Este trabalho foi financiado pelo NIH subvenções EY019312 e EY021126 (VJK), EY002687 ao Departamento de Oftalmologia e Ciências Visuais na Universidade de Washington, e pela Research para prevenir a cegueira.

Todos os protocolos experimentais foram de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório e foram aprovados pelo Comitê de Estudos de Animais institucional na Universidade de Washington. 1. Preparação de perfusão e Porta-amostra <ol><li> Preparar a solução de perfusão para a retina fresco no dia da experiência. Use água destilada e deionizada. Use um dos três seguintes soluções. <ol><li> Prepara-bicarbonato contendo &#39;solução (1 L): 1 garrafa de Ames Ames meios e 1,9 g de NaHCO 3, </li><li> Preparar a solução de Locke (em mM): 112,5 de NaCl, 3,6 KCl, 2,4 MgCl2, 1,2 de CaCl2, 10,0 de HEPES, 20,0 NaHCO3, 3,0 Na succinato, 0,5 Na glutamato, 0,02 de EDTA e 10,0 de glucose, 0,1% de vitaminas MEM e amino ácidos </li><li> Preparar solução de Ringer tamponada com HEPES (em mM): 133,3 de NaCl, 3,3 KCl, 2,0 MgCl2, 1,0 de CaCl2, 10,0 de glucose, 0,01 de EDTA, 12,0 HEPES, pHajustado para 7,5 com ~ 5,8 mL de NaOH 1 M, adicionar 0,72 g / L de meio de cultura de Leibovitz L-15. Use 20 - 50 pM de DL-AP4 e 50 - 100? M BaCl2 para isolar a resposta de fotorreceptores com qualquer meio de perfusão. </li></ol></li><li> Preparar a solução de eléctrodos de 28 (em mM): NaCl 140,0, KCl 3,6, MgCl2 2,4, CaCl2 1,2, HEPES 3, 0,01 de EDTA e ajustar o pH a 7,4-7,5 com NaOH. Eléctrodo solução pode ser armazenada à temperatura ambiente durante vários meses. </li><li> Preparar e testar o suporte da amostra. <ol><li> Cole papel de filtro preto / cinza em cima das cúpulas da parte inferior do suporte da amostra (ver Figura 1A). Espalhe de dois componentes de cola epóxi 5 min cuidadosamente em torno dos bordos das partes superiores planas das cúpulas. Se necessário, faça-o sob dissecção microscópio. </li><li> Aguarde até que a cola estiver quase seco (cerca de 4 min) e pressione o filtro de papel nas cúpulas usando um item de plano. Papel de filtro de cola, pelo menos, um dia antes da experiência. Opapel de filtro pode ser utilizada várias vezes, mas deve ser substituído após um mês de gravações. Use etanol a 70% para limpar cuidadosamente cúpulas de qualquer resíduo de cola antes de instalar o papel de substituição. </li><li> Preencha os canais de eletrodos com uma solução de eletrodo tentando evitar as bolhas de ar e parafuso eletrodos de pelotização fechados com um adaptador de rosca para os canais de eletrodo (ver figuras 1A e B). Ligar as peças superior e inferior do suporte da amostra com quatro parafusos e encher as linhas de perfusão com solução de eléctrodo. </li><li> Medir a resistência e tensão entre os condutores de cada par de eléctrodos usando um multímetro (Figura 1B). A resistência deve ser inferior a 100 Ωk ea tensão abaixo de 10 mV se os canais estão sem bolhas e os eletrodos estão em boas condições. </li></ol></li><li> Pour 400-700 ml de meio de perfusão no frasco de vidro. Separadas mais 300 ml para ser utilizada na dissecção das retinas e srasgou-o em uma geladeira. Definir a tubagem de perfusão no bloco permutador de calor e colocar o bloco de pré-aquecido sobre a placa de aquecimento (ver Figura 1D). </li><li> Coloque o frasco com uma tampa que tem ligações para o CO 2 / O 2 gás (se Ames ou Locke é usado) e para a tubagem de perfusão (ver Figura 1D). Pré-aqueça o frasco com a mídia para 37 ° C e coloque-o sobre a placa de aquecimento ou na parte superior da unidade de estímulo de luz no banho-maria regulado para 37-39 o C 17. <ol><li> Primeiro as linhas de perfusão, preenchendo-as com meios de perfusão para iniciar o fluxo orientado a gravidade. <ol><li> No sistema LKC, colocar a garrafa no topo da unidade estimulador 17 para proporcionar um escoamento gravitacional significativa que é, então, ajustado por reguladores de caudal sem ser influenciado