Apoyado por NEI conceder EY014850.

1. Preparación de Sylgard cubiertos platos, cámaras experimentales, y hojas de afeitar <ol><li> Falcon 35 mm placas de Petri recubiertas con elastómero Sylgard son necesarios para la tala de un adecuado retina aislada para obtener células fotorreceptoras sola. El elastómero se prepara según las instrucciones del proveedor y una pequeña cantidad se vierte en cada plato para cubrir el fondo con una capa. Vuelva a colocar el plato cubre después del recubrimiento y almacenarlos. En unos pocos días &quot;el elastómero se endurece y los platos están listos. </li><li> Fotorreceptores aislados que atenerse a la parte inferior de la cámara experimental a fin de que se inmovilizan en el transcurso de un experimento de imágenes. Esto se logra mediante el recubrimiento de la parte inferior de las cámaras con poli-L-lisina o poli-L-ornitina. Añadir 200 l de 0,01% de solución de uno por cada cámara, y la cubierta de las cámaras con una toalla de papel para protegerlos del polvo. Después de que la solución se haya secado, lavado de las cavidades con agua destilada y almacenar en una caja cerrada. Su uso dentro de 2 semanas. </li><li> Para la limpieza de las cámaras al final de un experimento, lávelos con etanol al 100% para eliminar el aceite de la lente de inmersión en aceite y restos celulares. Para eliminar los restos celulares, el uso bastoncillos de algodón y frote con cuidado la parte inferior de la cámara. A continuación, lavar con agua destilada y dejar que las cámaras de secar antes de volver a capa. </li><li> Cortar hojas de doble filo navaja de afeitar en pedazos pequeños (8 a partir de una hoja) con un cortador de metal. </li></ol> 2. Preparación de soluciones <ol><li> La composición de las soluciones depende de la especie. Para los anfibios, el Ringer tiene (en mmol / L): 110 NaCl, 2,5 KCl, 1,6 MgCl 2, 1 CaCl 2, 5 HEPES, pH = 7,55. El pH debe ser ajustado al valor final con NaOH. Para los mamíferos, la de Ringer tiene (en mmol / L): 130 NaCl, 5 KCl, 0,5 MgCl2, CaCl2 2, 25 hemisodium-HEPES, pH = 7,40. La solución de Ringer se puede mantener bien tapado a temperatura ambiente durante unos meses. </li><li> Stock de solución de glucosa, 1 mol / L, se mantiene a -20 ° C para evitar el crecimiento bacteriano. </li><li> En el día del experimento, añadir la glucosa a la solución de Ringer a una concentración final de 5 mmol / L. Al final del día, deseche el Ringer que contiene glucosa, ya que podría crecer la bacteria. </li></ol> 3. El aislamiento de la retina <ol><li> Para la escisión correcta de la retina que es importante que el animal esté adaptada a la oscuridad durante al menos 2-3 horas antes del sacrificio. Los animales deben estar adaptados a la oscuridad en un recipiente adecuado con ventilación en el cuarto oscuro. </li><li> Llenar dos cajas de Petri de 35 mm hasta la mitad con solución de Ringer. </li><li> El sacrificio del animal en tenue luz roja y eliminar los ojos. Posteriormente, todos los procedimientos se realizan bajo la luz infrarroja usando un microscopio de disección o una cámara con un monitor de video. </li><li> Remover el tejido sobrante de la superficie externa del ojo. Cortar y quitar la parte anterior, a continuación, transferir el ocular en una de las placas de Petri llenas de Ringer. </li><li> Extraer el humor vítreo y la retina cuidadosamente separada del resto de la ojera por pellizcar o cortar los archivos adjuntos. Levante suavemente la retina y la separan por completo de la ojera. </li><li> La transferencia de la retina a la segunda placa de Petri con una pipeta de transferencia de plástico. Mantenga el recipiente que contenga la retina en una caja a prueba de luz. </li></ol> 4. El aislamiento de las células fotorreceptoras sola <ol><li> Todos los procedimientos se realizan bajo la luz infrarroja. Corte un pedazo pequeño de la retina y la transferencia con una pipeta de plástico a un plato cubierto de Sylgard. El volumen de la solución que contiene la pieza de retina debe ser alrededor de 250 mL. </li><li> Coge un trozo de hoja de afeitar con el soporte de la cuchilla - el borde de la hoja debe estar en ángulo de aproximadamente 45 a su titular. Aplanar la pieza de la retina en la capa de Sylgard y el uso de la hoja de cortar la pieza de la retina finamente mientras que lo mantiene pegado a la capa de Sylgard. </li><li> Transferencia de 200 l de la solución que contiene las células a una cámara experimental - salir de cualquier parte restante de la retina en el plato Sylgard cubiertos. Mantenga la cámara con las células aisladas en una caja a prueba de luz. </li><li> Espere 