This work was supported by the Deutsche Forschungsgemeinschaft DFG FOR701 and FE464/11-1 to A.F. The authors want to acknowledge the excellent technical assistance of Andrea Nerz.

Todos los procedimientos se llevaron a cabo de acuerdo con las directrices para los experimentos con animales emitidos por la República Federal de Alemania. NOTA: Debido a que el tejido de la retina esté desprovisto de oxígeno durante prolongados partes de este procedimiento, todos los pasos que deben llevarse a cabo lo más rápido posible. Los ratones deben ser adaptados a la oscuridad, al menos 3 horas antes de la disección. Para evitar la adaptación a la luz de la retina, la preparación y el corte debe ser llevado a cabo bajo la luz roja tenue. 1. Preparación de los medios de comunicación y otros productos químicos <ol><li> Preparar Ames &#39;Medium 6. Después de la disección, sumergir tejido de la retina en el medio de Ames &#39;en todo momento. <ol><li> Disolver 2,2 g de Ames medio y 298 mg en HEPES 250 ml de agua destilada. La concentración de HEPES será de 5 mM. Agitar suavemente hasta que se disuelven todas las partículas. </li><li> Medio burbuja Ames &#39;a temperatura ambiente con carbógeno (95% de oxígeno, 5% cochedióxido de Bon) durante al menos 5 min. carbógeno de alimentación con una pipeta Pasteur de vidrio a la solución. Conectar la pipeta Pasteur través de un tubo apropiado a la salida del regulador de gas. </li><li> Añadir 475 mg de sodio-bicarbonato a la solución. La adición de bicarbonato después de burbujeo evita la precipitación de carbonato de calcio. </li></ol></li><li> Preparar agarosa de baja temperatura de gelificación. Se requiere de agarosa para incrustar de tejido de la retina durante el corte vibratome. <ol><li> Disolver 180 mg de agarosa en 9 ml de medio de Ames y 1 ml de agua destilada. Utilizar un microondas para calentar lentamente la solución de agarosa hasta que se disolvieron todos los sólidos. El calor dentro de 5 - 10 seg incrementos para evitar el desbordamiento. Solución debe ser clara y transparente. </li><li> Tienda disolvió agarosa en baño de agua a 37 ° C durante la duración del procedimiento. </li></ol></li></ol> 2. Configuración de la Vibratome NOTA: Todas seccionamiento se ha llevado a cabo con lavibratome enumerados en la Tabla de Materiales. Los ajustes indicados se refieren a este tipo de solamente vibratome. Para más detalles sobre vibratome seccionamiento de tejido neural por favor refiérase a la referencia 7. <ol><li> Coloque la cuchilla vibratome inyector en soporte de la cuchilla. </li><li> Medir la desviación vertical de la hoja con el dispositivo &quot;VibroCheck&quot;. Utilice el tornillo de ajuste en el soporte de la cuchilla para inclinar la pala hasta que quede paralela al plano de corte. La posición exacta de la hoja es necesaria para evitar efectos de persianas venecianas en la superficie de sección y para producir una sección plana que contiene células vivas en su superficie. </li><li> Establecer vibratome a los siguientes parámetros: amplitud, 1,00; velocidad, 0,20; espesor, 180-200 micras. </li></ol> 3. La disección de la retina del ratón <ol><li> Anestesiar a adultos C57BL / 6 ratones por vaporización de aproximadamente 1 ml de isoflurano en un recipiente hermético. Cuando la anestesia es completa, la eutanasia a los animales por dislocación cervical. losratón no debe ser mayor de 3 meses, de lo contrario la calidad de las rodajas se deteriorará. </li><li> Retire la bola del ojo mediante la colocación de una pinza curva debajo del ojo. levantando suavemente la bola del ojo expondrá el nervio óptico. Cortar el nervio óptico con una tijera primavera y transferir la bola del ojo a una placa de Petri de 35 mm que contiene 2 ml de Ames Medium. </li><li> Llevar a cabo todos los pasos subsiguientes en el escenario de un microscopio estereoscópico. Ajuste el aumento a un valor que permite un control visual óptimo de los siguientes procedimientos (pasos 3.4 a 3.9). </li><li> Perforar la córnea en la transición a la esclerótica con unas pinzas de punta o una aguja 20 G. Durante este paso, estabilizar el globo ocular con otros fórceps. Tenga cuidado de no dañar la retina con el dispositivo de punción. </li><li> Inserte las tijeras de primavera en el pequeño orificio realizado en el paso 3.3 y cortar un círculo alrededor de la córnea. Una vez más, estabilizar la bola del ojo con pinzas durante el corte. Mantenga la incisión muy cerca de la superficie a fin de no cut en la retina. </li><li> Retire cuidadosamente la córnea, el cristalino y el cuerpo vítreo con una pinza. El cuerpo vítreo comprende sólo una fina capa transparente en la parte posterior del ojo. Sin embargo, es esencial para vibratome seccionamiento que el cuerpo vítreo es completamente eliminado. </li><li> En la placa de Petri, la posición de la copa ocular de una manera que se mira a través del agujero hacia abajo sobre la retina. El borde de la abertura debe consistir solamente tejido de la esclerótica. Si el corte circular (paso 3.4) se ha llevado a cabo demasiado cerca del ecuador de la copa ocular, tejido de la retina también estará presente cerca de la incisión. Tenga cuidado de no dañar a continuación, la retina en el paso 3.8. </li><li> Coge la esclerótica con unas pinzas y romper cuidadosamente el tejido de separación. Es esencial no se rompa la retina durante este paso. Si es necesario, separar la retina de la ora serrata estabilizando la parte escleral