pelo nível de redução da solução de perfusão na garrafa durante a experiência. </li><li> No sistema Ocuscience, coloque o perfusion garrafa no interior da gaiola de Faraday, de modo a minimizar o ruído e colocar linhas de saída longo de perfusão de suporte da amostra (ver passo 2.8) bem abaixo do nível do suporte da amostra (e do frasco de perfusão) para aumentar a movimentação gravitacional da solução. Proteger essas linhas de saída e ligue a blindagem à terra do amplificador para impedir o acoplamento do ruído eletromagnético ao sinal ERG. </li></ol></li></ol></li><li> Ajustar a perfusão de 3-5 ml / min usando reguladores de caudal. Conecte-se 5% de CO2 / 95% de O 2 de gás de um cilindro com regulador apropriado e ajustar a taxa de fluxo para garantir borbulhamento constante da mídia na garrafa através de uma pedra de ar. </li></ol> Preparação 2. Amostra <ol><li> Montar instrumentos de dissecção limpas e nítidas incluindo microtesoura reta de lâmina, um ou dois 45 o pinças, lâmina de barbear e um pedaço retangular de papel de filtro. </li><li> Despeje cerca de 200 ml de perfu friosion solução numa placa de Petri grande, de modo que toda a parte inferior do suporte de amostra (incluindo as cúpulas) pode ser imersa na solução. Este passo torna-se importante ao montar as retinas sobre as cúpulas (veja o passo 2.6). Embora algumas soluções foram desenvolvidas para ser saturada com carbogéneo (5% de CO2 / 95% de O 2), isto não é essencial para os fins de dissecação e não foi feito nas experiências descritas aqui. </li><li> Para uma experiência típica, manter os animais em 12/12 horas escuro ciclo de luz / escuridão e-adaptá-los para 6-12 horas antes das gravações. Eutanásia do animal por inalação de CO2 seguido por deslocamento cervical sob luz vermelha ofuscante e fazer todas as seguintes operações sob luz vermelha ou IR dim (usar por exemplo, filtro vermelho na frente da fonte de luz do microscópio). <ol><li> Puxe os olhos para fora usando uma pinça e colocá-los na mídia. Coloque um olho de cada vez em um pequeno pedaço de papel (por exemplo, alguns papel de filtro regular) e fazer comoiluminado é aproximadamente o nível de ora serrata, mantendo o olho com uma pinça (isto é feito fora da solução aqui). </li></ol></li><li> O corte ao longo da ora serrata (ou mais perto do equador do olho) com microtesouras e remover a córnea e da lente. Coloque o copo olho na mídia frio na grande placa de Petri e repita o mesmo procedimento com o outro olho. </li><li> Corte de uma pequena incisão da parte superior da taça dos olhos no sentido do nervo óptico, mantendo a tesoura entre a retina e esclera, a fim de manter a retina como intacta quanto possível. Aderência da esclerótica de ambos os lados da incisão usando duas pinças e puxar as pinças longe um do outro para separar a retina. <ol><li> Cortar o nervo óptico e tentar isolar a retina com uma quantidade mínima de contacto físico com a superfície distai. RPE, na sua maioria separar automaticamente a partir da retina durante o processo de dissecção. É mais importante para evitar a perturbação mecânica da retina do que para realizar odissecção rapidamente, em geral, as retinas podem ser incubadas pelo menos 30 minutos na solução, sem efeitos significativos sobre as propriedades de resposta. </li></ol></li><li> Monte as retinas sobre as cúpulas de suporte para amostra (Figura 1C). Mergulhe a parte inferior do suporte da amostra na placa de Petri com as retinas dissecadas. Deslize da retina, fotorreceptores lado (a superfície convexa da retina isolado) para cima, por cima da cúpula e levantar o suporte da amostra, de modo que a retina atribui no papel de filtro. Repetir o procedimento para o outro retina. </li><li> Secar a placa suporte com cuidado para evitar interferência elétrica entre as retinas, bem como o ruído eo sinal de manobra. O suporte de amostras tem anéis de vedação em torno das cúpulas parte do fundo