Este trabajo fue apoyado por el NIH subvenciones EY019312 y EY021126 (VJK), EY002687 al Departamento de Oftalmología y Ciencias Visuales de la Universidad de Washington, y por la investigación para prevenir la ceguera.

Todos los protocolos experimentales fueron de acuerdo con la Guía para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por la Comisión de Estudios de Animales institucional de la Universidad de Washington. 1. Configuración de la perfusión y de muestras <ol><li> Preparar la solución para perfusión retina fresco en el día del experimento. Utilice agua destilada y desionizada. Utilice una de las tres soluciones siguientes. <ol><li> Prepare Bicarbonato que contiene &quot;solución (1 L): 1 botella de Ames Ames medios de comunicación y 1,9 g de NaHCO 3, </li><li> Preparar la solución de Locke (en mM): 112,5 NaCl, 3,6 KCl, 2,4 MgCl 2, 1,2 CaCl 2, 10,0 HEPES, 20,0 NaHCO 3, 3,0 Na succinato, 0,5 Na glutamato, 0,02 EDTA, y 10,0 glucosa, 0,1% de vitaminas MEM y amino ácidos </li><li> Solución de Ringer tamponada con HEPES Preparar (en mM): NaCl 133,3, KCl 3,3, 2,0 MgCl2, 1,0 CaCl 2, 10,0 glucosa, 0,01 EDTA, 12,0 HEPES, pHajustado a 7,5 con ~ 5,8 ml de NaOH 1 M, añadir 0,72 g / L de medio de cultivo Leibovitz L-15. Utilice 20 - 50 M DL-AP4 y 50 - 100 mM BaCl 2 para aislar la respuesta de los fotorreceptores con cualquier medio de perfusión. </li></ol></li><li> Preparar la solución para los electrodos 28 (en mM): NaCl 140,0, KCl 3,6, 2,4 MgCl2, 1,2 CaCl 2, 3, HEPES 0,01 EDTA y ajustar el pH a 7.4 a 7.5 con NaOH. Solución de electrodo se puede almacenar a temperatura ambiente durante varios meses. </li><li> Preparar y poner a prueba el soporte de la muestra. <ol><li> Pegue el papel de filtro negro / gris en la parte superior de las cúpulas de la parte inferior del soporte de la muestra (ver Figura 1 A). Spread de dos componentes 5 min pegamento epoxi cuidadosamente alrededor de los bordes de las tapas planas de las cúpulas. Si es necesario, hacerlo bajo microscopio de disección. </li><li> Espere hasta que el pegamento es casi seco (unos 4 minutos) y pulse el papel de filtro en las cúpulas utilizando un elemento plano. Papel de filtro pegamento al menos un día antes del experimento. Lospapel de filtro se puede utilizar varias veces, pero debe ser reemplazado después de un mes de grabaciones. Utilice etanol al 70% para limpiar cuidadosamente las cúpulas de cualquier residuo de pegamento antes de instalar el papel de sustitución. </li><li> Llenar los canales de electrodos con una solución de electrodo tratando de evitar las burbujas de aire y el tornillo electrodos de pellets cerrados con un adaptador roscado en los canales de los electrodos (ver figuras 1A y B). Conecte las piezas superior e inferior del soporte de la muestra con cuatro tornillos y llenar las líneas de perfusión con solución de electrodo. </li><li> Medir la resistencia y la tensión entre los conductores de cada par de electrodos con un multímetro (Figura 1B). La resistencia debe ser inferior a 100 Ωk y la tensión por debajo de 10 mV si los canales son sin burbujas y los electrodos están en buenas condiciones. </li></ol></li><li> Verter 400 a 700 ml de medio de perfusión en la botella de vidrio. Separar otros 300 ml a utilizar en la disección de las retinas y sla rompió en una nevera. Ajuste el tubo de perfusión en el bloque intercambiador de calor y coloque el bloque precalentado en la placa de calefacción (ver Figura 1D). </li><li> Incluya la botella con una tapa que tiene conexiones a CO 2 / O 2 gas (si se utiliza de Ames o Locke) y para la tubería de perfusión (ver Figura 1D). Precaliente la botella con los medios de comunicación a 37 o C y colocarlo en la placa de calentamiento o en la parte superior de la unidad de estimulación de la luz en el baño de agua a 37-39 ° C 17. <ol><li> Primer de las líneas de perfusión llenándolos de medios de perfusión para iniciar el flujo por gravedad. <ol><li> En el sistema de LKC, colocar la botella en la parte superior de la unidad estimuladora 17 para proporcionar un flujo gravitacional significativo que se ajusta entonces por los reguladores de caudal sin ser afectado por el nivel de