del tejido con las pinzas y con cuidado despegando de la retina con las otras pinzas. Mantener las pinzas cerradas; Nunca manejar la retina con pinzas ya que el tejido es muy suave y dará paso inmediatamente. NOTA: En este paso, la retina y el resto de la copa ocular se conecta sólo a la cabeza del nervio óptico. </li><li> Cortar el sitio de unión en la cabeza del nervio óptico con las tijeras de primavera. La retina ahora debe estar flotando libremente como una sola pieza de tejido en el medio de Ames. </li><li> Cortar la retina en 4 - 6 pequeños rectángulos de aproximadamente 23 mm utilizando una hoja de bisturí curvo mediante la celebración de la retina a lo largo de los bordes con el fórceps, para manipularlo en su posición. Evitar pellizcar el tejido de la retina dentro de los rectángulos. Tome las piezas de la parte central de la retina, no muy lejos de la periferia. </li><li> Transferencia de piezas de la retina con un diámetro grande pipeta de plástico Pasteur a una placa petri de plástico 35 mm, el fondo de la cual ha sido sustituida por una malla con tamaño de malla de menos de 1 mm. Fijar la placa de Petri en el tercio superior de un matraz de vaso de precipitados o cónica y sumergir piezas de la retina en bu Medium Ames &#39;bbled con Carbógeno. </li></ol> 4. Incorporación de la Retina Piezas de agarosa <ol><li> transferir cuidadosamente 2 - 3 piezas de la retina en una placa Petri de plástico de 35 mm junto con algunos de los Ames &#39;Medium. Es más conveniente usar un plástico de gran diámetro pipeta Pasteur para la transferencia. En este paso, la orientación exacta de las piezas de la retina no importa. </li><li> Eliminar la mayor cantidad posible de Medio el restante Ames &#39;con una pipeta Pasteur de vidrio. Tan pronto como el fluido ha sido retirado continúe con el paso 4.3. No deje que la retina seco del tejido. </li><li> Inmediatamente cubrir piezas de la retina con 2 ml de agarosa disuelto (37 ° C). Dado el tamaño mencionado en el paso 3.10, las piezas no se cerrarán hasta dentro de la agarosa. </li><li> Dentro de la agarosa líquido, empujar suavemente piezas de la retina a la parte inferior de la placa de Petri. La capa de células ganglionares debe estar boca abajo. Use un microscopio estereoscópico para la determinación de la posición exacta. Para el posicionamiento, manejar pi retinaeces con una pequeña espátula. Coloque la retina tan rápido y preciso como sea posible ya que la agarosa se solidifica rápidamente. <ol><li> Organizar piezas retinianas paralelos al fondo de la placa de vidrio; de lo contrario secciones serán tangencial u oblicua. Las piezas individuales no deben tocarse entre sí, sino que además no deben estar demasiado separados. Trate de colocar las piezas de la retina más o menos en el mismo nivel horizontal. </li></ol></li><li> Ponga el vidrio placa de Petri con piezas de retina incrustados en el refrigerador (4 - 6 ° C) durante al menos 2 minutos. Esto se endurecerá rápidamente la agarosa para su posterior procesamiento. Tan pronto como la agarosa ha polimerizado proceder con el paso siguiente. </li></ol> 5. Montaje de la Retina Piezas <ol><li> Retirar con cuidado el bloque de agarosa que contiene piezas de la retina de la placa de Petri. Utilice una espátula o un dispositivo similar para levantar el bloque de agarosa del vidrio. Debido a las fuerzas de adhesión, esto puede llegar a ser bastante difícil. Sin embargo, no romper labloque de agarosa. Llevar a cabo esta y todas las etapas posteriores a la temperatura ambiente. </li><li> Coloque el bloque de agarosa sobre un cubreobjetos de vidrio y ajustar su tamaño con una hoja de bisturí. Deja de 1 - 2 mm de agarosa en cada lado de las piezas de la retina. El tamaño final del bloque debe ser alrededor de 1,066 mm (10 mm se refiere a la altura del bloque, 6 mm a la longitud de la arista). </li><li> Pegue el bloque de agarosa para el soporte de la muestra de la vibratome utilizando un adhesivo instantáneo. piezas de retina se debe colocar en la parte superior de la alta bloque de 10 mm y paralelos orientados a su superficie, mientras que la parte inferior está fijada en el soporte. </li><li> Cubrir el bloque inmediatamente vertiendo el Medio Ames previamente burbujeado &#39;en la bandeja de amortiguamiento de la vibratome. </li></ol> 6. Preparación de Secciones Vibratome Horizontal <ol><li> Empezar a cortar. En primer lugar, establecer el grosor de cortar manualmente hasta fragmentos de tejido de la retina aparecen en el corte. A continuación, cambie a la configuración en el paso 2.3. Dado que laretina del ratón es de sólo 200 m de espesor, habrá no más de 2-3 rebanadas por bloque de agarosa. </li><li> Retire las secciones de la bandeja de tampón con una varilla de vidrio y transferirlos a 2 ml de medio de Ames &#39;en una placa de Petri de 35 mm. </li><li> Secciones de las tiendas inmediatamente en una incubadora a 37 ° C (5% CO2 / 55% O2). La agarosa que rodea las secciones permanecerá sólido en la incubadora y puede ser usado para manipular las secciones sin dañar la retina. se obtiene una mejor calidad de la rebanada al almacenar a 37 ° C dentro de la atmósfera respectivo en comparación con Medio oxigenada Ames &#39;a TA. Las secciones se pueden utilizar inmediatamente o se pueden almacenar hasta 4 horas en la incubadora con buenos resultados fisiológicos. </li><li> Incrustar siguientes piezas de la retina en agarosa y repetir las Secciones 4 - 6. </li></ol>

