Este trabajo fue financiado por becas de los Institutos Nacionales de Salud: EY015748, EY017964 (MEZ), y EY019517 (ASV). Este trabajo también fue apoyado por la investigación para prevenir la ceguera sin restricciones de subvención al Departamento de Oftalmología y los Leones de la central de Nueva York. También nos gustaría dar las gracias a nuestro técnico en animales, Matthew Mellini, por su excelente cuidado de los animales y por intervenir para protagonizar un video.

Cuidado de los Animales La Xenopus laevis renacuajos utilizadas en este estudio fueron cultivados, criados y tratados de acuerdo con los procedimientos aprobados por el Upstate Medical University IACUC y la Guía para el Cuidado y Uso de Animales de Laboratorio. . 1 Configuración Pre-comportamiento: Los renacuajos <ol><li> Obtener embriones de Xenopus fertilizados de una fuente disponible comercialmente o in vitro fertilizar oocitos, como se describió anteriormente 6. </li><li> Coloque blástula a embriones en estadio de la placa neural a 18 ˚ C en 60 mm placas de Petri que contienen 0.1X MMR [10 Modificado solución (MMR) de las acciones de Ringer X Marc (10 mM MgCl 2, 20 mM KCl, 20 mM CaCl 2, 50 mM HEPES; 1 M de NaCl; se ajustó a pH 7,5, sometido a autoclave y se almacenó a temperatura ambiente)] con antibiótico gentamicina 50 mg / ml. Cambie 0.1X MMR cada día y quitar cualquier embriones muertos. </li><li> Una vez que los embriones alcanzan el estadio Nieuwkoop y Faber ~ 27 al 30 julio, cambiar the embriones en 0.1X MMR sin antibiótico para permitir el crecimiento bacteriano intestinal. </li><li> Crecer los renacuajos hasta que alcanzan la etapa de alimentación (etapa 45) y moverlos a 100 mm placas de Petri llenas de 0.1X MMR. Aliméntelos sobrenadante polvo de ortiga cada dos días durante una semana, el cambio de la 0,1 X MMR en el día intermedio. </li><li> Después de una semana en el plato de Petri de 100 mm, desplazar a los animales a medio galón tanques llenos de agua rana [0,5 g / L océano instantánea y 2 mM de sodio fosfato dibásico (Na 2 HPO 4: mw 141.96) pH 6,8]. Darles de comer como en el paso 2.4, pero cambie el agua sólo 2-3 veces por semana, cuando el agua está turbia. Mantener a los animales en un ciclo de luz-oscuridad de 12 horas light/12 con las luces de encender a las 6 am. </li><li> Coloque el depósito de medio galón con los sujetos de prueba en una superficie blanca, como un underpad mesa de laboratorio, por lo menos 12 hr O / N, antes de la prueba. </li><li> Renacuajos de prueba en las etapas 45 y 50 tal como se describe en el paso 3 a continuación. </li></ol> . 2 Configuración Pre-comportamiento: Equipot <ol><li> Ponga a un lado un área de laboratorio para el ensayo de comportamiento. Debe estar en una zona tranquila y con poco tráfico con iluminación fluorescente estándar. </li><li> Preparar los bancos de ensayo para los animales. Los tanques de prueba de medio galón anidados se componen de dos partes: un tanque interno que contendrá el agua y renacuajos; y un tanque exterior con el estímulo visual. <ol><li> Para el interior del tanque, hacer una pequeña marca en las esquinas exteriores con un Sharpie, 5 cm desde la parte inferior; esta es la línea de agua (Figura 1A, línea punteada verde). </li><li> También, para el tanque interior, llena los divets en el tanque (en las esquinas y el centro) con un compuesto inerte, como Sylgard elastómero. NOTA: Los renacuajos se quedarán en estas zonas si no se llenan pulg </li><li> Para el tanque exterior, cubra exactamente la mitad de la parte exterior de un tanque con cinta aislante color negro y la otra mitad con el tejido blanco de papel Figura 1B. </li></ol></li><li> Coloque los tanques en ella inoculación de la placa giratoria. </li><li> Configuración de la cámara web / trípode de modo que está por encima de los bancos de ensayo. Ajustar la cámara para permitir la visualización de la zona de pruebas, como se muestra en la figura 1C. </li><li> Conecte la cámara web a un ordenador con el software QuickTime Player instalado 5. Encienda la cámara. En QuickTime Player, en Archivo, seleccione &#39;Nueva grabación de la película. &quot; La configuración del ensayo de comportamiento será visible en el equipo de inmediato. </li><li> Coloque un paño de algodón ligero en toda la instalación para reducir las señales externas que podrían afectar el comportamiento de