10 minutos para que las células de resolver, a continuación, agregar 2.3 mL de Ringer. En esta etapa, las células aisladas pueden ser cargados con un tinte fluorescente en particular (por ejemplo Fura-2) de acuerdo con el protocolo de carga de tintes. </li><li> Ahora las células se pueden tomar para la platina del microscopio para realizar experimentos. </li></ol> 5. Fluorescencia de imagen <ol><li> La transferencia de la cámara a la platina del microscopio de epifluorescencia. Ajuste solución de perfusión, las sondas de temperatura, iluminación infrarroja, etc </li><li> Cierre las cortinas y comenzar a experimentar. Encienda la luz infrarroja dentro de la jaula microscopio, se centran en la parte inferior de la cámara experimental, y mover el escenario en busca de las células. </li></ol> 6. Representante de Resultadoss: Fig. 1 muestra la morfología de la barra saludable aislados y los conos obtenidos con este protocolo de una salamandra (Ambystoma tigrinum) retina. Las células de salamandra se han utilizado ampliamente para los estudios de una sola celda de imágenes de fluorescencia debido a su gran tamaño y su capacidad de sobrevivir durante varias horas después del aislamiento de la retina. Además, a partir de una retina una salamandra con regularidad puede obtener tanto en la barra y los conos. Un criterio importante para la salud de las células es la presencia de un elipsoide intactos (Fig. 1), la parte de la célula donde se concentran las mitocondrias. Cuando las células son vistos bajo la óptica DAPI, esta concentración de mitocondrias da una fuerte señal de fluorescencia (Fig. 2) debido a la presencia de NADH. La falta de un elipsoide intacta es una señal de una célula dañada, por lo general no aptos para el experimento. Fig. 3 muestra una barra de fotorreceptores dañados salamandra, con un cuerpo celular hinchada y un núcleo de condensación. Estas células de visualización mucho más bajos de fluorescencia bajo la óptica DAPI, pero visto bajo la óptica FITC mostrar una señal fuerte FAD en la región del elipsoide (que se origina a partir de nucleótidos de flavina oxidado y flavoproteínas). Otro criterio para la salud de los fotorreceptores aislados es su capacidad de generar todo-trans retinol (vitamina A) en sus segmentos externos a la estimulación por la luz. La generación de la vitamina A requiere grandes cantidades de NADPH, que depende de una maquinaria metabólica intacto. Las figuras 4 y 5 muestran la formación de vitamina A en los segmentos externos de la rana intacta y los fotorreceptores de ratón varilla respectivamente. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="Figura 1"/> Figura 1. Fotorreceptores sanos sola varilla y el cono. Las células fueron aisladas de una retina salamandra tigre. Fototransducción tiene lugar en el segmento externo y el elipsoide está densamente poblada por las mitocondrias. Los bastones son responsables de la visión tenue luz, los conos para la visión de luz brillante. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="Figura 2"/> Figura 2. La fluorescencia de la vida salamandra varilla y el cono. Estos son adaptados a la oscuridad las células, que presentan una fluorescencia NADH fuerte en su elipsoides respectivos y no significativa de vitamina A de fluorescencia en sus segmentos externos. La captura de la imagen de fluorescencia representa su primera exposición a la luz visible después del período de adaptación a la oscuridad. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="Figura 3"/> Figura 3. Dañado salamandra varilla de fotorreceptores. El cuerpo de la célula hinchada y el núcleo condensado son indicativos de daño. La célula se oxida y no hay un mínimo de señal de NADH (óptica DAPI), pero la señal más fuerte FAD (FITC óptica). <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="Figura 4"/> Figura 4. Rana de la barra con el NADH y el retinol. Esto es un fotorreceptor rana barra saludable que presentan una fluorescencia NADH fuerte en la región elipsoide. Luz antes de la exposición hay un mínimo de fluorescencia en el segmento externo. A raíz de exposición a la luz, hay un aumento significativo de la fluorescencia de los segmentos externos debido a la formación de vitamina A. <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="Figura 5"/> Figura 5. Ratón varilla con retinol. Esto es un fotorreceptor del ratón la barra saludable que presentan una fluorescencia significativa de los segmentos externos después de exposición a la luz debido a la formación de la vitamina A. Las regiones elipsoide de fotorreceptores de ratón no muestran una fuerte señal de fluorescencia.