para impedir que a solução derrame entre a parte inferior e superior das peças do suporte, bem como para ajudar o isolamento eléctrico dos fotorreceptores e células ganglionares lados das retinas individuais. Fixe a parte superior do suporte com os quatro parafusos (ver Figura 1B) e encher os canais de perfusão com solução de perfusão utilizando uma seringa e uma agulha. </li><li> Transferir o porta-espécime ao lado do bloco do permutador de calor e conectar as linhas de entrada e saída de perfusão para o suporte da amostra (Figura 1D). Ligar os eléctrodos ao amplificador ERG (top eletrodos ligar para o chão / menos pino no amplificador) e anexar a unidade estimulador / controle sobre o suporte da amostra usando um adaptador ou deslizar a almofada de aquecimento na unidade estimulador dependendo de qual in vivo ERG sistema é utilizado. </li></ol> 3. Gravações <ol><li> Configurar as definições do amplificador e de estímulo usando o software do sistema in vivo. Defina a freqüência de aquisição a um valor entre 1 e 10 kHz e filtragem passa-baixo a 300 Hz. Não usar a filtragem passa-alta. Use 60 Hz (ou 50 Hz na Europa) entalhe filtro, se necessário. </li><li> Ficha de linha de base sem estimulação luminosa e sagacidadeh estimulação dim (eg., luz verde de -35 dB ou 0,3 mCd sm-2) para testar a boa conexão elétrica entre os eletrodos e amostra retina. Espera 10-20 min antes de iniciar a recolha de dados de modo a que a temperatura e a função da retina alcançar um estado estável. <ol><li> Escolha parâmetros de estímulo de acordo com qualquer experiência específica e comece a coleta de dados. Por exemplo, usar a luz verde de -40 até 0 dB ou de 0,1 até 1000 mCd sm -2 para uma família de resposta escotópica. Use cerca de 2 a 3 unidades de log mais brilhante flashes em gravações fotópicas onde hastes têm de ser suprimida pela luz de fundo (3-30 Cdm -2) ou um flash sonda (sm cerca de 1,3 -2 Cd = 1 dB, veja a Figura 3). </li><li> No entanto, lembre-se que os valores exatos de intensidade de luz pode ser um pouco dependente de sua calibração do sistema e as condições experimentais. Como uma regra geral, as intensidades da escala para baixo por 5-10 vezes, em comparação com in vivo </em&gt; gravações ao usar luz verde 17. Uma tampa preta com aberturas acima retinas (incluído no sistema adaptador comercial) pode ser usada para reduzir a dispersão de luz no suporte de amostras e facilitar estimulação homogénea e igual de ambas as retinas pela esfera Ganzfeld da comercial em sistemas in vivo. NOTA: Para gravações de retina de adaptação ao escuro, as intensidades de flashes de teste utilizadas numa experiência de branqueamento típico apenas uma fracção insignificante do pigmento, de modo que a falta de regeneração orientada para o EPR não é um problema. Além disso, foi anteriormente demonstrado que a regeneração cone de pigmento podem ainda ter lugar na retina isolada através do ciclo visual célula Müller 20. </li></ol></li><li> Como experiências durar tipicamente de 30 minutos até várias horas, monitorizar a deriva da linha de base, o nível de ruído e estabilidade de resposta durante a experiência como alterações na estes podem indicar problemas técnicos. NOTA: Aumento ou diminuição de ruídoamplitudes de resposta d pode indicar bolhas de ar nos canais de perfusão ou eletrodos, muito baixa ou alta temperatura, a solução de vazamento / derramamento ou deslocamento da retina. </li></ol> 4. Limpeza <ol><li> Retire o suporte da amostra a partir das linhas de perfusão, abra-o e lave as retinas do papel de filtro. Retirar os eléctrodos e lavá-los com água destilada (não use etanol). Limpar o suporte de amostras (incluindo os canais de perfusão) com etanol e / ou água destilada. Tubo de perfusão Lave cuidadosamente com etanol&gt; 70%. </li><li> Lavar o frasco de perfusão com água destilada (não use detergentes). O etanol pode também ser usado para limpar o frasco. </li></ol>

