Nos gustaría agradecer a los miembros actuales y anteriores del laboratorio de Enlace que ayudó a desarrollar los protocolos descritos aquí, particularmente de Gin Fonte, quien desarrolló el procedimiento original. Este trabajo ha sido apoyado por los premios de la NIH (AG12423) y la Asociación de Alzheimer (Premio Zenith) para CDL

1) Preparación de los nematodos medios de cultivo para el ensayo de placas de la parálisis. Los ensayos se llevan a cabo una parálisis en el estándar de crecimiento de los medios de nematodos (NGM) placas de procedimientos que para C. elegans propagación y la genética (Stiernagle, 2006). <ol><li> Autoclave la solución que contiene NaCl NGM, y Agar peptona en un erlenmeyer y añadir las cantidades adecuadas de estéril de CaCl2, uracilo, colesterol, 1M MgSO4 y 1M KPO 4. Alícuota de 10 ml de líquido en cada NGM 60 mm x 15 mm placa de Petri. Permitir NGM para solidificar la noche a temperatura ambiente. </li><li> Si la prueba de los efectos de los compuestos / extractos, alícuota del extracto en cada plato y el uso de un esparcidor para distribuir uniformemente el extracto sobre la superficie de la placa. Dejar que el extracto seco de la noche a temperatura ambiente. Volúmenes de más de 1 mL requerirá más tiempo para secarse. </li><li> Lugar cada placa con 250 l de E. OP5O coli cepa crecido en medio LB durante la noche para una densidad óptica de 0,4-0,6. Permitir que las bacterias que se seque durante la noche a temperatura ambiente. </li></ol> Variables relevantes La edad de las placas tiene un efecto significativo en el ensayo de la parálisis, como placas más viejas tienden a secarse. Use placas de vertido dentro de una semana de ensayo de la parálisis. Para resultados óptimos, verter en las placas de 3-4 días antes del inicio de las poblaciones de gusanos sincrónico [ver (2) más abajo]. Para cualquier experimento, sólo el uso de placas de vertido del mismo lote. La modificación del tamaño del césped (es decir, visto vs difusión) y / o el tipo de bacteria también modificar el comportamiento de los gusanos. Los ensayos de la parálisis se puede realizar utilizando la alternativa E. coli como fuente de alimento (por ejemplo, la cepa HT115 utilizado en experimentos de alimentación RNAi), pero esto puede cambiar la cinética de la parálisis, por lo que requieren controles internos apropiados. 2) Preparación de las poblaciones de gusanos edad síncrono Cepa CL4176 (SMG-1 (cc546 ts) I; dvIs27 [mio-3 / Aß minigene + rol-6 (su1006) gen marcador] X es más comúnmente utilizado para el ensayo de Aß la parálisis inducida por esta cepa y otros modelos transgénicos están disponibles. a los investigadores académicos por un precio nominal del Centro de Genética Caenorhabditis (http://www.cbs.umn.edu/CGC/). <ol><li> Para maximizar la sincronía edad de la población de prueba, es preferible comenzar con una generación de sincronizar los padres. Una semana antes del inicio de la prueba de la parálisis de la población de prueba, utilice un selector de gusano de platino para la transferencia de 20 a 30 adultos grávidas en varias placas de 10 cm NGM se extendió con OP50 por un huevo de dos horas estaba a 16 ° C (la temperatura permisiva para CL4176 ). Retire los adultos grávidas y permitir que la progenie de crecer durante 7 días, momento en el cual se &quot;segundo día&quot; adultos grávidas. </li><li> (Día 1) Los adultos días grávidas segundos se utilizan para preparar la prueba de las poblaciones de edad sincrónica. Para cada condición experimental el objeto es la generación de placas por triplicado, que contiene 50 a 75 años-sincrónica gusanos. Mediante un selector de gusano de platino, la transferencia de 10 a 12 del día 2 adultos grávidas en 60 mm (10 ml de volumen) NGM placas con manchas OP50. Permitir a los gusanos a poner huevos a los 16 ° C durante 2 horas, y luego escoger de los adultos y permitir que los huevos para incubar y crecer a 16 ° C. En este punto, lo mejor es tener a alguien que no será anotar las placas para el código, por lo que el marcador se ciega a las condiciones experimentales. </li></ol> Variables relevantes La etapa del desarrollo embrionario, cuando los huevos son puestos (~ la etapa de 26 células para los gusanos de tipo salvaje en condiciones óptimas) es una función de la edad adulta y la nutrición. Si los adultos grávidas utilizado para la preparación de la población de prueba son mayores o muerto de hambre, los huevos en una etapa posterior se establecerán, la reducción de la sincronía de la edad de la población y la reproducibilidad de la cinética de la parálisis. Es esencial que los mono-axénicos (es decir, sólo OP50 bacterias) los cultivos se utilizan (los contaminantes microbianos pueden afectar seriamente a este ensayo), y la reproducibilidad es mayor si las placas por triplicado tienen números similares de gusanos. 