<ol>
	<li>DeVries, S. H., Li, W., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+two+transmitter+microenvironments+at+the+cone+photoreceptor+synapse.">Parallel processing in two transmitter microenvironments at the cone photoreceptor synapse.</a> Neuron. 50, 735-748 (2006).</li><li>Jackman, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+synaptic+ribbon+in+transmitting+the+cone+light+response.">Role of the synaptic ribbon in transmitting the cone light response.</a> Nature Neuroscience. 12 (3), 303-310 (2009).</li><li>Rabl, K., Cadetti, L., Thoreson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+exocytosis+is+faster+in+cones+than+in+rods.">Kinetics of exocytosis is faster in cones than in rods.</a> J. Neurosci. 25 (18), 4633-4640 (2005).</li><li>Singer, J. H., Lassov&#225;, L., Vardi, N., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+multivesicular+release+at+a+mammalian+ribbon+synapse.">Coordinated multivesicular release at a mammalian ribbon synapse.</a> Nature Neuroscience. 7 (8), 826-833 (2004).</li><li>Peichl, L., Gonz&#225;lez-Soriano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+types+of+horizontal+cell+in+rodent+retinae:+a+comparison+of+rat,+mouse,+gerbil,+and+guinea+pig.">Morphological types of horizontal cell in rodent retinae: a comparison of rat, mouse, gerbil, and guinea pig.</a> Vis. Neurosci. 11, 501-517 (1994).</li><li>Ames, A., Nesbett, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+retina+as+an+experimental+model+of+the+central+nervous+system.">In vitro retina as an experimental model of the central nervous system.</a> J. Neurochem. 37 (4), 867-877 (1981).</li><li>Mathis, D. M., Furman, J. J., Norris, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+acute+hippocampal+slices+from+rats+and+transgenic+mice+for+the+study+of+synaptic+alterations+during+aging+and+amyloid+pathology.">Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology.</a> J. Vis. Exp. (49), (2011).</li><li>Peichl, L., Sandmann, D., Boycott, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+and+function+of+mammalian+horizontal+cells.">Comparative anatomy and function of mammalian horizontal cells.</a> Development of the Retina. , (1998).</li><li>Feigenspan, A., Babai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+spontaneous+excitatory+currents+and+encoding+of+light/dark+transitions+in+horizontal+cells+of+the+mouse+retina.">Functional properties of spontaneous excitatory currents and encoding of light/dark transitions in horizontal cells of the mouse retina.</a> Eur. J. Neurosci. 42 (9), 2615-2632 (2015).</li><li>Enoki, R., Jakobs, T. C., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+slice+preparation+of+the+retina.">Horizontal slice preparation of the retina.</a> J. Vis. Exp. (1), e108 (2006).</li><li>Babai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+complexin3+and+complexin4+at+mouse+photoreceptor+ribbon+synapses.">Functional roles of complexin3 and complexin4 at mouse photoreceptor ribbon synapses.</a> J. Neurosci. 36, 6651-6667 (2016).</li><li>Thoreson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+slices+of+mouse+retina+expose+horizontal+cells+and+their+properties+(Commentary+on+Feigenspan+&amp;+Babai).">Horizontal slices of mouse retina expose horizontal cells and their properties (Commentary on Feigenspan &amp; Babai).</a> Eur. J. Neurosci. 42 (9), 2613-2614 (2015).</li></ol>