<ol>
	<li>Armington, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Electroretinogram. , (1974).</li><li>Einthoven, W., Jolly, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+form+and+magnitude+of+the+electrical+response+of+the+eye+to+stimulation+by+light+at+various+intensities.">The form and magnitude of the electrical response of the eye to stimulation by light at various intensities.</a> Q J Exp Physiol. 1, 43 (1908).</li><li>Granit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+components+of+the+retinal+action+potential+in+mammals+and+their+relation+to+the+discharge+in+the+optic+nerve.">The components of the retinal action potential in mammals and their relation to the discharge in the optic nerve.</a> J. Physiol. 77, 207-239 (1933).</li><li>Penn, R. D., Hagins, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transmission+along+retinal+rods+and+the+origin+of+the+electroretinographic+a-wave.">Signal transmission along retinal rods and the origin of the electroretinographic a-wave.</a> Nature. 223, 201-204 (1969).</li><li>Stockton, R. A., Slaughter, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-wave+of+the+electroretinogram.+A+reflection+of+ON+bipolar+cell+activity.">B-wave of the electroretinogram. A reflection of ON bipolar cell activity.</a> J. Gen. Physiol. 93, 101-122 (1989).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+linearity+and+kinetics+of+the+cat+retina:+the+bipolar+cell+component+of+the+dark-adapted+electroretinogram.">Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.</a> Vis. Neurosci. 12, 837-850 (1995).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dissection+of+the+electroretinogram+from+the+isolated+rat+retina+with+microelectrodes+and+drugs.">A dissection of the electroretinogram from the isolated rat retina with microelectrodes and drugs.</a> Vis. Neurosci. 16, 727-741 (1999).</li><li>Steinberg, R. H., Schmidt, R., Brown, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+responses+to+light+from+cat+pigment+epithelium:+origin+of+the+electroretinogram+c-wave.">Intracellular responses to light from cat pigment epithelium: origin of the electroretinogram c-wave.</a> Nature. 227, 728-730 (1970).</li><li>Wilson, W. S., Shahidullah, M., Millar, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bovine+arterially-perfused+eye:+an+in+vitro+method+for+the+study+of+drug+mechanisms+on+IOP,+aqueous+humour+formation+and+uveal+vasculature.">The bovine arterially-perfused eye: an in vitro method for the study of drug mechanisms on IOP, aqueous humour formation and uveal vasculature.</a> Curr. Eye Res. 12, 609-620 (1993).</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>Donner, K., Hemila, 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=Temperature-dependence+of+rod+photoresponses+from+the+aspartate-treated+retina+of+the+frog+(Rana+temporaria).">Temperature-dependence of rod photoresponses from the aspartate-treated retina of the frog (Rana temporaria).</a> Acta Physiol. Scand. 134, 535-541 (1988).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+properties+of+a+new+isolated+rat+retina+preparation.">Electrophysiological properties of a new isolated rat retina preparation.</a> Vision Res. 39, 2165-2177 (1999).</li><li>Hamasaki, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+ion+concentration+on+the+electroretinogram+of+the+isolated+retina+of+the+frog.">The effect of sodium ion concentration on the electroretinogram of the isolated retina of the frog.</a> J. Physiol. 167, 156-168 (1963).</li><li>Luke, M., 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=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function.</a> Brain Res. Brain Res. Protoc. 16, 27-36 (2005).</li><li>Bastian, B. L., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+adaptation+in+toad+rods:+requirement+for+an+internal+messenger+which+is+not+calcium.">Light adaptation in toad rods: requirement for an internal messenger which is not calcium.</a> J. Physiol. 297, 493-520 (1979).</li><li>Arden, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+gradients+across+the+receptor+layer+of+the+isolated+rat+retina.">Voltage gradients across the receptor layer of the isolated rat retina.</a> J. Physiol. 256, 333-360 (1976).