<ol>
	<li>Burns, M. E., 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=Beyond+Counting+Photons:+Trials+and+Trends+in+Vertebrate+Visual+Transduction.">Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction.</a> Neuron. 48, 387-401 (2005).</li><li>Ebrey, T., 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=Vertebrate+Photoreceptors.">Vertebrate Photoreceptors.</a> Prog Retin Eye Res. 20, 49-94 (2001).</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>Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptor+Rhodopsin.">G Protein-Coupled Receptor Rhodopsin.</a> Annu Rev Biochem. 75, 743-767 (2006).</li><li>Saari, 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=Biochemistry+of+Visual+Pigment+Regeneration:+The+Friedenwald+Lecture.">Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 41, 337-348 (2000).</li><li>Lamb, T. D., 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=Dark+Adaptation+and+the+Retinoid+Cycle+of+Vision.">Dark Adaptation and the Retinoid Cycle of Vision.</a> Prog Retin Eye Res. 23, 307-380 (2004).</li><li>Imanishi, Y., Lodowski, K. H., 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=Two-Photon+Microscopy:+Shedding+Light+on+the+Chemistry+of+Vision.">Two-Photon Microscopy: Shedding Light on the Chemistry of Vision.</a> Biochemistry. 46, 9674-9684 (2007).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., 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=Bleached+Pigment+Produces+a+Maintained+Decrease+in+Outer+Segment+Ca2++in+Salamander+Rods.">Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods.</a> J Gen Physiol. 111, 53-64 (1998).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., 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-Dependent+Changes+in+Outer+Segment+Free-Ca2++Concentration+in+Salamander+Cone+Photoreceptors.">Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors.</a> J Gen Physiol. 113, 267-277 (1999).</li><li>Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., 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=Measurement+of+Cytoplasmic+Calcium+Concentration+in+the+Rods+of+Wild-Type+and+Transducin+Knock-out+Mice.">Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice.</a> J Physiol. 542, 843-854 (2002).</li><li>Leung, Y. T., Fain, G. L., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Measurement+of+Current+and+Calcium+in+the+Ultraviolet-Sensitive+Cones+of+Zebrafish.">Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish.</a> J Physiol. 579, 15-27 (2007).</li><li>Matthews, H. R., 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=Laser+Spot+Confocal+Technique+to+Measure+Cytoplasmic+Calcium+Concentration+in+Photoreceptors.">Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors.</a> Methods Enzymol. 316, 146-163 (2000).</li><li>Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Calcium+Stores+Regulates+Calcium+Influx+and+Signal+Transmission+in+Rod+Photoreceptors.">Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors.</a> J Physiol. 586, 4859-4875 (2008).</li><li>Krizaj, D., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+Calcium+Extrusion+Mechanisms+in+the+Outer+and+Inner+Segments+of+Photoreceptors.">Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors.</a> Neuron. 21, 249-256 (1998).</li><li>Chen, C., Nakatani, K., 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=Free+Magnesium+Concentration+in+Salamander+Photoreceptor+Outer+Segments.">Free Magnesium Concentration in Salamander Photoreceptor Outer Segments.</a> J Physiol. 553, 125-135 (2003).</li><li>Chen, C., Jiang, Y., 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=Dynamic+Behavior+of+Rod+Photoreceptor+Disks.">Dynamic Behavior of Rod Photoreceptor Disks.</a> Biophys J. 83, 1403-1412 (2002).</li><li>Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+Microfluorometric+Studies+of+Reduction+and+Clearance+of+Retinal+in+Bleached+Rod+Photoreceptors.">Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors.</a> J Gen Physiol. 124, 429-443 (2004).</li><li>Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle:+Dependence+of+Retinol+Production+and+Removal+on+Photoproduct+Decay+and+Cell+Morphology.">Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology.</a> J Gen Physiol. 128, 153-169 (2006).</li><li>Koutalos, Y., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluorometric+Measurement+of+the+Formation+of+All-Trans-Retinol+in+the+Outer+Segments+of+Single+Isolated+Vertebrate+Photoreceptors.">Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors.</a> Methods Mol Biol. 652, 129-147 (2010).</li><li>Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., 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=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., 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=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

