Name: in vivo imaging of dauer-specific neuronal remodeling in c. elegans
Uploaded: 2014-09-04T15:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1. crudo Dauer Pheromone Producción <ol><li> Crecer OP50 E. coli a partir de una sola colonia O / N a 37 ° C mientras se agita en 100 ml de caldo LB a 200 rpm. NOTA: Este cultivo O / N se puede hacer con antelación y se almacena a 4 ° C. </li><li> Crecer N2 C. elegans en junio 15 a 20 cm placas de Petri con agar NGM (ver receta en la Tabla 1) se sembró con 50 l de E. OP50 coli hasta que las bacterias se ha consumido casi por 19. </li><li> Hacer 5x 250 ml de S medios (Ver receta en la Tabla 1) en 1 L matraces Erlenmeyer de 19. NOTA: Esta solución se puede hacer con antelación. </li><li> Cultivar una cultura de OP50 E. coli en 4 L de caldo LB O / N mediante la inoculación con 1 ml de OP50 en E. coli y agitación a 37 ° C durante 16 horas. </li><li> Lavar los nematodos de las placas (paso 1,2) con 1 ml de solución de S basal (ver receta en la Tabla 1) y nematodos de transferencia 4-5 placas en cada uno de 4 1 matraces Erlenmeyer de L con los medios de comunicación S preparado en la etapa 1.3 (4 5 placaspara cada matraz) 19. </li><li> Centrifugar la OP50 desde el paso 1.4 a 650 xg durante 10 min y retirar el sobrenadante. Resuspender cada sedimento bacteriano en 10 ml de solución de S basal. Transfiera la misma cantidad de bacterias resuspendidas a cada frasco de nematodos. </li><li> Colocar los matraces en un agitador orbital a temperatura ambiente (20 - 25 ° C) y 100 - 150 rpm durante 4 - 5 días hasta que la solución comienza a limpiar, lo que indica un agotamiento de los alimentos. </li><li> Una vez que el alimento se agota, crecer otro cultivo O / N de OP50 E. coli en 4x 1 L de caldo LB. Centrifugar las bacterias a 650 xg durante 10 min y añadir una pastilla de un frasco a cada uno de los matraces S de medios con nematodos. Devolver los frascos S de medios a la agitación, como en el paso 1.7, de 3-4 días hasta que la comida se agota de nuevo. </li><li> Tome un ~ 1 ml alícuota de solución de cada matraz y estimar la concentración de nematodos por sub-muestreo de 1 l de cada alícuota de 1 ml y contando el número de nematodos. NOTA: Lapoblación de nematodos debe ser 50 000 - 75 000 nematodos / ml. A esta concentración la mayoría de los nematodos son dauers. </li><li> Deseche todos los frascos contaminados. NOTA: La contaminación se manifiesta con frecuencia como hifas fúngicas visto durante el paso 1.9. </li><li> Nematodos centrifugar a 4000 xg durante 10 min a 4 ° C. Deseche el sedimento nematodo y retener el sobrenadante. Como alternativa, vea la discusión para el uso de estos animales para hacer una feromona de baja potencia. </li><li> Filtrar el sobrenadante a través de # 1 papel filtro Whatman. NOTA: Un frasco de vacío acelerará este proceso. </li><li> Filtrar aún más el sobrenadante a través de 0,45 micras unidades de filtración de vacío estéril. NOTA: El filtrado se puede mantener O / N a 4 ° C o proceder a 1,14. </li><li> Añadir el filtrado a un vaso de precipitados de 2 l con una barra de agitación. Coloque en una campana extractora en una placa calefactora revuelo. Llevar a ebullición mientras se agita y se evapora hasta aproximadamente 200 ml. Transfiera la solución a un vaso de 500 ml. Continuar la ebullición hasta aproximadamente 50 ml son left. NOTA: Se debe tener cuidado durante este paso, ya que es posible que la solución a hervir. El color de la final de 50 ml es un marrón oscuro. En cualquier momento durante el proceso de cocción, el calor puede ser apagado y el filtrado almacena O / N a temperatura ambiente o 4 ° C. </li><li> Dejar enfriar y centrifugar durante 5 min a 1000 x g. Verter el sobrenadante a un vaso de 100 ml y desechar el sedimento. </li><li> Hervir el sobrenadante a una corteza de color marrón. Retire del fuego y romper la corteza con una espátula. Añadir suficiente etanol prueba 200 para cubrir la corteza. Cubrir el vaso con Parafilm y dejar que repose O / N a temperatura ambiente. Verter el etanol en un vaso de precipitados limpio y poner a un lado. Añadir etanol fresco para cubrir la corteza. Repita extracciones O / N 3 veces. Reunir las fracciones. </li><li> Utilice una bomba de vacío para secar los extractos de etanol. Si una bomba de vacío no está disponible, añadir el extracto de etanol a un vaso de precipitados limpio y cálido a 50 ° C mientras se agita en una campana de humos. NOTA: Una vez que el etanol se ha evaporado un residuo pegajoso de lde color marrón de vuelo debe permanecer. Si se utiliza calentamiento, se debe tener cuidado para no quemar la solución por calentamiento durante demasiado tiempo. </li><li> Disolver residuo en 10 ml de agua destilada estéril. Filtro esterilizar esta solución con un filtro de jeringa de 0,22 micras y se almacenan a -20 ° C en alícuotas de 1 ml. NOTA: 1 ml de agua se utiliza para disolver el residuo por cada 100 ml del cultivo de nematodos líquido original. Por ejemplo, si uno de los matraces de cultivo líquido se descarta debido a la contaminación, a continuación, ajustar la cantidad de agua utilizada para disolver el residuo. </li></ol> 2. Determinación de Pheromone Respuesta a la dosis <ol><li> Prepare feromona