Apoiado pelo Prémio Carreira de Desenvolvimento de Pesquisa para a Prevenção da Cegueira, NIH concede EY19312 e EY19543 (VJK), bem como pela concessão irrestrita de Pesquisa para a Prevenção da Cegueira e EY02687 (Departamento de Oftalmologia e Ciências Visuais da Universidade de Washington).

1. Fazendo Eletrodos <ol><li> Prepare-se eletrodos de vidro. Pesar 120 mg de agar e misturá-lo em 10 mL de água destilada (concentração final de ágar 1,2%). Derreta a solução ágar-ágar em banho de água quente. Encha capilares de vidro (usamos Instruments Presision palavra TW100-4 capilares com as seguintes dimensões: comprimento = 100 mm, OD / ID = 1/0.75 mm, eo volume interno = uL 44) com a solução de agar usando uma seringa de plástico. Solidificar o ágar, à temperatura ambiente durante 10 min. Assim, até ~ 200 capilares podem ser preenchidos (o resto da mistura de ágar pode ser armazenada a -4 ° C e re-usados ​​várias vezes). </li><li> Cortar os capilares em metades, utilizando uma faca de diamante. </li><li> Embeber os eléctrodos de vidro resultantes em solução eléctrodo rato durante pelo menos 24 horas a 4 ° C. Armazenar os eléctrodos a 4 ° C e utilizá-los ao longo do período de 3-4 meses. </li></ol> 2. Configurando o Experimento <ol><li> Dark-se adaptar um mouse durante a noite. Use tipo selvagem (por exemplo, C57Bl / 6) ratos para vara-driven gravações transretinal e alfa T - / - ratos haste falta de sinalização 8 para gravações de cone transretinal. </li><li> Preparar uma solução de perfusão L, 100 mL solução eléctrodo, e 20 mL solução de incubação (ver abaixo) e filtrar a solução de perfusão através de um Millipore de 0,45 ou 0,22 mM de filtro (opcional). </li><li> Calibrar a fonte de luz (505 nm LED ou estimulador de halogénio lâmpada óptico), utilizando um fotómetro colocado no plano da retina. No caso da fonte de luz de LED, usar um difusor óptico para alcançar a uniformidade da luz. </li><li> Prepare a câmara de perfusão para a experiência (construções de câmara pode variar). A nossa câmara é feita de 3 Plexiglas mm, com uma parte inferior feita a partir da tampa de uma placa de Petri de plástico pequeno, e tem um volume de perfusão activa de ~ 400 uL (L = 25 mm, W = 8 mm, H ≈ 2 mm). Encher o espaço eléctrodo inferior da câmara de eléctrodo com uma solução contendo 2 mM de L-glutamato e 10 mM de BaCl 2. Usando tubos de plástico e um capilar de vidro, fazer uma ligação entre a solução no espaço inferior do eléctrodo (feita de ligador de tubagem de plástico ligado à parte inferior da câmara) e do suporte de eléctrodo. Certifique-se que não há bolhas no eletrodo, o tubo de ligação, ou o titular do eletrodo (purgar a solução com seringa de plástico, se necessário). </li><li> Montar a câmara de gravação sobre a fase do microscópio (opcional: o microscópio pode ser montado sobre uma mesa de anti-vibração e blindado dentro de uma gaiola de Faraday para minimizar o ruído mecânica e electromagnética). Ligue o suporte inferior do eléctrodo para o pólo headstage positivo do amplificador diferencial (por exemplo, Warner Instruments amplificador DP-311). Centro e alinhar o eletrodo câmara inferior de abertura com o ponto de luz de estímulo. Ligue a linha de perfusão para a câmara de gravação e transformar a perfusão no. </li><li> Solução de bolha a perfusão com 95% O2 / 5% CO 2, enquanto incubando-lo em 40 ° C watbanho de er. CO 2 ajusta o pH da solução para 7,2. A solução flui a partir do frasco para a câmara de gravação por gravidade passando através de uma resistência de cerâmica de reaquecimento para 36-37 ° C. Para reduzir a perda de calor, o aquecedor deve estar localizado mais próximo da câmara de registo quanto possível, preferencialmente na fase do microscópio. </li><li> Com um regulador de fluxo ajustar a taxa de fluxo de até cerca de 1 mL / min. </li><li> Ligue o eléctrodo superior ao pólo (negativo) do segundo headstage usando um suporte de eléctrodo segundo. Se ligam a sonda do termopar ao eléctrodo superior usando um O-ring. Assegure-se que as pontas de eléctrodos e termopar estão juntos de modo a que a leitura da temperatura é tomado como perto do centro da retina quanto possível. Mergulha-se o ponta do eléctrodo superior na solução de perfusão e coloque-o no centro da câmara. Permitir 10-15 min para a estabilização de linha de base. </li><li> Ajustar a tensão de CC para o aquecedor de modo que a temperatura da solução é de 36 -37 ° C. </li></ol> 3. Isolando a retina do rato <ol><li> Sob a luz vermelha ofuscante, a eutanásia o mouse de adaptação ao escuro com CO 2 e deslocamento cervical. Opcional: fazer uma marca de referência em cada olho do rato por cauterização do ponto mais dorsal da esclera com uma caneta de bisturi ou pino dissecando aquecida. Todos os procedimentos subsequentes são realizados sob a luz infravermelha utilizando conversores de imagem de infravermelhos ajustado a um microscópio de dissecação. </li><li> Dissecar os olhos com tesouras curvas através da aplicação de uma ligeira pressão e esticar a pele sobre os lados de cada olho. Hemisect ambos os globos oculares sob iluminação infravermelha usando microtesoura ou uma lâmina de barbear. </li><li> Remover o córnea e do cristalino. Remover o máximo do vítreo quanto possível. </li><li> Peel a retina primeiro a partir da camada de epitélio pigmentar e, se necessário, limpar cuidadosamente com uma pinça para remover os grânulos permanência do epitélio do pigmento. Estamos tipicamente usando retina todo para ambos haste e transret conegravações Inal. No entanto, se você deseja gravar especificamente da parte dorsal da retina do rato que tem mais M / L de cones 9, antes de descascar a retina remover a parte ventral da ocular com uma lâmina de barbear ou microtesoura usando a marca de cauterização como uma