Si las células sanas aisladas no se obtienen, el problema radica ya sea con el aislamiento o la salud de la retina o con su tala. Por lo general, después de retirar la parte frontal del ojo y el vítreo, la retina se despega con facilidad el epitelio pigmentario. Si no es así, trate de despegar a partir de la periferia de la ojera. Si todavía es difícil de separar, una posibilidad probable es que el animal no ha sido adaptada a la oscuridad por un período de tiempo adecuado, o la luz roja es demasiado brillante. G...

<table border="1"><tbody><tr><th> Nombre </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Cuarto oscuro (100-150 m 2) </td><td></td><td></td><td></td></tr><tr><td> Las luces rojas 19 </td><td></td><td></td><td> Tiendas en línea </td></tr><tr><td> Fuentes de luz infrarroja y los espectadores de la imagen infrarroja </td><td> FJW Optical Systems, Inc. </td><td></td><td></td></tr><tr><td> Microscopio de disección 19 </td><td></td><td></td><td> Equipado con los espectadores de infrarrojos </td></tr><tr><td> Microscopio de epifluorescencia encerrada en una jaula a prueba de luz 19 </td><td></td><td></td><td></td></tr><tr><td> Herramientas de disección (tijeras, pinzas, porta-cuchilla) </td><td> Roboz o herramientas de Bellas Ciencia </td><td></td><td></td></tr><tr><td> Sylgard elastómero </td><td> Essex (Charlotte, Carolina del Norte) </td><td> Sylgard 184 elastómero kit </td><td></td></tr><tr><td> Poli-L-ornitina (0,01%) </td><td> Sigma-Aldrich </td><td> P4957 </td><td></td></tr><tr><td> Poli-L-lisina (0,1%) </td><td> Sigma-Aldrich </td><td> P8920 </td><td> Diluir a 0,01% </td></tr><tr><td> Cámaras experimentales </td><td> Warner Instruments (Hamden, CT) </td><td> D3512P </td><td></td></tr><tr><td> Placas de Petri, pipetas de plástico </td><td> Fisher Scientific </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