Dauer haciendo 6x 5 ml de medio NGM con agar Noble, sin peptona, y se suplementó con 50 mg / ml de sulfato de estreptomicina y una gama de feromona Dauer (véase la receta en la Tabla 1) 4, 19. Mezclar componentes en un 13x100 mm tubo de cultivo de vidrio, hervir sobre un mechero Bunsen, y luego verter en placas de Petri de 3 cm. </li><li> Pesar amtubo icrocentrifuge. Añadir 1 ml de cultivo O / N del E. coli OP50 al tubo y centrifugar a ~ 17.000 xg en un tubo de microcentrífuga. Retire todo el sobrenadante. Pesar el tubo de nuevo y determinar el peso de la pella bacteriana. Resuspender el sedimento con tampón M9 (ver receta en la Tabla 1) para una concentración final de 4% (w / v). Alícuota de 10 l de la suspensión bacteriana en el centro de cada placa de feromona 4. Placas Tienda O / N a temperatura ambiente. </li><li> Escoja 10-15 grávidas N2 hermafroditas de tipo salvaje un día después de la muda hasta la edad adulta en las placas de feromonas 4. Almacenar las placas a 25 ° C durante 3 - 4 horas. </li><li> Escoger de todos los adultos hermafroditas. Anote el número de huevos puestos por cada placa. Sellar las placas con Parafilm y volver a 25 ° C durante 3 días 17. </li><li> Cuente el porcentaje de dauers (Figura 2). Nota: Para ambos métodos es importante tener en cuenta que dauers (y en menor extienda de campaña no dauers) trepará por las paredes y en la tapa de platos de Petri. Por tanto, es importante examinar todas las superficies del plato para los nematodos. </li><li> Ajustar los datos a una curva dosis-respuesta y calcular la concentración EC 90 de feromona (feromona suficiente para inducir 90% de los animales para formar dauers) como se muestra en la Figura 3 Nota:. Normalmente utilizamos un modelo de regresión logística para estimar la CE 90 , pero diferentes paquetes de software estadísticos pueden tener métodos alternativos que producen estimaciones similares. </li></ol> 3. imágenes en vivo de Dauer IL2 Neuronas <ol><li> Preparar platos de feromonas dauer como en el protocolo 2 utilizando el valor EC 90 determinada en el paso 2.6. </li><li> Inducir dauers utilizando los métodos en el protocolo 2 con una cepa con un reportero transgén integrado IL2 (myIs13 [P klp-6 :: GFP], myIs14 [P klp-6 :: GFP], myIs16 [P <em&gt; klp-6 :: tdTomato] o qIs56 [P desfase-2 :: GFP]) 12, 20, 21. NOTA: Si se utiliza q Is56, expresión de GFP no será visible en los IL2Qs hasta varias horas en el muda Dauer y algunos de los eventos iniciales de remodelación no serán visibles. Además, mientras que la señal fluorescente del P lag-2 :: GFP transgén es en última instancia, más brillante en los IL2s durante dauer que los KLP-6 reporteros fluorescentes P, el P lag-2 :: GFP transgén también expresa débilmente en los músculos de la cabeza que puede enmascarar las ramas finas IL2. </li><li> Después de la puesta de huevos, retire los adultos hermafroditas, sellar las placas con Parafilm y se incuba durante 34 horas a 25 ° C 17. NOTA: La longitud de tiempo para la muda Dauer varía ligeramente entre los individuos. El período más rápido de la arborización dendrítica comienza 4-5 horas después del inicio de la muda dauer (39-44h después de la puesta de huevos a 25 ° C) 12. </li><li> El día antes de formación de imágenes, conforman una solución de 10% (w / v) de agarosa en tampón M9 con feromona Dauer a la concentración CE 90. Alícuota de aproximadamente 100 l de agarosa en un portaobjetos de microscopio y de forma rápida, pero suavemente, coloque otra diapositiva en la parte superior para crear una superficie plana de agarosa. Después de la agarosa se ha solidificado, envuelva las diapositivas en papel film y guardar en una cámara de humedad (caja de puntas de pipeta con toallas de papel húmedas). </li><li> Tras el período de incubación 34 horas, examinar las placas de feromonas para nematodos que han dejado de bombeo de la faringe. Diapositivas separadas de tal manera que sólo una superficie de una diapositiva tiene agarosa en él. Elija uno o más nematodos en una almohadilla de agarosa al 10% y 1 μ l de 0,1 micras perlas de poliestireno 22. NOTA: Con una población bien sincronizado, la mayoría de los nematodos en el plato va a ingresar en la muda de L2d a dauer. Sin embargo, puede haber variación entre ias personas. Por tanto, es importante tener en cuenta características adicionales (oclusión de estoma, la remodelación de la faringe) para estimar la cantidad de tiempo que los animales hayan estado dentro de la muda Dauer (ver resultados representativos, por ejemplo). </li><li> Determine la orientación (izquierda / derecha, dorsal / ventral) del nematodo. Captura imágenes Z-stack de un dendrita IL2Q a 100 aumentos con la intensidad de la fluorescencia bajo posible cada 15 minutos o cuando sea necesario para el experimento específico. Continuar la captura de imágenes hasta que el nematodo se ha separado de la cutícula L2d y la inmovilización es imposible o si el animal se recupera de Dauer (faringe comienza a bombear). Cuando no la formación de imágenes de forma activa, coloque el portaobjetos en una cámara húmeda. NOTA: Nosotros usamos un microscopio de epifluorescencia con un contraste de interferencia diferencial escenario y motorizado (DIC) óptica. </li></ol>