referência. </li><li> Coloque o globo ocular segundo hemisected numa placa de Petri com solução de incubação pequeno e incubar a preparação de uma caixa à prova de luz saturado com oxigénio puro até à utilização. Tipicamente, nestas condições a ocular do rato segundo pode ser armazenada durante várias horas à temperatura ambiente e utilizada para gravações. </li><li> Usando uma pipeta de vidro ou de plástico transferir a retina isolado para uma gota de solução de perfusão (300 uL ca.) sobre a tampa de uma placa de Petri. Adicionar 5-6 pequenas gotas de solução de eléctrodo contendo BaCl 2 a pré-incubar o retina com BaCl 2. Coloque um pedaço quadrado (mm 5x5 ca.) do lado do canto-untada (usamos Dow Corning lubrificante válvula 111 e selante) papel de filtro Millipore (0,45 mm Harg tipo) com pré-fabricados furo no seu centro (2-2,5 mm de diâmetro) para a gota mesma solução. Usando posição pinça da retina no topo do papel de filtro (fotoreceptor lado para cima) e pressionar os seus bordos na periferia à secção plana-montá-lo. A retina deve ser centrado sobre a abertura no papel de filtro. </li><li> Utilizando uma pinça transferir o papel de filtro com a retina para a câmara de perfusão e colocá-lo acima do estímulo spot-alinhados espaço eléctrodo inferior. Ligeiramente pressionar as bordas untadas do papel de filtro para ligação com a câmara. </li><li> Coloque o eletrodo superior sobre o centro da retina (ligeiramente tocar na camada dos fotorreceptores). Centrando o eléctrodo superior sobre a retina aumenta a amplitude do sinal (até 20-40% ~, em comparação com colocando-o mais perto da borda da retina). Embora a localização do eletrodo como distorce um pouco a intensidade do estímulo eficaz luz que atinge um pequeno setor da retina (~ 12-15% de sua área total), ainda é a melhor maneira de colocar a elecatropelou. A preparação está agora pronto para gravações transretinal. </li></ol> 4. Gravações Transretinal <ol><li> Dependendo da sua linha de mouse, registro haste ou M / L-cone-driven respostas flash de teste de fraca a intensidade de luz brilhante nm calibrado 505 fornecidos a partir de fonte de luz LED ou estimulador óptico. No caso de a nossa fonte de luz de LED, a intensidade do flash é controlada por uma combinação de controlo computadorizado de tensão de entrada de LED, resistores comutáveis, e um conjunto de filtros de densidade neutra (usamos E Cor-# 211 0,9 filtros ND película de Rosco Laboratories ), colocada entre o LED ea retina. A duração do teste LED de flash (20 ms) é controlado por um computador. Se usando uma lâmpada de halogéneo de estimulador óptico, teste de intensidade de flash e comprimento de onda pode ser controlada por um conjunto de filtros de densidade neutra e filtros de interferência de banda estreita, respectivamente. Teste de duração do flash pode ser controlado por computador orientadas persianas. </li><li> Opcional: para a manutenção eficientesupressão do componente glial de photoresponse com BaCl 2 ao longo do dia, mudar a solução eléctrodo inferior, ocasionalmente (por exemplo, antes do ensaio, cada retina) pela purga-lo usando uma seringa de plástico cheio com solução fresca. </li></ol> 5. Os resultados representativos Soluções <ol><li> Rato solução de perfusão retina: 112,5 mM de NaCl, 3,6 mM de KCl, 2,4 mM de MgCl2, 1,2 mM de CaCl2, 10 mM de HEPES (pH 7,2), 20 mM NaHCO3, 3 mM Na succinato, 0,5 mM de glutamato, de Na 0,02 mM de EDTA, e 10 mM de glucose. Além disso, a solução é suplementado com 2 mM de L-glutamato e 10 uM de DL-AP-4 para bloquear os componentes de ordem superior do photoresponse 10,11, e com 0,1% de vitaminas MEM e soluções de MEM de aminoácidos (Sigma) para melhorar a retina viabilidade. </li><li> Solução rato eléctrodo: 140 mM de NaCl, 3,6 mM de KCl, 2,4 mM de MgCl2, 1,2 mM de CaCl2, 3 mM de HEPES (pH 7,4, NaOH). A solução na parte inferior do elementoctrode espaço também contém 2 mM de L-glutamato de bloquear a transmissão sináptica 10 e, além disso, 10 mM de BaCl 2 para suprimir o componente glial do photoresponse 3,12. </li><li> Solução de incubação retina: dissolver 272 mg de L-15 médio (Sigma) e 20 mg de BSA em 20 mL de água desionizada. Final L-15 e as concentrações de BSA são 13,6 mg / mL e 1 mg / mL, respectivamente. Ajustar o pH para 7,4 com 1 M de HCl. </li></ol><img alt="Figura 1." src="/files/ftp_upload/3424/3424fig1.jpg" /> Figura 1. Diagrama esquemático, mostrando o isolamento de retina do rato para gravações transretinal ERG. <img alt="Figura 2." src="/files/ftp_upload/3424/3424fig2.jpg" /> Figura 2. Fotografia da câmara de perfusão e eletrodos de registro com seus titulares. As setas vermelhas indicam a direção do fluxo de perfusão. <img alt="Figura 3." src="/files/ftp_upload/3424/3424fig3.jpg"/&gt; Figura 3. Diagrama esquemático da montagem experimental para as gravações transretinal ERG. As setas vermelhas indicam a direção do fluxo de perfusão. <img alt="Figura 4." src="/files/ftp_upload/3424/3424fig4.jpg" /> Os resultados representativos Figura 4:. Gravado família de rato haste-driven respostas transretinal. Photoresponses foram filtrada passa-baixa de 30 Hz (8-pólo Bessel), digitalizados em 1 kHz e armazenadas em um computador para análise posterior. Traços apresentados representam médias de 5-6 respostas em intensidades de luz ténue e 2-3 respostas a intensidades luminosas saturantes. <img alt="Figura 5." src="/files/ftp_upload/3424/3424fig5.jpg" /> Figura 5: Os resultados representativos. Família gravada do rato-driven cone respostas transretinal. Photoresponses foram filtrada passa-baixa de 30 Hz (8-pólo Bessel), digitalizados em 1 kHz e armazenadas em um computador para análise posterior. Traços são mostrados averages de respostas 5-10 em intensidades de luz ténue e 3-5 respostas a intensidades luminosas saturantes.

