1. Instrumentation 
<ol>
<li>To measure the response of olfactory sensory neurons to photolysis of caged compounds we use a flash lamp in combination with a typical patch-clamp recording system including: a patch-clamp amplifier, a recording electrode and a reference electrode connected to the head-stage of a patch-clamp amplifier, a digitizer, a computer, software for data acquisition, micro-manipulators, an epifluorescence microscope, a perfusion system, an anti-vibration table and a Faraday cage (Figure 2).</li>
<li...

<ol>
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Flash fotólise de compostos enjaulado combinado com patch-clamp gravações é uma técnica útil para obter saltos rápidos e local da concentração de moléculas ativas fisiologicamente dentro e fora das células. Vários tipos de compounds1 enjaulado foram sintetizados, e esta técnica pode ser aplicada a vários tipos de células, incluindo células cultivadas expressar canais iônicos que podem ser ativadas ou moduladas por fotólise de alguns dos compostos disponíveis enjaulado 11. <p class="jove...

<table border="1"><tbody><tr><th> Equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Adaptador módulo da lâmpada de flash para microscópio </td><td> Rapp optoeletrônicos </td><td> FlashCube 70 </td><td></td></tr><tr><td> Air tabela </td><td> TMC </td><td> MICRO-g 63-534 </td><td></td></tr><tr><td> Digitizer </td><td> Axon Instruments </td><td> Digidata 1322A </td><td></td></tr><tr><td> Aquisição de Dados Software </td><td> Axon Instruments </td><td> pClamp 8 </td><td></td></tr><tr><td> Software Análise de dados </td><td> WaveMetrics </td><td> Igor </td><td></td></tr><tr><td> Espelho para módulo adaptador </td><td> Rapp optoeletrônicos </td><td> M70/100 </td><td></td></tr><tr><td> Suporte do eletrodo </td><td> Axon Instruments </td><td> 1-HL-U </td><td></td></tr><tr><td> Gaiola de Faraday </td><td> Feitos </td><td></td><td></td></tr><tr><td> Cubo de filtro </td><td> Olimpo </td><td> U-UTH </td><td> Filtro de excitação removido </td></tr><tr><td> Lâmpada de flash </td><td> Rapp optoeletrônicos </td><td> JML-C2 </td><td></td></tr><tr><td> Fórceps Dumont # 55 </td><td> Precision Instruments mundo </td><td> 14099 </td><td></td></tr><tr><td> Capilares de vidro </td><td> Precision Instruments mundo </td><td> PG10165-4 </td><td></td></tr><tr><td> Prato fundo de vidro </td><td> Precision Instruments mundo </td><td> FD35-100 </td><td></td></tr><tr><td> Iluminador </td><td> Olimpo </td><td> Destaque 3100 </td><td></td></tr><tr><td> Microscópio invertido </td><td> Olimpo </td><td> IX70 </td><td></td></tr><tr><td> Micromanipuladores </td><td> Luigs &amp; Neumann </td><td> SM eu </td><td></td></tr><tr><td> Micropipeta Puller </td><td> Narishige </td><td> PP-830 </td><td></td></tr><tr><td> Monitor </td><td> HesaVision </td><td> MTB-01 </td><td></td></tr><tr><td> Os filtros de densidade neutra </td><td> Omega Optical </td><td> varia </td><td></td></tr><tr><td> Objetivo 100X </td><td> Zeiss </td><td> Fluar 440285 </td><td> Ou Zeiss ou Olympus </td></tr><tr><td> Objetivo 100X </td><td> Olimpo </td><td> UPLFLN 100XOI2 </td><td> Ou Zeiss ou Olympus </td></tr><tr><td> Ópticos UV shortpass filtro </td><td> Rapp optoeletrônicos </td><td> SP400 </td><td></td></tr><tr><td> Amplificador de patch-clamp </td><td> Axon Instruments </td><td> Axopatch 200B </td><td></td></tr><tr><td> Foto de díodo </td><td> Rapp optoeletrônicos </td><td> PDA </td><td></td></tr><tr><td> Guia de luz de quartzo </td><td> Rapp optoeletrônicos </td><td> varia </td><td> Usamos 600 mM de diâmetro </td></tr><tr><td> Fio de prata </td><td> Precision Instruments mundo </td><td> AGT1025 </td><td></td></tr><tr><td> Prata chão pellet </td><td> Warner instrumentos </td><td> 64-1309 </td><td></td></tr><tr><td> Xenon arco da lâmpada </td><td> Rapp optoeletrônicos </td><td> XBL-JML </td><td></td></tr></tbody></table> <table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BCMCM gaiolas cAMP </td><td> Biolog </td><td> B016 </td></tr><tr><td> Albumina de soro bovino (BSA) </td><td> Sigma </td><td> A8806 </td></tr><tr><td> CaCl2 solução padrão 0,1 M </td><td> Fluka </td><td> 21059 </td></tr><tr><td> Ca Caged: DMNP-EDTA </td><td> Invitrogen </td><td> D6814 </td></tr><tr><td> Cisteína </td><td> Sigma </td><td> C9768 </td></tr><tr><td> Concanavalina A V tipo (ConA) </td><td> Sigma </td><td> C7275 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C4036 </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D8418 </td></tr><tr><td> DNAse I </td><td> Sigma </td><td> D4527 </td></tr><tr><td> EDTA </td><td> Sigma </td><td> E9884 </td></tr><tr><td> EGTA </td><td> Sigma </td><td> E4378 </td></tr><tr><td> Glicose </td><td> Sigma </td><td> G5767 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td></tr><tr><td> KCl </td><td> Sigma </td><td> P3911 </td></tr><tr><td> KOH </td><td> Sigma </td><td> P1767 </td></tr><tr><td> Leupeptin </td><td> Sigma </td><td> L0649 </td></tr><tr><td> MgCl2 </td><td> Fluka </td><td> 63020 </td></tr><tr><td> Papaína </td><td> Sigma </td><td> P3125 </td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P1274 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S9888 </td></tr><tr><td> NaOH </td><td> Sigma </td><td> S5881 </td></tr><tr><td> NaPyruvate </td><td> Sigma </td><td> P2256 </td></tr></tbody></table>

