Este trabalho foi apoiado por R01 DC013339, R03 DC011373 e Ruth L. Kirschstein Serviço Nacional Research Award T32 DC000014. Uma parte do trabalho foi realizada através do Monell Chemosensory Receptor Signaling Core, que é suportado, em parte, pelo financiamento do P30 DC011735 NIH-NIDCD Núcleo Grant. Os autores agradecem C. Sezille para obter ajuda com a coleta de dados.

1. Cultura de Células Hana3A <ol><li> Prepare a mídia M10, completando meio mínimo essencial (MEM) com 10% (v / v) de FBS. </li><li> Cultura de manutenção <ol><li> Manter as células em meios de M10. NOTA: Os vetores de expressão para RTP1L, RTP2, REEP1 e G αolf conferir resistência puromycin às células Hana3A, mas mantendo as células com este antibiótico não afeta significativamente os resultados do ensaio. </li><li> Subcultura a uma proporção de 1:08 em placas de 10 cm a cada 2-3 dias. </li><li> Incubar a 37 ° C com 5% de CO 2. </li></ol></li></ol> 2. Células Plating para transfecção <ol><li> Mídia aspirado de 100% confluente 10 centímetros prato de células Hana3A. </li><li> Lave as células pela adição de 10 ml de PBS, agitando o prato, e aspirar o PBS. </li><li> Adicionar 3 ml de 0,05% de tripsina / EDTA e esperar para dissociar as células (cerca de 1 minuto). </li><li> Inativar a tripsina, adicionando 5 ml M10 e quebrar aglomerados de células por trituração aproximadamente 10x eº 160, com uma pipeta de 10 ml. Pipeta com cuidado para evitar a introdução de bolhas de ar dentro da mídia. </li><li> Para cada placa de 96 poços, a transferência de um ml de células para um tubo de 15 ml, centrifugar a 200 xg durante 5 min, e aspirar o sobrenadante sem perturbar o sedimento celular. </li><li> Ressuspender as células em 6 ml de M10 por 1 ml de células transferidas no passo 2.5. </li><li> Pipetar 50 ul de células para cada poço de uma placa de 96 cavidades e incubar durante a noite a 37 ° C com 5% de CO 2. </li></ol> 3. Transfecção de Receptores olfativos <ol><li> Preparação do ADN O plasmídeo <ol><li> Preparar DNA de plasmídeo, através de um protocolo livre de endotoxina. NOTA: preparação kits usam DNA de plasmídeo designado &quot;livre de endotoxina,&quot; ou adicionar um passo de extracção com fenol-clorofórmio, para o protocolo de preparação de ADN de plasmídeo. </li><li> Dilui-se o ADN a uma concentração de 100 ng / mL em tampão de TE. </li></ol></li><li> Observar células plaqueadas (Passo 2.7) para garantir uma confluência adequado de aproximaçãomadamente 30-50% por poço e voltar a incubadora. Nota: Embora esta confluência não é óptima para o reagente de transfecção de lípidos, uma confluência de 30-50% nesta etapa é óptima para a medição da actividade de luciferase 24 h após a transfecção. </li><li> Preparação da mistura A transfecção <ol><li> Pipeta RTP1S-PCI, M3-R-PCI, pcre-luc e pSV40-RL plasmídeos em meio MEM por os volumes detalhados na Tabela 1 para fazer a mistura do plasmídeo (volumes indicados são por placa de 96 poços). <table border="0" cellpadding="0" cellspacing="0" style="width:356px;"><tbody><tr height="22"><td colspan="3" height="22" style="height:22px;width:356px;"> Mix plasmídeo </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td> por poço </td><td> por placa de 96 poços </td></tr><tr height="21"><td height="21" style="height:21px;"> MEM </td><td> - </td><td> 500 ul </td></tr><trheight = &quot;21&quot;&gt; <td height="21" style="height:21px;"> RTP1S-PCI </td><td> 5 ng </td><td> 480 ng </td></tr><tr height="21"><td height="21" style="height:21px;"> M3-R-PCI </td><td> 2,5 ng </td><td> 240 ng </td></tr><tr height="21"><td height="21" style="height:21px;"> pcre-luc </td><td> 10 ng </td><td> 960 ng </td></tr><tr height="22"><td height="22" style="height:22px;"> pSV40-RL </td><td> 5 ng </td><td> 480 ng </td></tr></tbody></table> Tabela 1. Componentes da mistura. Plasmid por poço e por volumes de placas de 96 poços de RTP1S-PCI, M3-R-PCI, pcre-luc e pSV40-RL, e MEM. </li><li> Para cada placa de 96 poços, diluir 18 ul de reagente de transfecção de lípidos em 450 ul de meio MEM. </li><li> Pipeta plasmídeo mistura (do Passo 3.3.1), o receptor olfactivo marcaram-rodopsina no plasmídeo pCI (Rho-OR-PCI), e mistura de transfecção de lípidos (a partir do passo 3.3.2) para fazer o complexo em detalhada <strong&gt; Tabela 2. Misture a solução por meio de trituração e incubar à temperatura ambiente durante 15 min. Parar a reacção adicionando M10 de acordo com a Tabela 2 NOTA:. Esta reação é sensível ao tempo e não deve ser autorizado a continuar por mais de 30 min. O cálculo bem + 10% é importante para assegurar um volume suficiente para os passos subsequentes. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="21"><td colspan="3" height="21" style="height:21px;width:472px;"> Complexo </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td> por poço </td><td> por cavidade + 10% </td></tr><tr height="21"><td height="21" style="height:21px;"> Mix plasmídeo </td><td> 4,2 mL </td><td> 4,58 mL </td></tr><tr height="21"><td height="21" style="height:21px;"> Rho-OR-PCI </td><td> 0,05 ng </td><td> 0,06 ng </td></tr><trheight = &quot;21&quot;&gt; <td height="21" style="height:21px;"> Mistura de transfecção lipídica </td><td> 4,2 mL </td><td> 4,58 mL </td></tr><tr height="22"><td height="22" style="height:22px;"> M10 </td><td> 41,7 mL </td><td> 45,83 mL </td></tr></tbody></table> Tabela 2. Componentes complexos. Por poço e assim por + 10% de volume de mistura de plasmídeo (Tabela 1), o plasmídeo receptor olfactivo (Rho-OR-PCI), e mistura de transfecção lipídica. M10 é adicionado para parar a reacção após uma incubação de 15 min à temperatura ambiente. </li></ol></li></ol><ol start="4"><li> Toque fora da mídia sobre as placas de celulares. </li><li> Pipetar 50 ul de complexo a cada poço e incubar durante a noite a 37 ° C com 5% de CO 2. </li></ol> 4. Odor Estimulação <ol><li> Observar as células transfectadas para garantir uma adequada confluência de 50-80% por poço e voltar a incubadora. NOTA: Se as células são menos de 50% Confluentes, firefly luciferase e renilla luciferase leituras pode ser muito baixo para a medição de ativação do receptor. Considere descartando a placa. </li><li> Preparar soluções stock 1 M de cada odor em DMSO. </li><li> Preparar soluções de estimulação odor em meio CD293. <ol><li> Para experiências de rastreio, uma solução diluída de estoque de odor a 100 uM. Também preparar um controle sem odor (CD293 apenas), a fim de controlar ou a ativação de fundo. NOTA: Para os experimentos de triagem, cada par OR / odor é testada apenas uma vez por experiência. Porque alguma difusão de odor através poços é possível, recomenda-se a estimular com um odorante para cada placa. </li><li> Para as experiências de dose-resposta, preparar sete diluições em série de 10 vezes de uma solução de estoque de odor em triplicado a partir de 1 mM para cada receptor. Também preparar as mesmas diluições de odor, em triplicado, para as células transfectadas com vector vazio, a fim de controlar a activação fundo odor. NOTA: Para os experimentos de dose-resposta, cada um otratamento concentração dor deve ser realizado em triplicata. </li></ol></li><li> Toque fora da mídia sobre as placas de celulares. </li><li> Pipetar 25 ul de solução de estimulação odor a cada poço e incubar durante 4 horas. </li></ol> 5. Medição ou atividade através de ensaio de luciferase <ol><li> Ressuspender substrato firefly luciferase acordo com as instruções do fabricante e armazenar a -80 ° C. </li><li> Descongelar 1 ml de substrato de luciferase do pirilampo por placa de 96 poços. </li><li> Prepare a reacção da luciferase do pirilampo fresco quencher e reagente de substrato de luciferase de Renilla (5 ul da luciferase extintor / substrato de luciferase de Renilla por 1 ml de tampão). NOTA: Aproximadamente 1 ml de reagente é necessária por placa de 96 poços. </li><li> Prepare o leitor de microplacas luminescente. Abra o software leitor de microplacas. Dentro do ícone do sistema: <ol><li> De acordo com a &quot;pré-aquecimento&quot; Tab, marque a caixa &quot;ON&quot; e ajustar a temperatura da máquina a 25 °; C. </li><li> Na guia &quot;Dispenser&quot;, primo cada dispenser com 1.000 l de etanol 70%, seguido por 1000 mL de água destilada. NOTA: Utilize alíquotas separadas de álcool e água para cada distribuidor. O etanol é usado para desinfectar os dispensadores e água remove o etanol residual. </li><li> Primeiro cada dispenser com 1.500 l de ar (remover dispensadores de líquido). NOTA: priming com ar garante que substratos luciferase não são diluídos com água residual. </li><li> Primeiro um dispensador com 1080 uL de substrato de