<ol>
	<li>Yau, K. W., Lamb, T. D., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+fluctuations+in+membrane+current+of+single+toad+rod+outer+segments.">Light-induced fluctuations in membrane current of single toad rod outer segments.</a> Nature. 269, 78-80 (1977).</li><li>Nikonov, S. S., Kholodenko, R., Lem, J., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+features+of+the+S-+and+M-cone+photoreceptors+of+wild-type+mice+from+single-cell+recordings.">Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.</a> J. Gen. Physiol. 127, 359-374 (2006).</li><li>Nymark, S., Heikkinen, H., Haldin, C., Donner, K., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+responses+and+light+adaptation+in+rat+retinal+rods+at+different+temperatures.">Light responses and light adaptation in rat retinal rods at different temperatures.</a> J. Physiol. 567, 923-938 (2005).</li><li>Heikkinen, H., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cone+photoresponses+obtained+with+electroretinogram+from+the+isolated+retina.">Mouse cone photoresponses obtained with electroretinogram from the isolated retina.</a> Vision Res. 48, 264-272 (2008).</li><li>Frank, R. N., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+photoproducts:+effects+on+electroretinogram+sensitivity+in+isolated+perfused+rat+retina.">Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina.</a> Science. 161, 487-489 (1968).</li><li>Wang, J. S., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+pathway+mediates+the+mouse+and+human+cone+visual+cycle.">An alternative pathway mediates the mouse and human cone visual cycle.</a> Curr. Biol. 19, 1665-1669 (2009).</li><li>Kolesnikov, A. V., Tang, P. H., Parker, R. O., Crouch, R. K., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+cone+visual+cycle+promotes+rapid+M/L-cone+pigment+regeneration+independently+of+the+interphotoreceptor+retinoid-binding+protein.">The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.</a> J. Neurosci. 31, (2011).</li><li>Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+in+transgenic+mice+after+targeted+deletion+of+the+rod+transducin+alpha+-subunit.">Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 13913-13918 (2000).</li><li>Applebury, M. 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+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27, 513-523 (2000).</li><li>Sillman, A. J., Ito, H., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mass+receptor+potential+of+the+isolated+frog+retina.+I.+General+properties+of+the+response.">Studies on the mass receptor potential of the isolated frog retina. I. General properties of the response.</a> Vision Res. 9, 1435-1442 (1969).</li><li>Vinberg, F., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sets+the+physiological+value+of+the+dominant+time+constant+of+saturated+mouse+rod+photoresponse+recovery.">Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery.</a> PLoS One. 5, e13025-e13025 (2010).</li><li>Bolnick, D. A., Walter, A. E., Sillman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li></ol>