<ol>
	<li>West-Eberhard, M. 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> Developmental Plasticity and Evolution. , (2003).</li><li>Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dauerlarva,+a+post-embryonic+developmental+variant+of+the+nematode+Caenorhabditis+elegans.">The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.</a> Dev. Biol. 46, 326-342 (1975).</li><li>Riddle, D. L., Albert, P. S., Riddle , D. L., Blumenthal, T., Meyer, B. J., Priess, 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=.">.</a> C. elegans II. , 739-768 (1998).</li><li>Golden, J. W., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+dauer+larva:+developmental+effects+of+pheromone,+food,+and+temperature.">The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature.</a> Dev. Biol. 102, 368-378 (1984).</li><li>Riddle, D. L., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode C. elegans. , 393-412 (1988).</li><li>Fielenbach, N., Antebi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+dauer+formation+and+the+molecular+basis+of+plasticity.">C. elegans dauer formation and the molecular basis of plasticity.</a> Genes Dev. 22, 2149-2165 (2008).</li><li>Hu, P. 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> WormBook. , 1-19 (2007).</li><li>Golden, J. W., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pheromone+influences+larval+development+in+the+nematode+Caenorhabditis+elegans.">A pheromone influences larval development in the nematode Caenorhabditis elegans.</a> Science. 218, 578-580 (1982).</li><li>Butcher, R. A., Fujita, M., Schroeder, F. C., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+pheromones+that+control+dauer+development+in+Caenorhabditis+elegans.">Small-molecule pheromones that control dauer development in Caenorhabditis elegans.</a> Nature Chemical Biology. 3, 420-422 (2007).</li><li>Jeong, P., 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=Chemical+structure+and+biological+activity+of+the+Caenorhabditis+elegans+dauer-inducing+pheromone.">Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.</a> Nature. 433, 541-545 (2005).</li><li>Srinivasan, J., 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=A+blend+of+small+molecules+regulates+both+mating+and+development+in+Caenorhabditis+elegans.">A blend of small molecules regulates both mating and development in Caenorhabditis elegans.</a> Nature. 454, 1115-1118 (2008).</li><li>Schroeder, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer-Specific+dendrite+arborization+in+C.+regulated+by+KPC-1/Furin.">Dauer-Specific dendrite arborization in C. regulated by KPC-1/Furin.</a> Curr. Biol. 16, 1527-1535 (2013).</li><li>Procko, C., Lu, Y., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+delimit+shape+changes+of+sensory+neuron+receptive+endings+in+C.+elegans.">Glia delimit shape changes of sensory neuron receptive endings in C. elegans.</a> Development. 138, 1371-1381 (2011).</li><li>Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+alterations+in+sensory+neuroanatomy+of+the+Caenorhabditis+elegans+dauer+larva.">Developmental alterations in sensory neuroanatomy of the Caenorhabditis elegans dauer larva.</a> J. Comp. Neurol. 219, 461-481 (1983).</li><li>Vowels, J. J., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+chemosensory+control+of+dauer+formation+in+Caenorhabditis+elegans.">Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans.</a> Genetics. 130, 105-123 (1992).</li><li>Vowels, J. J., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+chemosensory+defects+in+daf-11+and+daf-21+mutants+of+Caenorhabditis+elegans.">Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans.</a> Genetics. 138, 303-316 (1994).</li><li>Ailion, M., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer+formation+induced+by+high+temperatures+in+Caenorhabditis+elegans.">Dauer formation induced by high temperatures in Caenorhabditis elegans.</a> Genetics. 156, 1047-1067 (2000).</li><li>Neal, S. J., Kim, K., Sengupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromone Signaling. , 273-283 (2013).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Ouellet, J., Li, S., Roy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+is+required+for+both+dauer+maintenance+and+recovery+in+C.+elegans.">Notch signalling is required for both dauer maintenance and recovery in C. elegans.</a> Development. 135, 2583-2592 (2008).</li><li>Blelloch, R., 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=The+gon-1+gene+is+required+for+gonadal+morphogenesis+in+Caenorhabditis+elegans.">The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans.</a> Dev. Biol. 216, 382-393 (1999).</li><li>Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, (2012).</li><li>Nelson, F. K., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+study+of+the+Caenorhabditis+elegans+secretory-excretory+system+using+laser+microsurgery.">Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.</a> J. Exp. Zool. 231, 45-56 (1984).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Singh, R., Sulston, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li></ol>