luciferase de pirilampo (do Passo 5.2). Primeiro distribuidor 2 com substrato de luciferase de Renilla (do Passo 5.3). NOTA: Tenha cuidado para não contaminar os substratos luciferase. Priming com substratos luciferase preenche o espaço morto nos dispensadores de reagentes. </li></ol></li><li> Configure o seguinte protocolo para ler tanto vaga-lume e Renilla luciferase luminescência. Dentro do software associado ao leitor de microplacas, under o menu &quot;Arquivo&quot;, clique em &quot;Nova Tarefa&quot;. Destaque &quot;Protocolos&quot; e clique em &quot;Criar novo&quot;. Na janela seguinte, o círculo ao lado de &quot;protocolo padrão&quot; deve ser selecionada. Clique em &quot;OK&quot;. Dê um duplo clique em &quot;Procedure&quot; no lado esquerdo da tela. <ol><li> Dispensar 10 ml de substrato luciferase de vaga-lume a todos os poços que utilizam distribuidor 1. Sob o menu &quot;Ações&quot;, clique em &quot;Dispensar&quot;. Na janela &quot;Dispensar Step&quot;, defina: &quot;Dispenser&quot; a 1 &quot;Priming&quot; a nenhum, &quot;Volume Dispense&quot; a 10 mL e &quot;Rate&quot; para 225 mL / seg. Clique em &quot;OK&quot;. </li><li> Agitar a placa por 30 seg. Sob o menu &quot;Ações&quot;, clique em &quot;Shake&quot;. Na janela &quot;Agite Step&quot;, defina &quot;Intensity&quot; para Médio e &quot;Duração&quot; a 0:30 MM: SS. Clique em &quot;OK&quot;. </li><li> Leia a luminescência de todos os poços para 0,5 seg por poço. Sob o menu &quot;Ações&quot;, clique em &quot;Leia&quot;,. Na janela &quot;Ler Step&quot;, defina: &quot;Método de detecção&quot; para luminescência, &quot;Ler Tipo&quot; para Endpoint, &quot;Integração Time&quot; para 00:00:50 MM: SS: ss, &quot;Conjuntos de Filtros&quot; para 1, &quot;Emissão&quot; para Hole, &quot;Optics Position&quot; para cima &quot;, Gain&quot; para 135, e &quot;Ler Altura&quot; a 1.00 mm. Clique em &quot;OK&quot;. </li><li> Dispensar 10 ml de substrato Renilla luciferase em todos os poços utilizando dispenser 2. Definir as condições como no Passo 5.6.1, exceto set &quot;Dispenser&quot; a 2. </li><li> Agite placa por 30 seg. Definir as condições como no passo 5.6.2. </li><li> Leia luminescência de todos os poços para 0,5 seg por poço. Definir as condições como no passo 5.6.3. </li></ol></li><li> Remover a tampa da placa de 96 poços e colocar a placa no leitor de microplacas. Inicie o programa criado no Passo 5.5 para ler placa de luminescência. </li><li> Limpe as bombas de doseamento de reagentes. A partir do ícone do sistema, na guia &quot;Dispenser&quot;: <ol><li> Purifica 1.000 l de fsubstrato de luciferase irefly do dispensador de luciferase do pirilampo para um tubo de recuperação. NOTA: a luciferase de pirilampo podem ser armazenadas a -80 ° C e reutilizada. </li><li> Primeiro cada dispensador com 1.000 mL de água destilada, seguido por 1,000 mL de etanol a 70% e, finalmente, 1,500 l de ar (remover distribuidores de líquido). NOTA: A água remove substratos luciferase das bombas reagentes, desinfecta etanol e ar seca qualquer etanol residual. </li></ol></li></ol> 6. Análise de Dados <ol><li> Exportação de Dados <ol><li> No software leitor de microplacas, clique duas vezes no &quot;Relatório / Exportação Builders&quot; no lado esquerdo da tela. </li><li> Clique no botão &quot;New Export para o Excel&quot; e clique em &quot;OK&quot;. </li><li> Destaque Export1 e clique em &quot;Editar&quot;. <ol><li> Em &quot;Content&quot; cheque &quot;Descrição do Sistema&quot;, &quot;Procedimento&quot;, &quot;Placa Descrição&quot; e &quot;Placa de layout Matrix&quot;. Incluir um &quot;dados brutos&quot;nd &quot;Calculado de dados&quot;. </li><li> Em &quot;fluxo de trabalho&quot;, marque a opção &quot;Executar Automaticamente após a conclusão do procedimento.&quot; Em Modo de Exportação, verifique &quot;Todas as placas na mesma pasta de trabalho&quot; e &quot;Como uma nova planilha&quot;. </li><li> Em &quot;File&quot; escolher o formato de nome de arquivo eo local do arquivo e clique em &quot;OK&quot;. </li><li> Feche a janela &quot;Relatório / Exportação Builders&quot;. </li></ol></li></ol></li><li> Para obter os valores normalizados luciferase, divida a luciferase de vaga-lume leitura de luminescência para cada poço (Passo 5.6.3) pela leitura de luminescência Renilla para cada poço (Passo 5.6.6). </li></ol>