reducción de la solución de perfusión en la botella durante el experimento. </li><li> En el sistema Ocuscience, coloque el perfusion botella dentro de la jaula de Faraday con el fin de minimizar el ruido y colocar líneas de salida largo de perfusión desde el portamuestras (véase el paso 2.8) muy por debajo del nivel del soporte de la muestra (y la botella de perfusión) para aumentar la unidad gravitatoria de la solución. Proteja estas líneas de salida y conectar el blindaje a tierra del amplificador para evitar el acoplamiento del ruido electromagnético a la señal de ERG. </li></ol></li></ol></li><li> Ajuste la perfusión de 3-5 ml / min mediante el uso de reguladores de caudal. Conecte 5% de CO 2/95% O 2 gas de un cilindro con regulador adecuado y ajustar el caudal para garantizar burbujeo constante de los medios de comunicación en la botella a través de un difusor de aire. </li></ol> Preparación 2. Muestra <ol><li> Ensamble instrumentos de disección limpias y afiladas incluyendo micro tijeras con filo recto, uno o dos de 45 o pinzas, cuchillas de afeitar y una pieza rectangular de papel de filtro. </li><li> Vierta aproximadamente 200 ml de perfu fríaSion solución en una gran placa de Petri de modo que toda la parte inferior del soporte de muestra (incluyendo las cúpulas) se puede sumergir en la solución. Este paso es importante cuando el montaje de las retinas de las cúpulas (véase el paso 2.6). Aunque algunas soluciones están diseñadas para ser saturado con carbógeno (5% de CO2 / 95% de O 2), esto no es esencial para los fines de disección y no se hizo en los experimentos descritos aquí. </li><li> Para un experimento típico, mantener a los animales en 12/12 horas ciclo de oscuridad / luz y la oscuridad-adaptarlos por 6 - 12 horas antes de las grabaciones. La eutanasia a los animales por inhalación de CO 2 seguido por dislocación cervical bajo tenue luz roja y hacer todos los siguientes procedimientos previstos en luz roja o infrarrojos dim (utilizar por ejemplo, el filtro rojo delante de la fuente de luz del microscopio). <ol><li> Tire de los ojos mediante el uso de pinzas y los puso en los medios de comunicación. Coloque un ojo a la vez en un pequeño pedazo de papel (por ejemplo, un poco de papel de filtro regular) y hacer comoiluminado aproximadamente en el nivel de ora serrata mientras sostiene el ojo con unas pinzas (esto se hace fuera de la solución aquí). </li></ol></li><li> Corte a lo largo de la ora serrata (o más cerca del ecuador del ojo) con micro tijeras y retire la córnea y el cristalino. Coloque el ocular en los medios de frío en la gran placa de Petri y repita el mismo procedimiento con el otro ojo. </li><li> Cortar una pequeña incisión de la parte superior de la copa para el ojo hacia el nervio óptico manteniendo las tijeras entre la retina y la esclerótica con el fin de mantener la retina tan intacto como sea posible. Grip la esclerótica de ambos lados de la incisión mediante el uso de dos pinzas y tire de las pinzas de distancia el uno del otro para separar la retina. <ol><li> Cortar el nervio óptico y tratar de aislar a la retina con una mínima cantidad de contacto físico con la superficie distal. RPE será principalmente separar automáticamente de la retina durante el proceso de disección. Es más importante para evitar la perturbación mecánica de la retina que para realizar ladisección rápidamente, generalmente retinas se puede incubar al menos 30 min en la solución sin efectos significativos sobre las propiedades de respuesta. </li></ol></li><li> Montar las retinas de las cúpulas del portamuestras (Figura 1C). Sumergir la parte inferior del portamuestras en la placa de Petri con las retinas disecadas. Deslizar la retina, lado fotorreceptor (la superficie convexa de la retina aislada) hacia arriba, por encima de la cúpula y levantar el soporte de la muestra de modo que la retina se fija en el papel de filtro. Repita el procedimiento para el otro retina. </li><li> Secar la placa de soporte con cuidado para evitar la diafonía eléctrica entre las retinas así como el ruido y la señal de derivación. Portamuestras tiene juntas tóricas alrededor de la parte inferior cúpulas para evitar