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Un examen de la neuroplasticidad-dauer específicos y otros cambios morfológicos dauer asociada requiere controlado y formación fiable de dauers ya sea a través de la mutación genética (es decir, mutantes, daf-c) o por la exposición de los animales de tipo salvaje de la feromona. Mientras que el uso de una mutación genética para inducir dauers es conveniente, puede confundir los resultados. Por lo tanto, los datos recogidos con mutaciones daf-c deben confirmarse posteriormente con animales de t...

Plasticidad fenotípica, incluyendo la plasticidad del desarrollo y del comportamiento, es importante para las adaptaciones a las condiciones ambientales adversas y se considera una fuerza impulsora en la evolución 1. C. elegans es un organismo modelo utilizado con frecuencia para estudios en el desarrollo y la neurobiología. En condiciones ideales, C. elegans se desarrolla a través de cuatro estadios larvarios antes de entrar en la etapa reproductiva de adultos. Sin embargo, en condiciones de baja disponibilidad de alimentos y las altas densidades de población, C. elegans puede someterse a un interruptor de desarrollo y entrar en una de larga duración y resistente a la tensión etapa Dauer 2. Dauers muestran varias diferencias morfológicas y de comportamiento de los no dauers que probablemente median esta resistencia al estrés. Las señales ambientales y los mecanismos genéticos que regulan la entrada dauer han sido ampliamente descrito 3, 4, 5, 6, 7. Sin embargo, los mecanismos moleculares que controlan los cambios fenotípicos individuales que ocurren ddauer formación urante son menos conocidos. El examen de los fenotipos dauer específica requiere la producción de animales dauer. Esto se puede lograr de tres maneras: 1) Levantar partir de un cultivo de C. hambre elegans, 2) uso de la formación de dauer constitutiva (daf-c) mutantes, 3) Inducción de la formación de dauer través de feromona purificado. Es relativamente sencillo para recoger dauers de una placa estándar NGM con una C. población elegans que ha agotado su suministro de alimentos-bacteriana. En nuestras manos, una placa estándar NGM originalmente se siembra con 50 l de OP50 Escherichia coli, se dejó crecer durante 3 días y posteriormente inoculadas con 3 - 4 adultos hermafroditas se mantuvo a 25 ° C, se agotará el suministro de alimentos dentro de una semana y producir varios miles dauers dentro de 3 días adicionales (además de numerosos no dauers hambre). Sin embargo, este método es insatisfactorio para examinar procesos que ocurren durante la muda en dauer. Es difícil determinar cuál de los varios miles de animales en un plato típico hambriento están entrando en dauer. Además, el uso de una placa de hambre no ofrece ninguna posibilidad para la sincronización. El uso de mutantes daf-c permite la sincronización y la generación fiable de dauers. Sin embargo, no hay garantía de que una mutación particular daf-c no afectará a otros fenotipos-dauer específica solo o en combinación con mutaciones adicionales. La presencia de una feromona dauer se implicó por primera vez por Cassada y Russell y más tarde demostró por Golden y Riddle. La feromona dauer desde entonces se ha caracterizado químicamente 2, 8, 9, 10. La feromona purificada consiste en una mezcla compleja de ascarosides, que en diferentes formas a regular los procesos conductuales y de desarrollo 9, 11. Uso de un extracto de feromona dauer crudo permite inducción fiable controlada de dauers sincronizados en un fondo de tipo salvaje. </p&gt; El sistema nervioso y las células gliales asociadas se someten a la remodelación durante dauer larva formación 12, 13, 14. Algunos de estos cambios son irreversibles, tales como la remodelación de las células de la glia durante dauer 13, 14. Sin embargo otros eventos de remodelación son reversibles en un retorno a la favorable condiciones ambientales. Por ejemplo, un conjunto de cuatro neuronas IL2Q se someten a la remodelación neuronal rápida y reversible durante la formación de dauer incluyendo: arborización dendrítica, un interruptor de una sola dendríticas a las neuronas y los axones multidendritic remodelación 12. Los métodos de la presente memoria permiten la formación de imágenes in vivo fiable de la neuroplasticidad inducida por estrés en un organismo modelo. Los métodos presentados para la producción y las pruebas de feromona dauer crudo se describen anteriormente y compilados a partir de varias fuentes de 4, 8, 15, 16, 17, 18.