flash photolysis of caged compounds in the cilia of olfactory sensory  neurons

Fotólise de compostos enjaulado permite a produção de aumentos rápidos e localizados na concentração de vários compostos fisiologicamente ativos 1. Caged compostos são moléculas feitas fisiologicamente inativo por uma gaiola química que pode ser quebrado por um flash de luz ultravioleta. Aqui, nós mostramos como obter patch-clamp gravações combinado com fotólise de compostos enjaulado para o estudo da transdução olfativos em dissociada do mouse neurônios sensoriais olfativos. O processo de transdução olfativos (Figura 1) ocorre nos cílios dos neurônios sensoriais olfativos, onde odorant ligação aos receptores leva ao aumento de cAMP que abre nucleotídeo cíclico-gated (CNG) canais 2. Ca entrada através de canais CNG ativa os canais de Ca-activated Cl. Mostramos como dissociar os neurônios do epitélio olfativo do mouse 3 e como ativar canais CNG ou canais de Ca-Cl ativado por fotólise de cAMP enjaulado enjaulados Ca 4 ou 5 &lt;/ Sup&gt;. Nós usamos uma lâmpada de flash 6,7 aplicar flashes ultravioleta à região ciliar para libertar da gaiola ou cAMP Ca enquanto patch-clamp gravações são tomadas para medir a corrente em toda a configuração de tensão de células-clamp 11/08.

Watch this Scientific Journal Video about Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons at JoVE.com

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

Fotólise de compostos enjaulado permite a produção de aumentos rápidos e localizados na concentração de vários compostos fisiologicamente ativos. Aqui, nós mostramos como obter patch-clamp gravações combinado com fotólise de cAMP gaiola ou enjaulado Ca para o estudo da transdução olfativos em dissociada do mouse neurônios sensoriais olfativos.

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. ...

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