derrame de solución entre la parte inferior y superior del soporte, así como para ayudar a la separación galvánica de los fotorreceptores y células ganglionares lados de las retinas individuales. Coloque la parte superior del soporte con los cuatro tornillos (véase la Figura 1B) y llenar los canales de perfusión con solución de perfusión utilizando una jeringa y una aguja. </li><li> Transferir el portamuestras junto al bloque intercambiador de calor y conectar las líneas de entrada y salida de perfusión en el soporte de la muestra (Figura 1D). Conectar los electrodos al amplificador ERG (electrodos superiores conectan al suelo / menos pasador en el amplificador) y adjuntar la unidad estimulador / control en el soporte de la muestra mediante el uso de un adaptador o deslice la almohadilla eléctrica en la unidad estimulador dependiendo de la in vivo se utiliza el sistema ERG. </li></ol> 3. Grabaciones <ol><li> Configure los ajustes del amplificador y de estímulo mediante el uso de software del sistema en vivo. Ajuste la frecuencia de adquisición a un valor entre 1 y 10 kHz y de paso bajo filtrando a 300 Hz. No utilizar el filtrado de paso alto. Utilice 60 Hz (o 50 Hz en Europa) muesca filtro si es necesario. </li><li> La línea de base de registro y sin estimulación de la luz y el ingenioh tenue estimulación (por ejemplo., luz verde de -35 dB o 0,3 mCd sm -2) para probar la buena conexión eléctrica entre los electrodos y muestra de retina. Espere 10 a 20 min antes de iniciar la recogida de datos de manera que la temperatura y la función de la retina llegan a un estado estable. <ol><li> Elija los parámetros de estímulo de acuerdo con cualquier experimento específico y comenzar la recolección de datos. Por ejemplo, utilice la luz verde de -40 hasta 0 dB o de 0,1 hasta 1000 mCd sm -2 para una familia respuesta escotópica. Utilice cerca de 2 a 3 log-unidades más brillantes destellos en grabaciones fotópicas donde las barras tienen que ser suprimida por la luz de fondo (3-30 Cdm -2) o un flash de la sonda (aproximadamente 1,3 Cd sm -2 = 1 dB, ver Figura 3). </li><li> Sin embargo, recuerde que los valores de intensidad de luz exactas podrían ser algo dependiente de la calibración del sistema y de las condiciones experimentales. Como regla general, las intensidades de escala hacia abajo por 5 a 10 veces en comparación con in vivo </em&gt; grabaciones cuando se utiliza luz verde 17. Una cubierta de negro con aberturas por encima de las retinas (incluidos en el sistema de adaptador comercial) se puede utilizar para reducir la dispersión de luz en el soporte de la muestra y facilitar la estimulación homogénea e igualitaria de ambas retinas por la esfera Ganzfeld del comercial en vivo sistemas. NOTA: Para grabaciones de retina adaptada a la oscuridad, las intensidades de destellos de prueba utilizados en un experimento típico blanqueador sólo una fracción insignificante del pigmento de modo que la falta de regeneración RPE-impulsado no es un problema. Además, se ha demostrado previamente que la regeneración pigmento cono todavía puede tener lugar en la retina aislado a través del ciclo celular Müller visual 20. </li></ol></li><li> Como experimentos duran típicamente de 30 min hasta varias horas, controlar la deriva de la línea de base, nivel de ruido y la estabilidad de respuesta durante el experimento como cambios en estos podrían indicar problemas técnicos. NOTA: El aumento o disminución de ruidoamplitudes de respuesta d pueden indicar burbujas de aire en los canales de perfusión o electrodos, demasiado baja o alta temperatura, solución de fugas / derrames o desplazamiento de la retina. </li></ol> 4. Limpieza <ol><li> Separe el soporte de la muestra de las líneas de perfusión, abrirla y vaciar las retinas del papel de filtro. Retire los electrodos y enjuague con agua destilada (no utilizar etanol). Limpiar el soporte de la muestra (incluyendo los canales de perfusión) con etanol y / o agua destilada. Tubo de perfusión Flush cuidadosamente etanol con&gt; 70%. </li><li> Enjuague la botella de perfusión con agua destilada (no usar detergentes). El etanol puede ser también utilizado para limpiar la botella. </li></ol>