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R., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+state+of+the+M3+muscarinic+acetylcholine+receptor+modulates+mammalian+odorant+receptor+signaling.">Activation state of the M3 muscarinic acetylcholine receptor modulates mammalian odorant receptor signaling.</a> Science signaling. 4 (155), (2011).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nature. 3 (9), 1402-1413 (2008).</li><li>Haddad, R., Khan, R., Takahashi, Y. K., Mori, K., Harel, D., Sobel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metric+for+odorant+comparison.">A metric for odorant comparison.</a> Nature methods. 5 (5), 425-429 (2008).</li><li>Veithen, A., Wilkin, F., Philippeau, M., Van Osselaer, C., Chatelain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Receptors:+From+basic+science+to+applications+in+flavors+and+fragrances.">Olfactory Receptors: From basic science to applications in flavors and fragrances.</a> Perfumer and Flavorist. 35 (1), 38-40 (2010).</li><li>Bozza, T., Feinstein, P., Zheng, C., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system.">Odorant receptor expression defines functional units in the mouse olfactory system.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 22 (8), 3033-3043 (2002).</li><li>Oka, Y., Katada, S., Omura, M., Suwa, M., Yoshihara, Y., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+map+in+the+mouse+olfactory+bulb:+in+vivo+sensitivity+and+specificity+of+receptor-defined+glomeruli.">Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli.</a> Neuron. 52 (5), 857-869 (2006).</li><li>Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+a+mammalian+odorant+receptor.">Functional expression of a mammalian odorant receptor.</a> Science. 279 (5348), 237-242 (1998).</li><li>Touhara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+identification+and+reconstitution+of+an+odorant+receptor+in+single+olfactory+neurons.">Functional identification and reconstitution of an odorant receptor in single olfactory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 4040-4045 (1999).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+accessory+factors+in+functional+expression+of+mammalian+odorant+receptors.">Synergism of accessory factors in functional expression of mammalian odorant receptors.</a> The Journal of biological chemistry. 282 (20), 15284-15293 (2007).</li><li>McRae, J. F., Mainland, J. D., Jaeger, S. R., Adipietro, K. A., Matsunami, H., Newcomb, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+odorant+receptor+OR2J3+is+associated+with+the+ability+to+detect+the+&amp;#34;grassy&amp;#34;+smelling+odor,+cis-3-hexen-1-ol.">Genetic variation in the odorant receptor OR2J3 is associated with the ability to detect the &amp;#34;grassy&amp;#34; smelling odor, cis-3-hexen-1-ol.</a> Chemical senses. 37 (7), 585-593 (2012).</li><li>Adipietro, K. A., Mainland, J. D., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evolution+of+mammalian+odorant+receptors.">Functional evolution of mammalian odorant receptors.</a> PLoS genetics. 8 (7), (2012).</li><li>Zhuang, H., Chien, M. -. S., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+functional+evolution+of+an+odorant+receptor+for+sex-steroid-derived+odors+in+primates.">Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21247-21251 (2009).</li><li>Oka, Y., Nakamura, A., Watanabe, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+odorant+derivative+as+an+antagonist+for+an+olfactory+receptor.">An odorant derivative as an antagonist for an olfactory receptor.</a> Chemical senses. 29 (9), 815-822 (2004).</li><li>Oka, Y., Omura, M., Kataoka, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+receptor+antagonism+between+odorants.">Olfactory receptor antagonism between odorants.</a> The EMBO journal. 23 (1), 120-126 (2004).</li><li>Fawcett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+ROC+analysis.">An introduction to ROC analysis.</a> Pattern Recognition Letters. 27 (8), 861-874 (2006).</li><li>Baghaei, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Receptors.">Olfactory Receptors.</a> Olfactory Recept. Methods Protoc. 1003, 229-238 (2013).</li><li>Dey, S., Zhan, S., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+surface+expression+of+chemosensory+receptors+in+heterologous+cells.">Assaying surface expression of chemosensory receptors in heterologous cells.</a> Journal of visualized experiments JoVE. (48), (2011).</li></ol>