3) transgénica de inducción y de ensayo Parálisis <ol><li> (Día 3) placas de cambio ascendente a 25 ° C 48 horas después del final del huevo sincrónica laicos, los gusanos deben estar en la tercera etapa larvaria (L3). Las placas deben ser dispuestas sin apilar en una incubadora de 25 ° C para permitir que todas las placas hasta alcanzar los 25 ° C, al mismo tiempo. </li><li> (Día 4) Comience a anotar la paralización de los gusanos (cepa CL4176) a las 18-20 horas después del inicio del cambio ascendente. En este punto, todos los gusanos de la población debería haber llegado a la larva de cuarto (L4) el escenario. Continuar con calificaciones dentro de dos horas hasta que todos los incrementos de los gusanos en cada una de las placas se encuentran paralizados. Por razones desconocidas, la parálisis no es uniforme en toda la longitud del cuerpo: la región de la cabeza es la última parte del gusano dejan de moverse. Por lo tanto, wORM que recientemente han iniciado la parálisis no se puede traducir en la placa, pero se puede mover la cabeza, lo que se allanaba las bacterias en torno a su anterior y dejando un &quot;halo&quot; de las bacterias despejado. Estos gusanos siempre será completamente paralizado, y por lo tanto, los gusanos con halos se clasifican como paralizado. Algunos gusanos no tienen halos, pero tampoco mostrar un movimiento espontáneo, que son probados por pinchar con el selector de gusano. Si un gusano pinchó no pueden someterse a una propagación de onda completa en el cuerpo insistencia, también se anotó como paralizado. Para la puntuación eficiente, es más fácil mover los gusanos paralizados a un sector de la mancha de la placa para que no se involuntariamente rescored el período de puntuación siguiente. (Esto también permite que mal categorizado gusanos para demostrar el movimiento, lo que permite una corrección de puntuación). </li><li> Para generar una curva de la parálisis, en cada momento la fracción de los gusanos en cada plato que no se han paralizado se convierte en un porcentaje, y el promedio &quot;unparalyzed&quot; porcentaje en función del tiempo desde el inicio del cambio ascendente. (La razón para el trazado de esta manera es que la curva resultante es formalmente análoga a una curva de supervivencia, y por lo tanto pueden ser analizados usando estándares estadística no paramétrica de la supervivencia.) </li></ol> Variables relevantes El cambio ascendente de los gusanos transgénicos myo-3/Aβ debe ocurrir antes de que lleguen a la etapa de mediados de L4 para la parálisis que se inició, como el mio-3 es promotor de desarrollo regulado, y ha reducido significativamente su expresión en L4 tarde o gusanos adultos. Del mismo modo, la expresión de este transgén se bloquea si los gusanos se privan, por lo que es esencial que los gusanos están bien alimentados durante todo el experimento. Nunca hemos observado un gusano paralizada a recuperar en cualquier situación, y después de 12-16 horas de cambio ascendente, todos los gusanos CL4176 se paraliza, incluso si son downshifted a 16 ° C. En la cepa CL4176, upregulation suficiente de expresión de los transgenes a causa de la parálisis puede ocurrir a temperaturas más bajas (es decir, 20-25 ° C), aunque el tiempo de parálisis se verá alterada. La tasa de parálisis depende de la cepa específica de transgénicos utilizados (se han construido cepas myo-3/Aβ con tasas más rápido y más lento que CL4176), por lo que con otras cepas de la ventana de puntuación puede ser necesario ajustar. 4) Los resultados representativos Se muestra en la Figura 1 es un gráfico de una curva de la parálisis de CL4176, tanto sin tratar y se expone a 6,27 mM de cafeína (concentración final en la placa de NGM). Este tratamiento da como resultado un retraso estadísticamente significativa en la parálisis de aproximadamente 4 horas, lo que se interpreta como indicativo de la supresión de la toxicidad del Aß en este modelo. Muestra en la Figura 2 es un experimento independiente analizando los efectos de cafestol, otro compuesto que se encuentra en el café. Este gráfico es típico de los tratamientos que no afectan la toxicidad Aß. Tenga en cuenta que en ambos experimentos alrededor de la mitad de la población no tratada CL4176 están paralizadas a las 24 horas, y la parálisis es completa por 28 horas. Estos son los plazos típicos de la parálisis de control CL4176 poblaciones. Desviaciones significativas de este momento son indicativos de alteración de las condiciones ambientales o la eliminación espontánea de copias del transgén. (CL4176 contiene una amplia transgénicos cromosómicamente integrado con varias copias del transgén myo-3/Aβ. A pesar de esta variedad transgénica suele ser bastante estable, la propagación de la deformación a temperaturas&gt; 16 ° C se selecciona para los gusanos raros que han sufrido un eliminación espontánea de los transgenes, resultando en variantes que se han reducido expresión Aß y más lento resultados parálisis). <img src="/files/ftp_upload/2252/2252fig1.jpg" alt="Figura 1" /> Figura 1. Curva de la parálisis de la cepa CL4176 gusanos tratados o expuestos a la cafeína 6,27 mM (Sigma). Los tiempos indicados son desde el inicio del cambio ascendente de la temperatura. Las barras de error = SEM <img src="/files/ftp_upload/2252/2252fig2.jpg" alt="Figura 2" /> Figura 2. Curva de la parálisis de CL4176 gusanos expuestos a 0,48 mm cafestol (Sigma). Las barras de error = SEM