renacuajo, así como, reducir la luz reflejada en la superficie del agua. </li><li> Asegúrese de que la luminancia bajo las medidas de tela entre 35-50 cd / m 2. </li></ol> 3. Ensayo de Comportamiento <ol><li> Llene el tanque de pruebas interior para la marca de 5 cm de agua realizada en el paso 1.2.1 con agua rana. </li><li> El uso de una pequeña red, mover suavemente al animal en el tanque de pruebas interno. </li><li> Abrir QuickTime software en la computadora y, usando el tanque exterior como guía, asegúrese de que la cámara es capaz de visualizar y grabar la zona de pruebas. </li><li> Escriba el nombre de un animal, la fecha y la hora en un pedazo de papel y grabar con la cámara. </li><li> Coloque el tanque de prueba en el tanque exterior con el lado negro de la cisterna a la derecha Figura 2. </li><li> Comience a grabar la película inmediatamente después de arreglar el tanque y ajustar el temporizador durante 2 min. </li><li> Cuando el temporizador de pitidos, retire el tanque de prueba interno, gire el depósito exterior 180 ° y coloque el tanque de prueba interna en el interior del tanque externo. Inicie el temporizador. Nota: el lado negro debe estar ahora en el otro lado. </li><li> Repita el paso 3.7, ocho veces más para un total de diez pruebas. Utilice la Figura 2 como guía, marcando cada ensayo. </li><li> Repita el ensayo de comportamiento en dos días separados. </li></ol> 4. Retina Axotomía <ol><li> Coloque animal en 0.02% tricaína hasta que no respondena una pizca de la cola con un # 3 pinzas. </li><li> Derretir 1% de agarosa en 0.1X MMR y luego añadir a un 60 mm placa de Petri. Una vez enfriado, hacer una pequeña chuleta rectangular en la agarosa y añadir el renacuajo a la chuleta con un poco de 0,02% tricaína, de modo que el animal está parcialmente sumergido en el líquido. </li><li> Pierce la piel en un ángulo de 45 grados detrás de la región dorsal del ojo con una aguja de 25 G, mientras sostiene el animal contra el fórceps sobre el lado opuesto. </li><li> Teniendo cuidado de no cortar la vena, que se encuentra al lado del nervio óptico, el alcance cuidadosamente en el agujero por el n º 5 de fórceps, cortar el nervio óptico y darle la vuelta a un lado. Si hay sangrado grave por accidente cortando la arteria, luego coloque rápidamente al animal en un 2% tricaína la eutanasia. PRECAUCIÓN: solución tricaína 2% puede causar entumecimiento en los seres humanos. Esta solución debe ser manejado con guantes. </li><li> Para recuperar el animal después de la cirugía, colocar animal en 100 mm de placa de Petri con 0.7X MMR y 50 mg / ml de gentamicina durante 20 min. Next, transferencia de renacuajo a depósito de recuperación con agua rana. Deje reposar animales O / N. </li><li> A la mañana siguiente, probar la función visual como se describió anteriormente (pasos 3.1 a 3.9). </li></ol> 5. Analizar Resultados <ol><li> Después de que se completen los ensayos, medir la cantidad de tiempo que el renacuajo se queda en el lado negro de la cisterna. <ol><li> Ver manual de los vídeos mediante un software que muestra la hora y permite pausas frecuentes. Mira el tiempo de visualización de vídeo. Anote el inicio de la prueba de dos minutos, que es cuando el tanque interior anida con éxito dentro del tanque exterior y se eleva al cuadrado en vista de la cámara. </li><li> Use la posición de los ojos del renacuajo de definir de qué lado del tanque de los animales están nadando en. Definir cruzar de un lado del tanque al otro sólo cuando ambos ojos han cruzado la línea de negro / blanco. </li></ol></li><li> Anote el comienzo y el final de cada intervalo (en segundos) que el animal pasa en el lado negro. Haga una pausa y volverenrollar el video para asegurar la exactitud. Sume el sec dentro de cada ensayo. </li><li> Entrada estos números en la hoja de trabajo Comportamiento adjunto. La hoja de trabajo calcula la relación entre el tiempo que el animal pasó por el lado blanco, restando el tiempo invertido en el lado negro del tanque de la prueba total de 120 segundos y se divide por el tiempo total en segundos [(120 - total gastado en segundos el lado negro) / 120 seg]. Los promedios de hoja de cálculo de este ratio de más de los 10 ensayos. </li><li> Utilice la prueba T de un estudiante, a la par, dos colas de distribución para determinar si los juicios entre los días son significativas (P ≤ 0,05). </li></ol>