</li><li>Vinberg, F., Kolesnikov, A. V., 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=Ex+vivo+ERG+analysis+of+photoreceptors+using+an+in+vivo+ERG+system.">Ex vivo ERG analysis of photoreceptors using an in vivo ERG system.</a> Vision Res. 101, 108-117 (2014).</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>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>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><li>Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+conductance+block+by+barium+in+amphibian+Muller+cells.">Potassium conductance block by barium in amphibian Muller cells.</a> Brain Res. 498, 308-314 (1989).</li><li>Oakley, B., Katz, B. J., Xu, Z., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+buffering+of+extracellular+potassium+by+Muller+(glial)+cells+in+the+toad+retina.">Spatial buffering of extracellular potassium by Muller (glial) cells in the toad retina.</a> Exp. Eye Res. 55, 539-550 (1992).</li><li>Nymark, S., Haldin, C., Tenhu, H., 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=A+new+method+for+measuring+free+drug+concentration:+retinal+tissue+as+a+biosensor.">A new method for measuring free drug concentration: retinal tissue as a biosensor.</a> Invest. Ophthalmol. Vis. Sci. 47, 2583-2588 (2006).</li><li>Walter, P., Luke, C., Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+light+responses+in+superfused+bovine+retina.">Antibiotics and light responses in superfused bovine retina.</a> Cell. Mol. Neurobiol. 19, 87-92 (1999).</li><li>Luke, M., 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=The+safety+profile+of+alkylphosphocholines+in+the+model+of+the+isolated+perfused+vertebrate+retina.">The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 248, 511-518 (2010).</li><li>Januschowski, 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=Electrophysiological+toxicity+testing+of+VEGF+Trap-Eye+in+an+isolated+perfused+vertebrate+retina+organ+culture+model.">Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.</a> Acta Ophthalmol. 92, e305-e311 (2014).</li><li>Kolesnikov, A. V., 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=Transretinal+ERG+recordings+from+mouse+retina:+rod+and+cone+photoresponses.">Transretinal ERG recordings from mouse retina: rod and cone photoresponses.</a> J Vis Exp. , (2012).</li><li>Koskelainen, A., Hemila, S., Donner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivities+of+short-+and+long-wavelength+sensitive+cone+mechanisms+in+the+frog+retina.">Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.</a> Acta Physiol. Scand. 152, 115-124 (1994).</li><li>Lyubarsky, A. L., Falsini, B., Pennesi, M. E., Valentini, P., 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=UV-+and+midwave-sensitive+cone-driven+retinal+responses+of+the+mouse:+a+possible+phenotype+for+coexpression+of+cone+photopigments.">UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.</a> J. Neurosci. 19, 442-455 (1999).</li><li>Lyubarsky, A. L., Daniele, L. L., 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=From+candelas+to+photoisomerizations+in+the+mouse+eye+by+rhodopsin+bleaching+in+situ+and+the+light-rearing+dependence+of+the+major+components+of+the+mouse+ERG.">From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.</a> Vision Res. 44, 3235-3251 (2004).</li><li>Azevedo, A. W., Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocols+alter+phototransduction:+the+implications+for+retinal+processing+at+visual+threshold.">Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold.</a> J. Neurosci. 31, 3670-3682 (2011).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+I.+Structural+analysis+using+light+and+electron+microscopy.">Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.</a> J. Comp. Neurol. 188, 245-262 (1979).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+vertebrate+photoreceptors.">Adaptation in vertebrate photoreceptors.</a> Physiol. Rev. 81, 117-151 (2001).</li><li>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends Cell Biol. 16, 560-568 (2006).</li><li>Schneeweis, D. M., Schnapf, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photovoltage+of+macaque+cone+photoreceptors:+adaptation,+noise,+and+kinetics.">The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics.</a> J. Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., 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=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

