteur pipeta com FBS. </li><li> Adicionar uma pipeta Pasteur cheio de L15 para a cônica e depois triturar delicadamente até obter uma solução homogênea, sem grumos visíveis. Evitar bolhas. </li><li> Prepare uma solução de 9% do Optiprep em L15. Prepare um tubo com 3 mL da solução Optiprep para cada dois embriões (se houver um número ímpar de embriões, arredondar para cima). </li><li> Gota a solução homogeneizada para a solução Optiprep, dividindo-o igualmente entre todos os tubos. Girar os tubos a 2000 RPM por 15 min. </li><li> Recolher com cuidado o topo 2 mL de cada tubo e piscina em um 50 mL cônico. Preencher o cônico com L15 e girar a 1000 RPM por 5 minutos para enxaguar as células do Optiprep. </li><li> Deitar fora o sobrenadante e ressuspender as células em uma pequena quantidade de media chapeamento (250-500 mL). Contar as células usando a exclusão de tripan azul para identificar as células vivas. Pule para o passo 10,1 </li></ol> 9. Sensoriais Neuron Processing (SN) 7 <ol><li> Despeje o DRG removido da medula espinhal e L15 em um tubo cônico e giram a 1000 RPM por 2-3 min para agregar as peças. </li><li> Deitar fora o sobrenadante e adicionar 3 mL de tripsina ao DRG peletizada. Incubar em banho-maria a 37 ° C por 10 min. </li><li> Adicionar 3 mL FBS a cônica e girar a 1000 RPM por 2-3 min para re-pellet. </li><li> Deitar fora o sobrenadante e casaco de um incêndio polido Pasteur pipeta com FBS. </li><li> Adicionar uma pipeta Pasteur cheio de L15 para a cônica e depois triturar delicadamente até obter uma solução homogênea, sem grumos visíveis. Evitar bolhas. </li><li> Girar o homogeneizado a 1000 RPM por 5 min. Deitar fora o sobrenadante e ressuspender as células em uma pequena quantidade de media plating SN (250-500 mL). Contar as células usando a exclusão de tripan azul para identificar as células vivas. Continue até a etapa 10,1 </li></ol> 10. Revestimento e Cultura <ol><li> Para SN dissociados ou cultura MN: <ol><li> Cobrir os substratos com algumas gotas de mídia em seguida, adicione um volume adequado de suspensão celular. Células dissociadas devem ser banhados com uma densidade baixa (25-50 células / mm 2). Permitir que as células a aderir, pelo menos, uma hora antes de inundar os poços com a mídia. </li></ol></li><li> DRG para a cultura de explantes: <ol><li> Cobrir os substratos com algumas gotas de mídia em seguida, adicione DRG para a mídia. Organizar o DRG para que eles estão uniformemente distribuídas em todo o substrato (aproximadamente 4 DRG vai caber em um pedaço 22x22 mm capa). Permitir que o DRG para aderir por pelo menos 4 horas antes da inundação dos poços com a mídia. </li></ol></li><li> Culturas pode ser mantido por até 4 dias sem mudança de mídia. Para culturas mais, as mudanças meia media deve ser feito com alimentação de mídia. Alimentação de mídia é a mesma composição que media plating menos a L-glutamina. </li></ol> 11. Imunocitoquímica 1,5,8 <ol><li> Fixação células: media Aspirar e substituir com calorosa PFA 4% (paraformaldeído) por 30 min. Em seguida, executar 3X 5 min lava 1x PBS. </li><li> Bloqueio / permeabilização: Prepare bloco / perm solução (1,25% de soro albumina bovina em 1X PBS, 0,05% Triton X-100 e 2,0% de soro de cabra normal). Aspirar PBS e aplicar bloco / perm solução para amostras por 30 minutos. Em seguida, executar 1X 5 min lava 1x PBS. </li><li> Anticorpo primário: Prepare a solução de anticorpo primário de trabalho (10% soro de cabra normal, 1,25% albumina sérica bovina, 0,1% Na azida, 0,05% Triton X-100 mL e 10 com 1x PBS). Adicionar a quantidade adequada do anticorpo primário (ver Tabela 2). Linha de vários pratos com parafilme. Coloque uma gota 200μl de solução de anticorpo primário sobre o parafilme para cada substrato. Inverter os substratos, flutuando-los lado célula para baixo sobre a solução de anticorpo primário trabalhando durante a noite (se o quarto tem ar condicionado ou particularmente seco, um pedaço de papel saturado de água pode ser adicionada a um canto do prato para evitar a evaporação da solução de trabalho) . </li><li> Anticorpo secundário: Remove os substratos do primário (a solução de