<ol>
	<li>Maurer, C. M., Huang, Y. Y., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zebrafish+oculomotor+behavior+to+model+human+disorders.">Application of zebrafish oculomotor behavior to model human disorders.</a> Rev Neurosci. 22 (1), 5-16 (2011).</li><li>Solessio, E., Scheraga, D., Engbretson, G. A., Knox, B. E., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+temporal+properties+of+the+rod+pathway+in+larval+Xenopus.">Circadian modulation of temporal properties of the rod pathway in larval Xenopus.</a> J Neurophysiol. 92 (5), 2672-2684 (2004).</li><li>Moriya, T., Kito, K., Miyashita, Y., Asami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+background+color+of+the+Xenopus+laevis+tadpole.">Preference for background color of the Xenopus laevis tadpole.</a> J Exp Zool. 276 (5), 335-344 (1996).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7 (8), (2009).</li><li>Choi, R. Y., 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=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 52 (1), 364-373 (2011).</li><li>Viczian, A. S., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+determination+using+the+animal+cap+transplant+(ACT)+assay+in+Xenopus+laevis.">Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.</a> J Vis Exp. 16 (39), (2010).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second-generation+device+for+automated+training+and+quantitative+behavior+analyses+of+molecularly-tractable+model+organisms.">A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.</a> PLoS One. 5 (12), (2010).</li><li>Rosemberg, D. B., 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=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS One. 6 (5), (2011).</li><li>Dong, W., 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=Visual+Avoidance+in+Xenopus+Tadpoles+is+Correlated+With+the+Maturation+of+Visual+Responses+in+the+Optic+Tectum.">Visual Avoidance in Xenopus Tadpoles is Correlated With the Maturation of Visual Responses in the Optic Tectum.</a> J Neurophysiol. 101 (2), 803-815 (2009).</li><li>Lan, 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=Noggin+Elicits+Retinal+Fate+In+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate In Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. 27 (9), 2146-2152 (2009).</li><li>De Robertis, E. M., Kuroda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+and+neural+induction+in+Xenopus+embryos.">Dorsal-ventral patterning and neural induction in Xenopus embryos.</a> Annu Rev Cell Dev Biol. 20, 285-308 (2004).</li></ol>

The authors have nothing to disclose.

Se presenta aquí un sencillo ensayo de comportamiento guiado por visión que puede ser fácilmente realizado en menos de una semana por personal de laboratorio mínimamente entrenados. Mientras otros ensayos requieren equipo especializado y la experiencia en el comportamiento animal, este ensayo permite una prueba rápida para determinar la función visual. Otro ensayo de comportamiento, el ensayo de evitación de Visual, se ha desarrollado para determinar cómo el tectum contribuye a la percepción visual en Xenop...