Os autores não têm nada a revelar.

O método de vara e cone-driven gravações transretinal ERG descrito acima está se tornando uma poderosa ferramenta para investigar a função dos fotorreceptores do mouse tanto no tipo selvagem e animais geneticamente modificados. Além disso para a caracterização das propriedades de fácil photoresponse básicos, esta técnica simples fornece estabilidade grande resposta de longa duração, durante experimentos realizados em preparações próxima do intactas da retina. Ambos de adaptação ao escuro haste amplitu...

<table border="1"><tbody><tr><td> Nome do material </td><td> Tipo </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentar </td></tr><tr><td> DL-AP-4 </td><td></td><td> TOCRIS biociência </td><td> 0101 </td><td></td></tr><tr><td> Todos os outros reagentes </td><td></td><td> Sigma-Aldrich </td><td></td><td></td></tr><tr><td> Capilares de vidro </td><td></td><td> Instrumentos de precisão do mundo </td><td> TW100-4 </td><td> Para fazer eletrodos </td></tr><tr><td> Papel-filtro </td><td> Harg </td><td> Millipore </td><td> HABG01300 </td><td></td></tr><tr><td> Fotômetro </td><td></td><td> Instrumentos da UDT </td><td> S350 </td><td> Para a calibração da luz </td></tr><tr><td> Sensor de silício radiométrica </td><td></td&gt; <td> Instrumentos da UDT </td><td> 221 </td><td> Para a calibração da luz </td></tr><tr><td> Anti-vibração tabela </td><td></td><td> Técnico Manufacturing Corporation </td><td> TMC 78-239-02R, TMC63-26012-01 </td><td> Para minimizar o ruído mecânico </td></tr><tr><td> Compressor de ar Panther P 15TC </td><td></td><td> Werther Internacional </td><td> P 15TC </td><td> Conectado a anti-vibração tabela </td></tr><tr><td> Estereomicroscópio </td><td></td><td> LEICA </td><td> MZ9.5 </td><td> Para dissecção olho do mouse </td></tr><tr><td> Infravermelhos conversores de imagem </td><td></td><td> BEMeyers </td><td> ProwlerTM </td><td> Obrigado a estereomicroscópio </td></tr><tr><td> Amplificador diferencial DP-311 </td><td></td><td> Warner Instruments </td><td> DP-311 </td><td></td></tr><tr><td> Axon Digidata 1440A Digitizer </td><td>; </td><td> Molecular Devices </td><td> 1440A </td><td></td></tr><tr><td> Dual Channel 8 pólos Filtro </td><td></td><td> Krohn-HITE Corporação </td><td> 3382 </td><td></td></tr><tr><td> Resistor de cerâmica </td><td></td><td> TE Conectividade </td><td> CGS SBCHE618RJ </td><td> Para reaquecer a solução de perfusão </td></tr><tr><td> Termopar </td><td> T </td><td> Physitemp Instruments </td><td> TI-18 </td><td></td></tr><tr><td> Monitor de temperatura </td><td></td><td> Ómega </td><td> DPi32 </td><td> Conectado ao termopar </td></tr><tr><td> LED 505 nm </td><td></td><td> TT Electronics / Optek Technology </td><td> Digi-chave P / N 365-1185-ND </td><td> Para aplicar flashes de ensaio ou de luz de branqueamento </td></tr><tr><td> Caneta Cauterização </td><td></td><td> Bovie </td><td> AA25 </td><td> Para marcar a parte dorsaldo globo ocular do mouse </td></tr><tr><td> pCLAMP 10 Eletrofisiologia de Aquisição de Dados e Análise de Software </td><td></td><td> Molecular Devices </td><td></td><td></td></tr></tbody></table>