<ol>
	<li>Arya, U., Dwivedi, H., Subramaniam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

El protocolo que hemos descrito puede ser utilizado con eficacia para analizar los efectos de los compuestos sobre la toxicidad de Aß. Ejemplos publicados incluyen los efectos de los componentes de extracto de Ginkgo biloba (Wu et al. 2006) y la reserpina (Arya et al. 2009). Compuestos que han sido de protección contra la toxicidad del Aß en otros sistemas también han demostrado tener un efecto protector en este modelo (por ejemplo, la memantina, nuestros resultados no publicados). Una razón importante para el establecimiento de este modelo es que las herramientas bien desarrolladas genéticos y moleculares disponibles en C. elegans pueden ser utilizados para investigar los mecanismos de toxicidad de Aß. Por lo tanto, las mutaciones de los efectos específicos sobre la toxicidad de Aß se puede probar (por ejemplo, el efecto de la reducción de la insulina como la señalización mediante la introducción de un daf-2 la pérdida de la función de la mutación, Florez-McClure et al. 2007), y este modelo también se puede se utiliza para examinar los efectos de los transgenes sobre-expresión (Fonte et al. 2008). Recientemente hemos hecho un amplio uso de este modelo para examinar los efectos sobre la toxicidad de una sola sustitución de aminoácido en Aß (datos no publicados). El modelo transgénico se describe aquí el efecto de los ensayos de expresión Aß agudos sobre la función de las células musculares. En el momento de las células musculares y parálisis de sus sarcómeros están intactos, el fenotipo de la parálisis no es el resultado de la muerte de las células musculares. Sin embargo, después de la parálisis (~ 48 horas después del cambio ascendente inicial) se produce una ruptura de la integridad del músculo y la eventual muerte de los gusanos. No hemos investigado el efecto aguas abajo de expresión Aß o lo han utilizado como un marcador de toxicidad. El modelo de expresión inducible Aß se describe aquí no es apropiado para la investigación de tratamientos que modulan la formación de β-amiloide en sí. Aunque los gusanos transgénicos con forma de expresión constitutiva depósitos de amiloide Aß fácilmente detectables (Fay et al, 1998;.. Link et al 2001), los gusanos transgénicos con expresión inducible Aß rara vez tienen los depósitos de amiloide y el fenotipo de la parálisis parece independiente de la deposición de amiloide (Drake et al. 2003). Nuestros resultados son consistentes con la opinión de que la toxicidad aguda inducida por los resultados de Aß expresión de la acumulación de Aß oligómeros solubles, no el depósito de amiloide.