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Identidade Odorant é codificada por padrões de ativação dos receptores olfativos, mas padrões de ativação dos receptores, incluindo os receptores que são ativados e em que grau, são conhecidos por menos de 6% dos receptores olfativos humanos 1-11. Os esforços para caracterizar receptores olfativos têm sido limitados por seus métodos de trabalho intensivo ou aplicabilidade a apenas um subconjunto da família de receptores olfativos 17,23,24,33,34. O sistema de expressão heterólogo Hana...

O sistema olfactivo dos mamíferos tem a capacidade de responder a um grande número de estímulos odoríferos, permitindo a detecção e a discriminação de milhares de odorantes. Receptores olfativos (RUP) são os sensores moleculares expressas pelos neurônios sensoriais olfativos no epitélio olfativo 12. Reconhecimento odor de mamíferos ocorre através da ativação diferencial das RUP por odores, ea família ou gene é extensa, com cerca de 1.000 murino e 400 receptores humanos 12-16. Análises funcionais anteriores do RUP em neurônios olfativos e em células heterólogas têm demonstrado que diferentes odores são reconhecidos pela única, mas a sobreposição de conjuntos RUP 10,17-20. Ligantes Correspondência para RUP é fundamental para a compreensão do código olfativo e essencial para a construção de modelos viáveis ​​de olfato. Devido às dificuldades de expressar RUP em sistemas heterólogos, bem como o grande número de ambos os odorantes e RUP, estes dados tem sido largamente ausente do field; de facto, menos de 6% de RUP humanos têm um ligando publicado 1-11. Este protocolo descreve o uso de um ensaio de luciferase para a caracterização do odorante / ou interacções. Este ensaio permite a caracterização de alto rendimento das RUP, uma tarefa que é essencial para a compreensão de odorante / ou interações, bem como o desenvolvimento de um modelo de codificação odor. Estudos de alto rendimento de RUP enfrentam três grandes desafios. Primeiro, RUP expressos em células heterólogas foi retida no ER e subsequentemente degradados no proteassoma 21,22, impedindo as RUP de interagir com odorantes no sistema de ensaio de 23-25. Este problema foi resolvido com a descoberta de proteínas acessórias que facilitam a expressão da superfície celular estável de uma ampla gama de RUP 19,26,27. Receptor-transportador proteínas 1 e 2 (RTP 1 e 2) promover a expressão ou da superfície celular e activação em resposta à estimulação do odorante 19. Com base neste trabalho, células HEK293T forammodificada para expressar de forma estável a longo RTP 1 (RTP1L) e RTP 2, receptor de aumentar a expressão da proteína 1, e L αolf, resultando na linha celular Hana3A 19,27. Além disso, o receptor de acetilcolina de tipo 3 muscarínica (M3-R) interage com o RUP na superfície da célula e aumenta a activação em resposta aos odores 26. A co-transfecção de um OU com RTP1S e M3-R em Hana3A células resulta na expressão robusta, consistente, e funcional de uma ampla gama de RUP na superfície da célula 27. Em segundo lugar, mamíferos ou repertórios são bastante grandes. Nos seres humanos, por exemplo, o repertório OR é uma ordem de magnitude mais numerosos do que o repertório de receptores gustativos, e duas ordens de magnitude mais numerosos do que o repertório de receptor visual. Embora a clonagem de um único OR é um protocolo relativamente simples, o esforço up-front significativa é necessária para gerar uma biblioteca abrangente. Em terceiro lugar, embora saibamos que na visão, comprimento de onda se traduz em cor ena freqüência de audição se traduz em campo, a organização de odores é mal compreendida, o que torna difícil para os pesquisadores para interpolar a partir de uma amostra representativa de odorantes. Apesar de alguns progressos foram feitos nesta frente 10,28, o mapa da paisagem olfativa permanece incompleta. Triagem