Universidade de Washington em St. Louis tem um acordo de licença com Xenotec, Inc. e pode receber royalties da venda do adaptador ex vivo.

<html> Nós demonstramos aqui os passos críticos para a obtenção de alta qualidade ex gravações vivo ERG simultaneamente a partir de duas rato retinas isoladas usando componentes in vivo do sistema ERG juntamente com um adaptador vivo ex ERG. Neste estudo, ambas as retinas perfundidos a partir do animal com a mesma solução (ou Ames, Locke ou riscas) mas também é possível perfundir cada retina com uma solução diferente, por exemplo, para fins de teste de drogas. Os ...

Electrorretinograma (ERG) é uma técnica bem estabelecida que pode ser utilizado para registar a actividade eléctrica da retina desencadeadas pela luz. O sinal é gerado principalmente ERG por mudanças de tensão provocadas por correntes radiais (ao longo do eixo dos fotorreceptores e células bipolares) que flui no espaço extracelular resistiva da retina. O primeiro sinal ERG foi gravado em 1865 por Holmgren a partir da superfície de um olho de peixe 1. Einthoven e Jolly 1908 2 dividido a resposta do ERG ao aparecimento de luz em três ondas diferentes, denominado A, B, e C-ondas, que são agora conhecidos, principalmente, para reflectir a actividade de fotorreceptores, em células bipolares, e epitélio pigmentado células, respectivamente 3-8. ERG pode ser gravado a partir dos olhos de animais anestesiados ou humanos (in vivo), de olho preparação isolada 9, em frente isolado retina intacta (ex vivo) ou através 3,10-15 camadas da retina específicas com microeletrodos (localERG) 4,16. Destes, in vivo ERG é atualmente o método mais utilizado para avaliar a função da retina. É uma técnica não invasiva que pode ser utilizada para fins de diagnóstico ou para seguir a progressão das doenças da retina em animais ou pacientes. No entanto, in vivo registos de ERG produzir um sinal complexo com vários componentes sobrepostos, muitas vezes contaminados por ruído extraocular fisiológico (por exemplo, a respiração e da actividade cardíaca). ERG local pode ser utilizado para gravar o sinal através de camadas específicas da retina, mas é mais invasiva e tem uma menor relação de sinal-para-ruído (SNR) em relação a outras configurações de gravação ERG. ERG local também é tecnicamente exigente e requer equipamento caro (por exemplo, microscópio e micromanipuladores). Transretinal ERG do intacta, isolado retina (ex vivo ERG) oferece um compromisso entre os métodos in vivo e ERG locais permitindo estável e higgravações h SNR de retinas intactas de animais ou seres humanos 17. Recentemente, este método tem sido utilizado com êxito para estudar a função de fotorreceptores cone e da haste em mamíferos, primatas e humanos retinas 18-20. Além disso, devido à ausência de epitélio pigmentar da retina ex vivo, o componente do sinal de ERG c-onda positiva é removida e um componente lento PIII negativos proeminentes é revelado nas gravações ex vivo. O componente PIII lenta tem sido demonstrado que se originam a partir da actividade de células da glia de Müller na retina 21-23. Assim, método ex vivo ERG também poderia ser usado para estudar as células Müller na retina intacta. Vários estudos também demonstraram que os registos de ERG ex vivo pode ser usada para medir a concentração de agentes farmacológicos em torno da retina 24 e testar a segurança e eficácia de drogas 25-27. Comercial múltipla em sistemas in vivo estão disponíveis eusado em muitos laboratórios que não têm necessariamente extensa experiência eletrofisiologia. Em contraste, ex vivo dispositivos não estavam disponíveis até recentemente 17 e, como resultado muito poucos laboratórios estão actualmente a tirar partido desta poderosa técnica. Seria benéfico para fazer gravações ex vivo ERG disponível para mais laboratórios, a fim de aumentar o nosso conhecimento sobre a fisiologia da retina e patologia, e para desenvolver novas terapias para doenças ofuscante. Nós demonstramos aqui um dispositivo simples e acessíveis ex vivo ERG 17 e mostram como ele pode ser utilizado em combinação com vários sistemas in vivo ERG disponíveis comercialmente para gravar e sinalização mediada rod- de cone (a- e b-ondas) e a função de células de Müller (retardar PIII) de íntegras do tipo selvagem do rato retinas.