Xenopus laevis se han utilizado como un organismo modelo para estudiar la formación del ojo. Los ojos se desarrollan rápidamente, creciendo hacia la madurez en menos de una semana para los genes de prueba o vías que tienen un efecto sobre el desarrollo visual y la función. Para probar la función visual, la respuesta optocinético y optomotor se ha utilizado en el pez cebra y los renacuajos Xenopus, respectivamente 1,2. Debido a que los ojos de los renacuajos Xenopus son relativamente más pequeño que el pez cebra, este ensayo requiere el uso de equipo especializado y personal entrenado para detectar el comportamiento sutil cola-flip y de movimiento ocular en Xenopus. Un comportamiento más robusto en Xenopus es la preferencia para nadar en un tanque con un fondo blanco, que se describe en el presente documento 3. Al colocar un renacuajo en un medio tanque negro / mitad blanco, el renacuajo pre-metamórfica nada rápidamente hacia el lado blanco del tanque. Se utilizó previamente este ensayo para determinar si derivado de células pluripotentesojos eran funcionales 4. Aquí, se presenta una versión detallada de este ensayo, que se puede utilizar para probar la función visual de los renacuajos de Xenopus premetamórficas. Este ensayo es más sencillo que el ensayo de respuesta optomotor, ya que sólo requiere una cámara de vídeo montada digitales, software de la cámara digital y el equipo estándar en la mayoría de los laboratorios de Xenopus. Además, la respuesta registrada no requiere una formación especial a los resultados de conteo. Nuestros resultados representativos muestran que el mismo grupo de renacuajos, después de haber sido sometidos a axotomía de la retina doble, nadar al azar alrededor del tanque. También hemos incluido los resultados del ensayo de comportamiento de un renacuajo representante, mostrando como uno de los ojos puede ser probado para la respuesta visual. Una hoja de cálculo se ha incluido para que los números adquiridos durante el ensayo pueden ser insertados y se analizaron. Esta hoja de trabajo se puede utilizar para determinar si los renacuajos probados tienen una respuesta visual.

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			<td height="21" style="height:21px;width:216px;">1/2-gal. Flex-Tank with Cover</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">SB19271M</td>
			<td style="width:225px;">size: 5-3/8&#34; x 7&#34; x 3-3/4&#34;</td>
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			<td height="21" style="height:21px;width:216px;">Black electrical tape</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">White tissue paper</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large Inoculating Turntable</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">50809-022</td>
			<td>size: Dia 114.3 x&#160; H 76.2 mm (4 1/2&#160; x&#160; 3&#34;)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Durasorb Underpad</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">82004-836</td>
			<td>size: 43.2 x 60.1 cm (17 x 24&#34;)</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Kim wipe</td>
			<td style="width:95px;">Krackeler Scientific, Inc.</td>
			<td style="width:119px;">1945-34155-CS</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Tripod</td>
			<td style="width:95px;"></td>
			<td style="width:119px;">various</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iSight camera or webcam</td>
			<td style="width:95px;">Apple</td>
			<td style="width:119px;">M8817LL/A</td>
			<td>Good for larger tadpoles but small ones are difficult to see</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable computer</td>
			<td style="width:95px;">Apple/PC</td>
			<td style="width:119px;">various</td>
			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MiniDV Handycam Camcorder</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCR-HC42</td>
			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Handycam Station</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCRA-C121</td>
			<td>This can be used for connecting firewire to camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuickTime Player software</td>
			<td style="width:95px;">Quicktime</td>
			<td style="width:119px;">Version 10.1</td>
			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">26 Gauge Needle (5/8&#34; length)</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">BD305115</td>
			<td></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Dumont #5 Forceps Biology</td>
			<td style="width:95px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
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			<td height="24" style="height:24px;width:216px;">Disposables</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamicin Sulfate [50 mg/ml]</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">17-528Z</td>
			<td>Stored at room temperature</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sylgard 184 Silicone Elastomer</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Instant Ocean</td>
			<td style="width:95px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td>Stock solution = 100 g/L stored at room temperature</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">S0876</td>
			<td>Stock solution = 0.4 M stored at room temperature</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tricaine (Ethyl-3-aminobenzoate methanesulfonate salt)</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td>Stock solution = 10% stored at -20&#730;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">LM00490MX</td>
			<td>100 embryos/unit</td>
		</tr>
	</tbody>
</table>