dezenas de milhares de moléculas contra centenas de RUP é uma tarefa difícil; telas de alto rendimento neste domínio exigem campanhas cuidadosamente definidos. Os principais desafios que restam são as de logística e custo, em vez de problemas inerentes à técnica. Apesar de triagem heteróloga não tem sido amplamente utilizada para identificar ligantes por grupos acadêmicos, uma empresa privada tem usado a mesma técnica para identificar ligantes para 100 RUP humanos 29. Infelizmente, esses dados são proprietários. O ensaio de luciferase de elevado rendimento descrita aqui tem várias vantagens sobre os métodos alternativos utilizados para avaliar ou activação. Embora a responses de neurônios olfatórios nativas foram medidos utilizando eletrofisiologia e de imagem de cálcio, estas técnicas têm dificuldade provocando além OR que leva à resposta de um neurônio devido à sobreposição de propriedades de resposta de neurônios olfativos. Embora a bater-se um tipo de receptor marcado com GFP 30,31, entregando receptores específicos através de adenovirus para murino neurónios olfactivos 32,33, ou realizando RT-PCR após gravações 17,24,33 pode ligar gravações de tipos de receptores individuais, estes métodos são baixo rendimento e não é adequado para telas de grande escala. Sistemas de rastreio heterólogas são mais escalável e duas formas principais são encontradas na literatura: repórteres via inositol trifosfato de acampamento e (IP3) repórteres da via. Após a estimulação de odor, RUP activar uma cascata de sinalização L transdução αolf que resulta na produção de AMP cíclico (AMPc) 12. Por co-transfecção de um gene repórter da luciferase de pirilampo sob o controlo de ACElemento de resposta a AMP (CRE), a produção de luciferase pode ser medido como uma função de resposta de odor, o que permite a quantificação de OU activação. OU a activação pode também ser ligada à via de IP3 por co-expressando proteínas-G tais como G α15/16 ou um L-α15 olf quimera 24,25,34. Nós escolhemos o ensaio aqui apresentado com base em três fatores: (1) a co-expressão da RTP1 com receptores olfativos Rho-tag melhora a expressão de receptores olfativos na superfície da célula 19,27; (2) utilização de um gene repórter de AMPc-responsiva permite a medição de OU activação através da via do segundo mensageiro canónica; e (3) o ensaio é adequado para telas de alto rendimento. Este ensaio de luciferase de alta produtividade é aplicável a uma variedade de estudos valiosos para o campo do olfacto. Primeiro, um grande número de RUP podem ser rastreados contra um único odorante, a fim de determinar o padrão de activação do receptor por um spodorante ESPECÍFICOS. Este tipo de estudo identificou OR7D4 como o OR responsável por responder ao androstenona odorante esteróide 8. Por outro lado, uma ou podem ser rastreados contra um painel de odores, a fim de determinar o perfil de resposta do receptor de 10. Quando o candidato olfactiva odorante / OU pares são identificados através dessas telas, a interacção pode ser confirmada através da realização de uma experiência de resposta à dose de exame da resposta do OR a concentrações crescentes de odorante. Curvas de resposta de dose também pode avaliar como a variação genética em um OR afeta em resposta odorante vitro 8,9,11,35, e esses estudos podem ser estendidos para interespecífica ou a variação, permitindo a análise da evolução receptor entre espécies e mutações causais em evolução 36,37, Finalmente, este ensaio pode ser usado para o rastreio de antagonistas de odor, que são capazes de antagonizar a resposta ao OU um odorante particular para um par odorante / receptor conhecido 38,39. Em resumo, esta altaEnsaio de luciferase-throughput é aplicável a uma gama de estudos que ajudarão caracterizar ou padrões de ativação e proporcionar uma melhor compreensão da codificação odor no sistema olfativo.