<table><tbody><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>OcuScience</td><td>HMsERG</td><td>&lt;a href=&quot;http://www.ocuscience.us/id77.html&quot;&gt;www.ocuscience.us/id77.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>LKC Technologies</td><td>UTAS-E 3000</td><td>&lt;a href=&quot;http://www.lkc.com/products/UTAS/bigshot.html&quot;&gt;www.lkc.com/products/UTAS/bigshot.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;Ex vivo&lt;/i&gt; adapter</td><td>OcuScience</td><td>Ex VIVO ERG adapter</td><td>&lt;a href=&quot;http://www.ocuscience.us/id107.html&quot;&gt;www.ocuscience.us/id107.html&lt;/a&gt;</td></tr><tr><td>Dissection microscope</td><td>North Central Instruments</td><td>Leica M80</td><td>&lt;a href=&quot;http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;amp;idCategory=27&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>IR emitter</td><td>Opto Diode Corp.</td><td>OD-50L</td><td>&lt;a href=&quot;http://www.optodiode.com/&quot;&gt;www.optodiode.com&lt;/a&gt;</td></tr><tr><td>Prowler Night Vision Scopes</td><td>B.E. Meyers Electro Optics</td><td>D4300-I</td><td>Military grade product.</td></tr><tr><td>Red filter</td><td>Rosco Laboratories</td><td>Roscolux #27 Medium Red</td><td>May be used instead of IR system</td></tr><tr><td>Red head light</td><td>OcuScience</td><td>ERGX011</td><td>&lt;a href=&quot;http://www.ocuscience.us/catalog/i29.html&quot;&gt;www.ocuscience.us/catalog/i29.html&lt;/a&gt;</td></tr><tr><td>Microscissors</td><td>WPI, Inc.</td><td>500086</td><td>&lt;a href=&quot;http://www.wpiinc.com/&quot;&gt;www.wpiinc.com/&lt;/a&gt;</td></tr><tr><td>Dumont tweezers #5</td><td>WPI, Inc.</td><td>14101</td><td></td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com&lt;/a&gt;</td></tr><tr><td>Scale</td><td>Metler Toledo</td><td>AB54-S/FACT</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>pH meter and electrode</td><td>Beckman Coulter</td><td>pHI 350</td><td>May use any brand</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60129</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63020</td><td>1.0 M solution</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>21114</td><td>1.0 M solution</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>431788</td><td>May use any brand</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>May use any brand</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>May use any brand</td></tr><tr><td>Ames medium</td><td>Sigma-Aldrich</td><td>A1420</td><td>May use any brand</td></tr><tr><td>BaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>B0750</td><td>May use any brand</td></tr><tr><td>DL-AP4</td><td>Tocris Bioscience</td><td>101</td><td>May use any brand</td></tr><tr><td>Succinic acid disodium salt</td><td>Sigma-Aldrich</td><td>224731</td><td>May use any brand</td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G2834</td><td>May use any brand</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>May use any brand</td></tr><tr><td>Leibovitz culture medium L-15</td><td>Sigma-Aldrich</td><td>L4386</td><td>May use any brand</td></tr><tr><td>MEM vitamins</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>MEM amino acids</td><td>Sigma-Aldrich</td><td>M5550</td><td></td></tr><tr><td>Carbogen</td><td>Airgas</td><td>UN3156</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr></tbody></table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Foram registrados respostas de flash a partir do tipo selvagem de adaptação ao escuro (WT) C57BL / 6 de rato retinas seguindo os protocolos experimentais descritos acima e ilustrados na Figura 1, usando diferentes soluções para perfusão padrão (Figura 2). As formas de onda de resposta e cinética, bem como sensibilidade dos bastonetes apareceu semelhante em Ames e mídia de Locke (Figura 2A e B). Por outro lado, sob a solução de Ringer tamponada...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K visualizações.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Vídeo: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Multifocal Electroretinograms

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1.
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.

The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

Eletrorretinograma multifocal

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity.  Generally, ERG results are ...

Medicina

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

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 whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

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 ...

Neurociência

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caracterização eletrofisiológica de GFP-Expressando populações de células na retina intacta

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

Eletroretinograma acoplado direto (DC-ERG) para gravar as respostas elétricas evocadas pela luz do epitélio pigmento de retina do rato

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

Química

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Usando o Eletrorretinograma na Avaliação da Função na Roedor Retina e os efeitos protetores do remoto Limb Pré-Condicionamento Isquêmico

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

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

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

Gravação simultânea de Eletrorretinografia e potenciais evocados visuais em ratos anestesiados

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

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Otimizando a configuração e as condições do eletrorretinograma ex vivo para estudar a função da retina em olhos pequenos e grandes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Explante Retiniano da Retina de Rato Adulto como Modelo Ex Vivo para o Estudo de Doenças Neurovasculares da Retina

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://appjove.unicartagenaproxy.elogim.com/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Simultâneo

Resumo

Explore mais vídeos

Capítulos neste vídeo