The authors have nothing to disclose.

En contraste con las rebanadas verticales, la morfología dendrítica de las neuronas orientados radialmente, como las células horizontales y amacrinas está bien conservado. En comparación con las preparaciones de retina enteros, estos tipos de células son fácilmente accesibles para los microelectrodos y pueden ser estudiados por ambas técnicas electrofisiológicas y de imagen. Aquí, nos centramos en la retina externa; Sin embargo, un enfoque similar se ha informado sobre todo para las células amacrinas de la re...

La información visual se codifica como un patrón espacio-temporal dinámica de la activación de los fotorreceptores, y la sinapsis cinta entre los fotorreceptores y neuronas de segundo orden determina la transferencia de aguas abajo de la información visual. La preparación rebanada vertical de la retina de mamífero es una herramienta muy valiosa para estudiar el procesamiento de señales en las vías verticales, es decir, entre los fotorreceptores y células bipolares, y entre las células bipolares y algunos tipos de células amacrinas 1, 2, 3, 4. Sin embargo, seccionamiento verticales siempre provoca el truncamiento grave de los campos dendríticas de muchas neuronas de la retina, lo que lleva a una pérdida considerable de los contactos sinápticos. Especialmente en la retina externa, las células horizontales se ven afectados debido a sus campos dendríticas extendidos lateralmente extendidas y sus sistemas de terminales del axón 5. En una línea verticalrodaja de preparación, la entrada sináptica de cientos de terminales de cono fotorreceptores a las dendritas de una célula horizontal se reduce a unos pocos contactos dispersos. Esto podría ser suficiente para estudiar las propiedades de las sinapsis individuales, pero lo hace de ninguna manera representan las capacidades de procesamiento de señal que subyacen a la activación sincronizada de las sinapsis cinta fotorreceptor. Por lo tanto, hemos desarrollado una preparación rebanada horizontal, lo que deja casi todo el fotorreceptor cono de entrada sináptica a las dendritas de células horizontales intacto y funcional. El plano de carreras de seccionado paralelo a la superficie de la retina, idealmente corte a través de la capa nuclear interna entre las células horizontales y células amacrinas. Por lo tanto, las rebanadas horizontales de la retina externa son creados que contienen, en el sitio presináptico, fotorreceptores con segmentos interior y exterior, así como terminales sinápticas, y células horizontales y células bipolares en el sitio postsináptica. Esta preparación es muy adecuado to investigar las entradas sinápticas coordinadas en las dendritas de células horizontales por todos los conos dentro de su campo dendríticas. Por lo tanto, podría resultar útil para comprender mejor el funcionamiento de la sinapsis cinta fotorreceptor, que es crucial para la transferencia de la información visual de la matriz de la activación de los fotorreceptores en una respuesta postsináptica.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; Medium</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A1420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbogen</td>
			<td style="width:125px;">Air Liquide</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A9045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">35-mm petri dish (plastic)</td>
			<td style="width:125px;">Nunc Thermofisher</td>
			<td style="width:119px;">150318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass petri dish</td>
			<td style="width:125px;">Chemoline</td>
			<td style="width:119px;">50-1470-40</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Isoflurane</td>
			<td style="width:125px;">Dispensary university hospital</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome injector blade</td>
			<td style="width:125px;">Biosystems</td>
			<td style="width:119px;">39053250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibratome</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;">VT1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrocheck</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microwave</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20-gauge needles</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spring scissors</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved scalpel blades</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of horizontal slices of adult mouse retina for electrophysiological studies