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="685">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">M3-R-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pCRE-luc</td>
			<td style="width:135px;">Agilent</td>
			<td style="width:119px;">219076</td>
			<td><a href="http://www.chem.agilent.com/store/ProductDetail.aspx?productID=219076%28Agilent%29">LUC</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pSV40-RL</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2231</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/prl-renilla-luciferase-control-reporter-vectors/">RL</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media, Eagle</td>
			<td>Sigma Aldrich</td>
			<td>M4655</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m4655?lang=en&amp;region=US">MEM</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/16000044?ICID=search-16000044">FBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (without Ca2+ and Mg2+)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td><a href="http://cellgro.com/products/balanced-salt-solutions/phosphate-buffered-saline/phosphate-buffered-saline-1x-2.html">PBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% Trypsin EDTA)</td>
			<td>Life Technologies</td>
			<td>25300</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product">Trypsin</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CD293</td>
			<td>Life Technologies</td>
			<td>11913-019</td>
			<td><a href="http://products.invitrogen.com/ivgn/product/11913019?ICID=search-11913019">CD293</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">96 well PDL white/clear plate</td>
			<td style="width:135px;">BD BioCoat</td>
			<td style="width:119px;">356693</td>
			<td><a href="http://www.thomassci.com/Supplies/Tissue-Culture-Cellware/_/BD-Biocoat-Poly-D-Lysine-Culture-Dishes?q=356693">plates</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Lipid transfection reagent: Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/11668019?ICID=search-11668019">Lipofectamine</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:265px;">Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2980</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_glo-luciferase-assay-system/?__utma=1.1932750276.1376486173.1376486173.1376486173.1&amp;__utmb=1.1.10.1376486173&amp;__utmc=1&amp;__utmx=-&amp;__utmz=1.1376486173.1.1.utmcsr=google|utmc">dual glo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy S2&#160;</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">SLAD</td>
			<td><a href="http://www.biotek.com/products/microplate_detection/synergy2_multimode_microplate_reader.html">BioTek S2</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Microplate reader software: Gen5 Data Analysis Software</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">Gen5</td>
			<td><a href="http://www.biotek.com/products/microplate_software/gen5_data_analysis_software.html">Gen5</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIOSTACK</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">BIOSTACK2WR</td>
			<td><a href="http://www.biotek.com/products/microplate_software/biostack_microplate_stacker.html">BioStack</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Multiflo</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">MFP</td>
			<td><a href="http://www.biotek.com/products/liquid_handling/multiflo_microplate_dispenser.html">MultiFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4414</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips ViaFlo96</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips 384 XYZ</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6403</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384ViaFlo</td>
			<td style="width:135px;">Integra</td>
			<td>6030</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/96_384_channel_pipette.html">384ViaFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td>Macherey Nagel</td>
			<td>740797.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&amp;region=US">DMSO</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td>Enzo Life Sciences</td>
			<td>BML-CN100-0010</td>
			<td><a href="http://www.thomassci.com/007635F0-AA41-4B24-B46A-05723D685997/_/FORSKOLIN-10MG?q=c002e24">FOR</a></td>
		</tr>
	</tbody>
</table>