trabalho podem ser recolhidos, armazenados a 4 ° C e reutilizados) e realizar 2X 5 min lava 1x PBS. Aplicar 200 mL de anticorpo secundário diluído 1:200 em PBS (também invertida em parafilme prato forrado) por 4 horas. </li><li> Retire do secundário em seguida, executar 2X 5 min lava 1x PBS. Substratos de montagem em lâminas com Prolong ouro contendo DAPI ou outro contador adequado nuclear mancha. </li></ol> 12. Resultados representativos: A morfologia típica de alinhados e el aleatóriaectrospun fibras são mostrados na Figura 1. Nós normalmente obter MN pureza&gt; 90% 7,8 neurônios com este protocolo e mantivemos DRG culturas durante o tempo que uma semana sem qualquer crescimento de fibroblastos significativa. A Figura 2 mostra a aparência típica MN em vidro e fibras após 24 horas de cultura e imunomarcação. DRG imunocoradas cultivados por três dias em vidro e fibras são representadas na Figura 3. <img src="/files/ftp_upload/2389/2389fig1.jpg" alt="Figura 1" /> Figura alinhamento Fiber 1. É manipulado por escolha de colecionador. A.) Alinhados fibras fiadas em um coletor roda girando e B.) fibras aleatórias girou em um coletor placa estacionária. Barra de escala = 20 mM. <img src="/files/ftp_upload/2389/2389fig2.jpg" alt="Figura 2" /> Figura 2. Neurônios motores Representante coradas para TuJ1 (verde) e DAPI (azul) após 24 horas de cultura na A. revestido PLL) fibras e B.) de vidro. Barra de escala = 20 mM. <img src="/files/ftp_upload/2389/2389fig3.jpg" alt="Figura 3" /> Figura 3. Manchada Representante DRG para neurofilamento (verde) após 3 dias de cultura em PLL A. revestido) de vidro e B.) fibras. Barra de escala = 200 mM. <table border="1"><tbody><tr><td> Solução estoque: </td><td> MN mídia: </td><td> DRG / SN mídia: </td></tr><tr><td> 10 mg mL -1 de albumina </td><td> 12,5 mL </td><td> - </td></tr><tr><td> 10 mg mL -1 apo-transferrina </td><td> 50 ul </td><td> 40 mL </td></tr><tr><td> 50 mg mL -1 β-estradiol </td><td> 2,7 mL </td><td> 2,2 mL </td></tr><tr><td> 0,1 mg mL -1 biotina </td><td> 50 ul </td><td> - </td></tr><tr><td> 16 mg mL -1 catalase </td><td> 8 mL </td><td> - </td></tr><tr><td> 15 mg mL -1 D-galactose </td><td> 50 ul </td><td> - </td></tr><tr><td> 50 mg mL -1 hidrocortisona </td><td> 3,7 mL </td><td> 2,9 mL </td></tr><tr><td> 0,63 mg mL -1 progesterona </td><td> 0,5 mL </td><td> - </td></tr><tr><td> 16 mg mL -1 putrescina </td><td> 50 ul </td><td> - </td></tr><tr><td> 50 mg mL -1 de selênio </td><td> 3,4 mL </td><td> 4,1 mL </td></tr><tr><td> 2,5 mg mL -1 superóxido dismutase </td><td> 50 ul </td><td> - </td></tr><tr><td> * 500 ng mL -1 fator de crescimento neural </td><td> - </td><td> 1 mL </td></tr><tr><td> * 100X PSN </td><td> 0,5 mL </td><td> 0,5 mL </td></tr><tr><td> * B27 </td><td> 1 mL </td><td> 1 mL </td></tr><tr><td> * 2 mM L-glutamina </td><td> 35 mL </td><td> 35 mL </td></tr><tr><td> * Neurobasal </td><td> a 50 mL </td><td> a 50 mL </td></tr></tbody></table> Tabela 1. Adicionar asterisco (*) componentes para a mídia pouco antes de usar. Os demais componentes podem ser preparados como solução estoque e mantido a -20 ° C até ser necessário 6,7. <table border="1"><tbody><tr><td> Primário (concentração) </td><td> Neurônios: </td><td> Glia: </td><td> Fibroblastos: </td></tr><tr><td> anti-β-tubulina (TuJ1) (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-neurofilamento (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-S-100 (1:250) </td><td> X </td><td> ✓ </td><td> ✓ </td></tr><tr><td> anti-NGFR p75 (1:500) </td><td> ✓ </td><td> ✓ </td><td> X </td></tr></tbody></table> Tabela 2. A seleção do anticorpo primário depende dos objetivos do pesquisador. O acima são vários anticorpos e as concentrações que temos utilizado com sucesso em nosso laboratório. S-100 é particularmente útil para identificar as células de Schwann e / ou verificar se há contaminação fibroblastos, mas note que ele deve ser usado em combinação com NGFR p75 distinguir entre glia e fibroblasts4. Temos notado também que NGFR p75 manchas neurites muito levemente, enquanto neurofilamento ou TuJ1 são excelentes opções, se o objetivo é visualizar neurites individual.