el tramo preparativos verticales están bien establecidos para estudiar los circuitos y la transmisión de la señal en la retina de mamíferos adultos. El plano de seccionamiento en estas preparaciones es perpendicular a la superficie de la retina, lo que es ideal para el estudio de las neuronas orientados radialmente como fotorreceptores y células bipolares. Sin embargo, las grandes cenadores dendríticas de las células horizontales, células amacrinas de campo amplio, y las células ganglionares son en su mayoría truncado, dejando marcadamente reducida la actividad sináptica en estas células. Mientras que las células ganglionares y células amacrinas desplazadas se pueden estudiar en una preparación de todo ello montado de la retina, células horizontales y células amacrinas ubicadas en la capa nuclear interna sólo son de difícil acceso para los electrodos en el tejido retina conjunto. Para lograr la máxima accesibilidad y la integridad sináptica, hemos desarrollado una rodaja de preparación horizontal de la retina del ratón, y estudiamos la transmisión de señales en la sinapsis entre los fotorreceptores y horizontalCélulas. seccionamiento horizontal permite (1) la identificación visual fácil e inequívoca de los cuerpos celulares horizontales para la orientación del electrodo, y (2) la preservación de los campos dendríticas de células horizontales extendidos, como requisito previo para el cono de entrada sináptica intacta y funcional a las dendritas de células horizontales. Las células horizontales de los cortes horizontales exhibieron actividad sináptica tónica en la oscuridad, y ellos respondieron a breves destellos de luz con una reducción de la actividad sináptica actual y la disminución hacia el interior. pruebas de inmuno indica que casi todos los conos dentro del campo de una célula dendrítica horizontal establecen sinapsis con las dendritas sus periféricos. La preparación rebanada horizontal es por lo tanto muy adecuado para estudiar las propiedades fisiológicas de las neuronas de la retina horizontalmente extendidos, así como la transmisión de la señal sensorial y la integración a través de las sinapsis seleccionados.

<html> En rebanadas horizontales de la retina del ratón, los cuerpos celulares horizontales podrían ser identificados fácilmente de acuerdo con su morfología única con varias dendritas primarias que salen de un cuerpo de células poligonales (Figura 1A). cuerpos celulares horizontales situados cerca de la superficie de la rebanada fueron rutinariamente accesible para los electrodos de patch-clamp. Durante las grabaciones, las células se llenaron con un colorante flu...

Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Hemos desarrollado una preparación rebanada horizontal de la retina de mamíferos adultos con el plano de seccionamiento en paralelo a la superficie de la retina. campos dendríticas de las neuronas de la retina orientadas nonradially no fueron truncados, lo que permite el estudio de las técnicas de procesamiento de señales con patch-clamp y de imagen.

Preparación de cortes horizontales de ratón adulto Retina de Estudios electrofisiológicos

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

preparation-horizontal-slices-adult-mouse-retina-for

Research

JoVE Journal

Neuroscience

10.0K vistas.  Friedrich-Alexander-University (FAU) Erlangen-Nürnberg.  Hemos desarrollado una preparación rebanada horizontal de la retina de mamíferos adultos con el plano de seccionamiento en paralelo a la superficie de la retina. campos dendríticas de las neuronas de la retina orientadas nonradially no fueron truncados, lo que permite el estudio de las técnicas de procesamiento de señales con patch-clamp y de imagen. 

Video: Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

Cultura organotípicos de espesor completo Retina adulto porcino

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Investigación

Neurociencias

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Time-lapse de imágenes de Migración neuroblasto en rodajas agudas del cerebro anterior ratón adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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 ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Mejora de la preparación y conservación de las rebanadas de hipocampo de un ratón de grabación muy estable y reproducible de la potenciación a largo plazo

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch Clamp grabación de células amacrinas Starburst en un plano de montaje Preparación de Deafferentated retina del ratón

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Preparación de mezcla de culturas gliales primarios de ratón adulto espinal tejido del cordón

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

Rebanadas de cerebro-pituitaria sanas para las investigaciones electrofisiológicas de las células hipofisarias en peces teleósteos

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...