high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity

Fragrâncias criar padrões únicos e sobrepostos de ativação do receptor olfativo, permitindo que uma família de aproximadamente 1.000 murino e 400 receptores humanos de reconhecer milhares de odorantes. Ligantes odorantes foram publicados para menos de 6% dos receptores humanos 1-11. Esta falta de dados é devido, em parte, às dificuldades funcionalmente que expressam estes receptores em sistemas heterólogos. Aqui, nós descrevemos um método para expressar a maioria da família do receptor olfactivo, em células Hana3A, seguido por uma avaliação de elevado rendimento de activação do receptor olfactivo utilizando um ensaio de repórter de luciferase. Este ensaio pode ser usado para (1) painéis de tela de odorantes contra painéis de receptores olfactivos; (2) confirmar a interação odorante / receptor via curvas de dose-resposta; e (3) comparar os níveis de ativação dos receptores entre variantes de receptores. Em nossos dados da amostra, 328 receptores olfativos foram triados contra 26 odorantes. Pares Odorant / receptores com diferentes contagens de resposta foram selected e testado em resposta à dose. Estes dados indicam que um ecrã é um método eficaz para enriquecer os pares odorante / receptoras que vai passar uma experiência de resposta a dose, isto é, os receptores que têm uma resposta bona fide para um odorante. Portanto, este ensaio de luciferase de alto rendimento é um método eficaz para caracterizar olfativo receptores de um passo essencial em direção a um modelo de codificação odor no sistema olfativo dos mamíferos.

Um ecrã primário testadas 328 RUP contra odores 26, a uma concentração de 100 uM. Esta concentração de odor tem sido demonstrada para activar de forma eficaz uma grande proporção de RUP com ligandos que se sabe 10. Primeiro, a actividade de luciferase normalizada foi calculada dividindo a leitura de luciferase de pirilampo pela da luciferase de Renilla leitura. Depois, os valores baseline foram calculados subtraindo as leituras de luciferase normalizada para o controle de odor a partir das le...

Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Padrões de ativação de receptores olfativos codificar identidade odor, mas a falta de dados publicados que identificam ligantes olfativos para receptores olfativos mamíferos impede o desenvolvimento de um modelo abrangente de codificação odor. Este protocolo descreve um método para facilitar a identificação de alto rendimento de ligandos de receptores olfactivos e quantificação da activação do receptor.

De alta capacidade de análise de mamíferos olfativas Receptores: Medição do Receptor de ativação via luciferase Atividade

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

high-throughput-analysis-mammalian-olfactory-receptors-measurement

Research

JoVE Journal

Neuroscience

12.3K visualizações.  Monell Chemical Senses Center.  Padrões de ativação de receptores olfativos codificar identidade odor, mas a falta de dados publicados que identificam ligantes olfativos para receptores olfativos mamíferos impede o desenvolvimento de um modelo abrangente de codificação odor. Este protocolo descreve um método para facilitar a identificação de alto rendimento de ligandos de receptores olfactivos e quantificação da activação do receptor. 

Vídeo: High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Preparação fatia, cultura de tecidos organotípicas e Gravação luciferase de Atividade Gene Relógio no núcleo supraquiasmático

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorante induzidas respostas registradas a partir de neurônios receptores olfativos utilizando a técnica de sucção da pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Interrogatório funcional do Adulto Hypothalamic Neurogenesis com Focal Radiológica Inibição

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

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 ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM Imaging and FIMtrack: Duas novas ferramentas que permitam de alta capacidade e Análise de Custo Efetivo Locomotion

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

A autorradiografia do receptor Protocolo para a visualização localizada da angiotensina II Receptores

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Viver-pilha medição de odorante Receptor de ativação usando um ensaio de campo em tempo real

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.