<table><tbody><tr><td>N2 &lt;em&gt;C. elegans&lt;/em&gt; Bristol strain</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>PT2660 myIs13[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] III</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>PT2762 myIs14[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] V</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>JK2868 qIs56[P&lt;sub&gt;lag-2&lt;/sub&gt;::gfp] V</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>OP50 E. coli</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Tritech Research</td><td>60 mm T3308, 35 mm T3501</td><td></td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>3 g NaCl, 17 g agar, 2.5 g peptone in 975 ml H&lt;sub&gt;2&lt;/sub&gt;O autoclaved cooled to 55&amp;deg;C followed by 1 ml of 5 mg/ml cholesterol in ethanol, 25 ml of 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 1 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>S Media</td><td>Various</td><td></td><td>S media consists of S basal solution (1.46 g NaCl, 0.25g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.5 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.25 ml of 5 mg/ml cholesterol in ethanol and H&lt;sub&gt;2&lt;/sub&gt;O to 250 ml. Autoclaved.) To the S basal solution add 2.5 ml of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate&lt;sub&gt;, &lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml, autoclaved), 2.5 ml of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.2 g MnCl2&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O, 0.29 g ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.025 g CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Autoclaved and stored in the dark), 0.75 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, and 0.75 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;.</td></tr><tr><td>Dauer pheromone plates for dose-response assay</td><td>Various</td><td></td><td>Mix 0.085 g Noble agar, 0.5 ml of 0.513 M NaCl and 5 &amp;micro;l of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 ml culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H&lt;sub&gt;2&lt;/sub&gt;O to bring the volume to 4.725 ml.&amp;nbsp; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &amp;deg;C. Add 125 &amp;micro;l 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 50 &amp;micro;l 0.1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 50 &amp;micro;l 0.1 M MgSO&lt;sub&gt;4&lt;/sub&gt; and 50 &amp;micro;l of 5 mg/ml streptomycin sulfate to each tube.&amp;nbsp; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td></tr><tr><td>Microsphere beads 0.1 micron</td><td>Polysciences Inc.</td><td>00876-15</td><td></td></tr><tr><td>M9 buffer</td><td>Various</td><td></td><td>Mix 3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 liter. Sterilize by autoclaving</td></tr></tbody></table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