<table border="1"><tbody><tr><td> Reactivos </td><td> Quantity/2L </td><td> Compañía / Catálogo # </td><td> Comentarios </td></tr><tr><td> NaCl </td><td> 6g </td><td> Sigma-Aldrich / S9888 </td><td></td></tr><tr><td> Agar </td><td> 40g </td><td> Sigma-Aldrich / A7002 </td><td></td></tr><tr><td> Peptona </td><td> 5g </td><td> Sigma-Aldrich / P0556 </td><td></td></tr><tr><td> ddH2O </td><td> 2L </td><td></td><td></td></tr><tr><td> 1M CaCl2 </td><td> 2 ml </td><td></td><td> 147.02mg/mL en ddH2O </td></tr><tr><td> Uracilo </td><td> 2 ml </td><td></td><td> 2mg/ml en ddH2O </td></tr><tr><td> Colesterol </td><td> 2 ml </td><td></td><td> 5mg/mL en etanol (no autoclave) </td></tr><tr><td> 1M MgSO4 </td><td> 2 ml </td><td></td><td> 246.5mg/mL en ddH2O </td></tr><tr><td> 1M KPO 4 </td><td> 50 ml </td><td></td><td> 98mg KH 2 PO 4. ML, 48mg K2HPO4/mL en ddH2O, pH </td></tr><tr><td> Erlenmeyer </td><td></td><td> VWR </td><td></td></tr></tbody></table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

La acumulación del péptido β-amiloide (Aß) que generalmente se cree que ser central en la inducción de la enfermedad de Alzheimer, pero el mecanismo correspondiente (s) de toxicidad aún no están claros. Aß también se deposita por vía intramuscular en miositis cuerpos de inclusión, una miopatía grave en el hombre. El estudió intensamente nematodo Caenorhabditis elegans gusano puede ser transgénica para expresar Aß humanos. Dependiendo del tejido o momento de la expresión Aß, gusanos transgénicos pueden tener fenotipos fácilmente mensurables que sirven como una lectura de la toxicidad del Aß. Por ejemplo, los gusanos transgénicos con expresión Aß pan-neuronal tienen defectos es el aprendizaje asociativo (Dosanjh y otros, 2009.), Mientras que los gusanos transgénicos con los músculos específicos de expresión constitutiva muestran un progresivo, dependiente de la edad fenotipo parálisis (Link, 1995; Cohen et al . 2006). Uno particularmente útil C. elegans modelo emplea una mutación sensible a la temperatura en el sistema de vigilancia de ARNm para el ingeniero de la temperatura muscular de expresión inducible de un transgén Aß, lo que resulta en un fenotipo parálisis reproducible en cambio ascendente de la temperatura (Link et al. 2003). Tratamientos que la toxicidad contra Aß en este modelo [por ejemplo, la expresión de un transgén de protección (Hassan et al. 2009) o la exposición a los extractos de Ginkgo biloba (Wu et al. 2006)] reproducible alterar la tasa de parálisis muscular causada por cambios ascendentes temperaturas de estos transgénicos gusanos. A continuación se describe el protocolo para la medición de la tasa de parálisis en el C. transgénicos modelo elegans, con especial atención a las variables experimentales que pueden influir en esta medición.

Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

El nematodo estudiado intensamente gusano<em&gt; Caenorhabditis elegans</em&gt; Puede ser transgénica para expresar lo humano β-amiloide (Aß). Indujo la expresión de Aß en<em&gt; C. elegans</emMuscular&gt; conduce a un fenotipo rápido, parálisis reproducible que puede ser usado para monitorear los tratamientos que modulan la toxicidad de Aß.

Ensayo β-amiloide toxicidad con un transgénico C. elegans Modelo

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

assaying-amyloid-toxicity-using-a-transgenic-c-elegans-model

Research

JoVE Journal

Neuroscience

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

25.2K vistas.  University of Colorado. El nematodo estudiado intensamente gusano Caenorhabditis elegans Puede ser transgénica para expresar lo humano β-amiloide (Aß). Indujo la expresión de Aß en C. elegans conduce a un fenotipo rápido, parálisis reproducible que puede ser usado para monitorear los tratamientos que modulan la toxicidad de Aß.

Video: Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Ensayo Actividad locomotora para el Estudio de los ritmos circadianos y del sueño en los parámetros Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Investigación

Neurociencias

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

En vivo láser axotomía en C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

La cuantificación automática de la fluorescencia en el Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Seguimiento y Medición del Comportamiento

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Una lectura molecular de adaptación a largo plazo olfativa en<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Ensayo de daño del ADN en las neuronas del hipocampo utilizando el ensayo cometa

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Formación de imágenes in vivo de calcio neuronal en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

La<em&gt; Caenorhabditis elegans</em&gt; Sistema Modelo para el Estudio Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizar Neuroblast Citocinesis Durante<em&gt; C. elegans</em&gt; Embriogénesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

En vivo de imágenes de Dauer-específica neuronal Remodelación en C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...