<ol>
	<li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>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. , (1998).</li><li>Kameda, Y. <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+glial+progenitor+markers+p75NTR+and+S100+protein+in+the+developing+mouse+parathyroid+gland.">Expression of glial progenitor markers p75NTR and S100 protein in the developing mouse parathyroid gland.</a> Cell Tissue Res. 327, 15-23 (2007).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Vincent, A. M., Mobley, B. C., Hiller, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-I+prevents+glutamate-induced+motor+neuron+programmed+cell+death.">IGF-I prevents glutamate-induced motor neuron programmed cell death.</a> Neurobiol. Dis. 16, 407-416 (2004).</li><li>Russell, J. W., Windebank, A. J., Schenone, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+prevents+apoptosis+in+neurons+after+nerve+growth+factor+withdrawal.">Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.</a> J. Neurobiol. 36, 455-467 (1998).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, 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=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

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Este protocolo tem várias etapas críticas. A primeira envolve a produção adequada dos substratos fibra electrospun. Em meio líquido, as fibras PLLA, PLGA cinema e fosso PLGA será separada da lamínula de vidro como uma única unidade. Se o PLGA é omitido, as fibras PLLA não continuará a ser uma folha plana - eles vão enrolar em um emaranhado inutilizável. Assim, o filme PLGA está incluído como um substrato adequado para manter o alinhamento da fibra durante a cultura. O fosso é adicionado após girar tanto para garantir que as fibras estão firmemente fixos ao filme PLGA e acrescentar rigidez estrutural para o filme. O fosso também serve como uma alça útil quando os substratos são manipuladas durante a fixação, coloração e montagem. Trituração é o segundo passo crítico. Todo esforço deve ser feito para evitar a formação de bolhas. Além disso, temos obtido rendimentos muito maior quando uma pipeta-fogo polido é utilizada para esta etapa. Ao fogo polonês, acender um bico de Bunsen e anexar uma lâmpada para a pipeta. Passe a ponta da pipeta rapidamente através da chama 2-3 vezes, enquanto continuamente apertando e soltando o bulbo - fluxo de ar através da pipeta ajudará a impedir que a ponta de fechar completamente. Examine a ponta da pipeta de modo que o buraco ainda está aberto e que as bordas ligeiramente arredondadas aparecem antes do uso. Eletrofiação é um processo muito versátil, os parâmetros electrospinning pode ser modificado para produzir fibras com uma variedade de morfologias. Tan et al. (2005) detalha um estudo sistemático dos parâmetros que afetam o diâmetro das fibras PLLA electrospun. Wang et al. (2009) apresenta um estudo detalhado dos parâmetros que influenciam o alinhamento das fibras electrospun PLLA.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

Eletrofiação é uma técnica para a produção de micro-escala nanométrica para fibras. Fibras podem ser electrospun com diferentes graus de alinhamento, de altamente alinhado completamente ao acaso. Além disso, as fibras pode ser girado de uma variedade de materiais, incluindo polímeros biodegradáveis, tais como poli-L-láctico (PLLA). Estas características tornam as fibras electrospun adequado para uma variedade de aplicações andaime em engenharia de tecidos. Nosso foco é sobre o uso de fibras electrospun alinhados para a regeneração do nervo. Nós temos mostrado previamente que as fibras alinhadas electrospun PLLA direcionar o crescimento de ambos os neurônios sensoriais e motoras primária in vitro. Sustentamos que o uso de um sistema primário de cultura de células é essencial quando se avalia biomateriais para os neurônios modelo real encontrada in vivo, tanto quanto possível. Aqui, descrevemos técnicas utilizadas em nosso laboratório para electrospin andaimes fibroso e cultura explantes gânglios da raiz dorsal, bem como dissociar os neurônios sensoriais e motoras, em andaimes electrospun. No entanto, o electrospinning e / ou técnicas de cultura aqui apresentados são facilmente adaptadas para uso em outras aplicações.

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Fibras alinhadas electrospun direcionar o crescimento de neurônios in vitro e são um componente potencial de andaimes regeneração nervosa. Nós descrevemos um procedimento para elaborar substratos fibra electrospun e da cultura livre de soro-sensorial primário de ratos E15 (DRG) e neurônios motores. Visualização de neurônios por imunocitoquímica também está incluído.

A Cultura da Motor Primário e neurônios sensoriais no Media Definidas em electrospun Poly-L-ácido lático nanofibras Andaimes

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

19.3K visualizações.  University of Michigan. Fibras alinhadas electrospun direcionar o crescimento de neurônios in vitro e são um componente potencial de andaimes regeneração nervosa. Nós descrevemos um procedimento para elaborar substratos fibra electrospun e da cultura livre de soro-sensorial primário de ratos E15 (DRG) e neurônios motores. Visualização de neurônios por imunocitoquímica também está incluído.

Vídeo: The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash Fotólise de Compostos Caged no Cilia de Olfactory neurônios sensoriais

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura de Dissociated neurônios sensoriais de Chick Embriões

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparação para gravações eletrofisiológicas da Spinal Sensorial e Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...