a simple behavioral assay for testing visual function in xenopus laevis

La medición de la función visual en los renacuajos de la rana Xenopus laevis, permite la detección de la ceguera en los animales vivos. La respuesta es un optocinético, comportamiento reflexivo basado en visión que se ha observado en todos los vertebrados examinados. Tadpole ojos son pequeños, así que utiliza la respuesta flip cola como medida alternativa, que requiere de un técnico capacitado para registrar la respuesta sutil. Hemos desarrollado un ensayo alternativo comportamiento basado en el hecho de que los renacuajos prefieren nadar en el lado blanco de un tanque cuando se coloca en un tanque con lados negros y blancos. El ensayo que aquí se presenta es una alternativa de bajo costo, simple que crea una respuesta que se mide fácilmente. La instalación consiste en un trípode, cámara web y tanques de prueba anidados, fácilmente disponible en la mayoría de los laboratorios de Xenopus. Este artículo incluye una película que muestra el comportamiento de los renacuajos, antes y después de cortar el nervio óptico. Con el fin de probar la función de uno de los ojos, también incluimos repreresultados representativos de un renacuajo en la que cada ojo se sometió a la axotomía de la retina en días consecutivos. Los estudios futuros podrían desarrollar una versión automatizada de este ensayo para probar la visión de muchos renacuajos a la vez.

Informes anteriores han demostrado que los renacuajos de Xenopus premetamórficas prefieren nadar en la parte blanca de un / tanque blanco negro y llamado a este ensayo, el fondo del ensayo preferencia de color 3 Hemos cambiado este ensayo con el fin de probar la función visual de uno de los ojos de los renacuajos en menos de una semana. De esta manera, sus ojos se pueden recoger para el examen histológico. Mostramos aquí cómo la respuesta se debe a las señales visua...

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A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis renacuajos prefieren nadar en la parte blanca de un / tanque blanco negro. Este comportamiento es guiado por su visión. Sobre la base de este comportamiento, se presenta un ensayo simple para probar la función visual de los renacuajos.

Un ensayo de comportamiento simple para Pruebas de función visual en<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

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Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.7K vistas.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  Xenopus laevis renacuajos prefieren nadar en la parte blanca de un / tanque blanco negro. Este comportamiento es guiado por su visión. Sobre la base de este comportamiento, se presenta un ensayo simple para probar la función visual de los renacuajos. 

Video: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

El grano y Pruebas de Manejo de Capellini: Simple medidas cuantitativas de la función de las patas delanteras diestro en ratas y ratones

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Investigación

Neurociencias

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un método general para la evaluación de incubación de sacarosa en ratas Deseo

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Los cultivos neuronales de explantes de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Disección, Cultura y Análisis de<em&gt; Xenopus laevis</em&gt; Tejido de la retina embrionaria

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Microinyección insulina hipocampo y<em&gt; En vivo</em&gt; Microdiálisis durante la prueba de memoria espacial

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Un peso ligero, Auriculares basada Sistema de Manipulación de retroalimentación auditiva en Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

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

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

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

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

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

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

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Un ensayo de conducta para Mechanosensation de clones en MARCM-basa<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Grabación de la actividad neuronal inducida por temperatura a través de la supervisión de cambios de calcio en el bulbo olfatorio de<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparaciones y protocolos para todo celular parche abrazadera de neuronas Tectal de Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...