transretinal erg recordings from mouse retina: rod and cone photoresponses

Existem duas classes distintas de imagem formadoras de fotorreceptores na retina de vertebrados: bastonetes e cones. Rods são capazes de detectar fótons individuais de luz, enquanto cones operar continuamente sob rápidas mudanças das condições de luz brilhante. A absorção de luz pela vara e cone específicos pigmentos visuais nos segmentos exteriores de fotorreceptores desencadeia uma cascata de fototransdução que conduz eventualmente a encerramento de nucleótidos cíclicos-gated canais na membrana plasmática e hiperpolarização da célula. Esta mudança induzida pela luz em corrente e potencial de membrana pode ser registrado como um photoresponse, por qualquer de sucção 1,2 eletrodo clássica técnica de gravação ou por gravações eletrorretinograma transretinal (ERG) de retinas isoladas com farmacologicamente bloqueados componentes da resposta pós-sinápticos 3-5. O último método permite a droga acessíveis de longa duração gravações de fotorreceptores do rato e é particularmente útil para a obtenção de photoresponses estáveis ​​from os cones do mouse escassos e frágeis. No caso de cones, tais experimentos podem ser realizados tanto em condições de adaptação ao escuro e iluminação intensa seguinte que branqueia essencialmente todo o pigmento visual, para acompanhar o processo de recuperação fotossensibilidade cone durante a adaptação ao escuro 6,7. Neste vídeo, vamos mostrar como realizar vara e M / L-cone-driven gravações transretinal de escuro-adaptado retina mouse. Gravações haste irá ser efectuada utilizando retina do tipo selvagem (C57BL / 6) ratinhos. Por simplicidade, as gravações de cone será obtida a partir geneticamente modificado haste transducina α subunidade knockout (Tα - / -) ratinhos que carecem de haste de sinalização 8.

Watch this Scientific Journal Video about Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses at JoVE.com

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

Nós descrevemos um método relativamente simples de transretinal eletrorretinograma (ERG) para a obtenção de gravações photoresponses haste e cone da retina do rato intacto. Esta abordagem tira vantagem do bloco de transmissão sináptica de fotorreceptores para isolar as suas respostas de luz e gravar os usando eléctrodos de campo colocados através da retina plana montado isolado.

Transretinal Recordings ERG de Retina do mouse: Rod e Photoresponses Cone

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

transretinal-erg-recordings-from-mouse-retina-rod-cone

Research

JoVE Journal

Neuroscience

17.7K visualizações.  Washington University School of Medicine. Nós descrevemos um método relativamente simples de transretinal eletrorretinograma (ERG) para a obtenção de gravações photoresponses haste e cone da retina do rato intacto. Esta abordagem tira vantagem do bloco de transmissão sináptica de fotorreceptores para isolar as suas respostas de luz e gravar os usando eléctrodos de campo colocados através da retina plana montado isolado.

Vídeo: Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Remendo Recordings Grampo de neurônios da retina do rato em um Preparação Slice Dark-adaptados

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Neurociência

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

As gravações eletrofisiológicas da via fibra Gigante da D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Grampo patch Gravações de Embryonic Cells Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Assessment of Vascular regeneração no SNC Usando o rato Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome Seccionamento Rato Retina de Preparar culturas Fotorreceptoras

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagem Ca<sup&gt; 2+</sup&gt; Dynamics nos terminais Cone fotorreceptores Axon do mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Onde cortar matérias: Uma dissecção e análise de guia para a orientação espacial da Retina do rato de Marcos Ocular

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

Gravações de EEG não invasiva de leitões movimentando-se livremente

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Visualizando o colapso do Cone de crescimento Axonal e primeiros efeitos β amiloide nos neurônios cultivados Mouse

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Transretinal Recordings ERG de Retina do mouse: Rod e Photoresponses Cone

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