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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>

Universidad de Washington en St. Louis tiene un acuerdo de licencia con Xenotec, Inc. y puede recibir una regalía por la venta del adaptador ex vivo.

<html> Estamos aquí demostrar los pasos críticos para la obtención de alta calidad ex grabaciones ERG vivo simultáneamente desde dos retinas de ratones aislados mediante el uso de componentes en vivo del sistema ERG, junto con un adaptador ex vivo ERG. En este estudio se perfundidos ambas retinas del animal con la misma solución (ya sea Ames &#39;, Locke o de Ringer), pero también es posible para perfundir cada retina con una solución diferente, por ejemplo, para fines ...

Electrorretinograma (ERG) es una técnica bien establecida que puede utilizarse para registrar la actividad eléctrica de la retina provocado por la luz. La señal de ERG se genera principalmente por cambios de voltaje causadas por las corrientes radiales (a lo largo del eje de los fotorreceptores y células bipolares) que fluye en el espacio extracelular resistiva de la retina. La primera señal de ERG fue grabado en 1865 por Holmgren desde la superficie de un ojo de pez 1. Einthoven y Jolly 1908 2 dividen la respuesta ERG a la aparición de la luz en tres ondas diferentes, llamado a-, b-, y c-ondas, que ahora se conoce para reflejar principalmente la actividad de los fotorreceptores, células bipolares EN, y el epitelio de pigmento células, respectivamente 3-8. ERG se puede grabar desde los ojos de animales o humanos (in vivo) anestesiados, desde aislada preparación ojo 9, a través de la retina intacta aislada (ex vivo) 3,10-15 oa través de las capas de la retina específicas con microelectrodos (localERG) 4,16. De estos, in vivo ERG es actualmente el método más utilizado para evaluar la función de la retina. Es una técnica no invasiva que se puede utilizar con fines de diagnóstico o para seguir la progresión de enfermedades de la retina en animales o pacientes. Sin embargo, en vivo grabaciones ERG producen una señal compleja con varios componentes superpuestos, a menudo contaminados por el ruido extraoculares fisiológico (por ejemplo, la respiración y la actividad cardíaca). ERG local se puede utilizar para grabar la señal a través de capas específicas de la retina, pero es el más invasivo y tiene la relación más baja de señal a ruido (SNR) en comparación con las otras configuraciones de grabación de ERG. ERG local también es técnicamente exigente y requiere equipos costosos (por ejemplo, microscopio y micromanipuladores). Transretinal ERG de la intacto, aislado retina (ex vivo ERG) ofrece un compromiso entre in vivo y métodos locales ERG permitiendo estable y higgrabaciones h SNR de retinas intactas de animales o seres humanos 17. Recientemente, este método ha sido utilizado con éxito para estudiar varilla y fotorreceptor cono función en retinas de mamíferos, primates y humanos 18-20. Además, debido a la ausencia de epitelio pigmentario de la retina ex vivo, se retira el componente de la onda c positivo de la señal de ERG y un componente prominente PIII lento negativo se revela en las grabaciones ex vivo. El componente lento PIII se ha demostrado que proceden de la actividad de las células gliales de Müller en la retina 21-23. Por lo tanto, ex vivo ERG método también podría ser usado para estudiar las células de Müller en la retina intacta. Varios estudios también han demostrado que los ex vivo grabaciones ERG se podrían utilizar para medir la concentración de agentes farmacológicos alrededor de la retina 24 y probar la seguridad y eficacia de los fármacos 25-27. Comercial múltiple en sistemas in vivo están disponibles yutilizado en muchos laboratorios que no necesariamente tienen amplia experiencia electrofisiología. En contraste, los dispositivos ex vivo no han estado disponibles hasta hace poco 17 y como resultado sólo muy pocos laboratorios están tomando actualmente ventaja de esta técnica de gran alcance. Sería beneficioso para hacer grabaciones ex vivo ERG a disposición de más laboratorios con el fin de avanzar en nuestro conocimiento sobre la fisiología de la retina y la patología, y desarrollar nuevas terapias para enfermedades que causan ceguera. Se demuestra aquí una sencilla y asequible dispositivo ex vivo ERG 17 y mostrar cómo se puede utilizar en combinación con varios sistemas vivo ERG disponibles en el mercado para grabar en la señalización de varilla y el cono mediada (a- y b-ondas) y la función de las células de Müller (lento PIII) desde intactas las retinas de ratones de tipo salvaje.

<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.

Se registraron las respuestas de flash de tipo salvaje adaptada a la oscuridad (WT) C57BL / 6 retinas de ratones siguiendo los protocolos experimentales descritos anteriormente e ilustrados en la Figura 1 mediante el uso de diferentes soluciones de perfusión estándar (Figura 2). Las formas de onda y la cinética de respuesta, así como la sensibilidad de los fotorreceptores de varilla parecían similares en Ames y los medios de Locke (Figura 2A y B). ...

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.

Simultáneo<em&gt; Ex vivo</em&gt; Pruebas funcionales de dos retinas por<em&gt; In vivo</em&gt; Sistema Electrorretinograma

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 vistas.  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.

Video: 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.

Electrorretinograma multifocal

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

Investigación

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 Grabaciones ERG de retina del ratón: Rod y el Cono 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 ...

Neurociencias

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.

Caracterización electrofisiológica de las buenas prácticas agrarias que expresan las poblaciones de células en la 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.

Electroretinograma de acoplamiento directo (DC-ERG) para registrar las respuestas eléctricas evocadas por la luz del epitelio del pigmento retinivo del ratón

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.

Utilizando el Electrorretinograma para evaluar la función en el Roedor Retina y los efectos protectores de distancia Limb Preacondicionamiento Isquémico

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.

Grabación simultánea de Electrorretinografía y los potenciales evocados visuales en ratas anestesiadas

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.

Imágenes transpupilares de dos fotones in vivo de la retina del ratón

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.

Optimización de la configuración y las condiciones del electrorretinograma ex vivo para estudiar la función de la retina en ojos pequeños y 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 de la retina de ratón adulto como modelo ex vivo para el estudio de enfermedades neurovasculares retinianas

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 ...

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