En la retina de los vertebrados, fototransducción, la conversión de la luz en una señal eléctrica, se lleva a cabo por la barra y conos fotorreceptores 1-4. Fotorreceptores de los bastones son responsables de la visión con poca luz, los conos de luz brillante. Fototransducción tiene lugar en el segmento externo de las células fotorreceptoras, un compartimento especializado que contiene una alta concentración de pigmento visual, el detector de luz principal. El pigmento visual se compone de un cromóforo, 11 - cis retinal, unido a una proteína, la opsina. Un fotón absorbido por el pigmento visual isomeriza el cromóforo de 11 - cis-trans de todos. Este fotoisomerización produce un cambio conformacional en el pigmento visual que inicia una cascada de reacciones que culminaron en un cambio en el potencial de membrana, y provocar la transducción de los estímulos de luz en una señal eléctrica. La recuperación de la célula de la estimulación de la luz consiste en la desactivación de los intermediarios activados por la luz, y el restablecimiento del potencial de membrana. Ca 2 + modula la actividad de varias de las enzimas involucradas en la fototransducción, y su concentración se reduce a la estimulación de luz. De esta manera, Ca 2 + juega un papel importante en la recuperación de la célula de la estimulación de la luz y su adaptación a la luz de fondo. Otra parte esencial del proceso de recuperación es la regeneración del pigmento visual que ha sido destruido durante la luz de la detección por el fotoisomerización de los 11 - cromóforo cis a trans-5.7. Esta regeneración se inicia con la liberación de todo-trans retinal por el pigmento fotoactivado, dejando atrás la opsina apo-proteína. La libertad a todos-trans retinal se reduce rápidamente en una reacción de la utilización de NADPH a todo-trans retinol y opsina se combina con el nuevo 11 - cis retinal trajo en el segmento externo de reforma del pigmento visual. Todo-trans retinol se transfiere fuera del segmento externo y en las células vecinas por la proteína portadora retinoides especializados Interphotoreceptor (IRBP). Imágenes de fluorescencia de las células fotorreceptoras solo puede ser usado para estudiar la fisiología y la biología celular. Ca 2 + sensibles a tintes fluorescentes se pueden utilizar para examinar en detalle la interacción entre los segmentos externos de Ca 2 + y la respuesta a los cambios de luz 08/12, así como el papel de segmento interno Ca 2 + en la homeostasis del Ca 2 + 13,14 . Los tintes fluorescentes también se puede utilizar para medir la concentración de Mg 2 + 15, el pH, y como trazadores de humor acuoso y compartimentos de membrana 16. Finalmente, la fluorescencia intrínseca de todo-trans retinol (vitamina A) puede utilizarse para controlar la cinética de su formación y la eliminación de las células fotorreceptoras solo 17-19.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Se describe un método para la preparación de las células vivas único fotorreceptor de diferentes especies de vertebrados para imágenes de fluorescencia. El método se puede utilizar para la imagen de la fluorescencia de los fluoróforos endógenos, como el NADH o vitamina A, o el de la adición exógena de tintes fluorescentes sensibles a Ca<sup&gt; 2 +</sup&gt; U otros factores.

Preparación de la vida aislada de Vertebrados células visuales para imágenes de fluorescencia

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

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JoVE Journal

Neuroscience

12.2K vistas.  Medical University of South Carolina. Se describe un método para la preparación de las células vivas único fotorreceptor de diferentes especies de vertebrados para imágenes de fluorescencia. El método se puede utilizar para la imagen de la fluorescencia de los fluoróforos endógenos, como el NADH o vitamina A, o el de la adición exógena de tintes fluorescentes sensibles a Ca 2 + U otros factores.

Video: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparación aislada de retina para registrar la respuesta de la luz a partir de semillas etiquetadas las células ganglionares retinianas

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Investigación

Neurociencias

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

La detección de la hipoxia Microrregional en corteza cerebral de ratón por la imagen de dos fotones de fluorescencia endógena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparación de agudos Slices zona subventricular for Imaging Calcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualización de la Dinámica en Endosoma Vivir terminaciones nerviosas con imágenes de fluorescencia en cuatro dimensiones

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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

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

Vibratome Seccionamiento retina del ratón para preparar cultivos de fotorreceptores

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

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

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

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

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

Imagen Ca<sup&gt; 2 +</sup&gt; Dinámica de Terminales cono fotorreceptor axón de la retina del ratón

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

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

Aislamiento confiable de microvasculares del sistema nervioso central en cinco grupos de vertebrados

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Espectrometría de masas de intercambio de hidrógeno/deuterio de milisegundos para el estudio de la dinámica estructural de la alfa-sinucleína en condiciones fisiológicas

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...