Los mecanismos que controlan el estrés inducido por la plasticidad fenotípica en animales son frecuentemente complejos y difíciles de estudiar in vivo. Un ejemplo clásico de la plasticidad inducida por el estrés es la etapa dauer de C. elegans. Dauers son una etapa larval de desarrollo alternativo formado bajo condiciones de bajas concentraciones de los alimentos bacteriana y altas concentraciones de una feromona Dauer. Dauers muestran amplia plasticidad del desarrollo y del comportamiento. Por ejemplo, un conjunto de cuatro cuadrantes interior-labial (IL2Q), las neuronas se someten a una amplia remodelación reversible durante la formación de Dauer. Utilizando la entrada vías ambientales de regulación Dauer bien conocido, un método previamente establecido para la producción de feromona Dauer crudo a partir de cultivos de nematodos líquido a gran escala se demuestra. Con este método, una concentración de 50.000 - 75.000 nemátodos / ml de cultivo líquido es suficiente para producir una muy potente feromona Dauer crudo. La potencia feromona crudo es determined mediante un bioensayo de respuesta a la dosis. Por último, se describen los métodos utilizados para in vivo time-lapse de los IL2Qs durante la formación de dauer.

La producción de resultados de feromonas dauer crudo en un líquido amarillo (Figura 1) que es tanto el calor como el frío estable. Alícuotas de feromona crudo se almacenan a -20 ° C indefinidamente sin pérdida evidente de la actividad. Después de la producción de feromona, un único bioensayo dosis-respuesta es suficiente para una estimación aproximada de la potencia. Sin embargo, para la mayoría de los experimentos es necesario repetir el bioensayo 2 - 3 veces y tomar un promedio de cada expe...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

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 ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

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

11.2K vistas.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos de microfluidos para<em&gt; In vivo</em&gt; Imágenes de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Y pez cebra

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Investigación

Bioingeniería

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In Vivo Calcium Imaging en C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Bioquímica

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, ...

Neurociencias

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microcámaras de agarosa para Imagen de calcio a largo plazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imágenes C. elegans Los embriones utilizando un microscopio de epifluorescencia y software de código abierto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biología

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Un chip microfluídico simple para el crecimiento a largo plazo y la obtención de imágenes de Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Evaluación de la dinámica y orientación de los microtúbulos en las neuronas caenorhabditis elegans

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Cuantificación de los niveles de alteración morfológica y degeneración de la neurona dopaminérgica en Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imágenes subcelulares del manejo neuronal del calcio in vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

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

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