Nos gustaría dar las gracias a Scott Hamilton, y los miembros del laboratorio O&#39;Halloran para la lectura de este manuscrito. También agradecemos a nuestro revisor anónimo por excelentes sugerencias y comentarios perspicaces.

1. Construcción de GFP Tagged EGL-4 animales que expresan <ol><li> Clonar la traslación de fusión GFP :: EGL-4 bajo el promotor para el gen ODR-3 (2678 pb uso directamente aguas arriba del codón de inicio): (p) ODR-3 :: GFP :: EGL-4. La expresión ODR-3 unidades promotoras en los pares de neuronas amphid: AWA, AWB, AWC, y débilmente en ASH. </li><li> Inyectar el plásmido (p) ODR-3 :: GFP :: EGL-4 de tipo salvaje (en N2) los animales en 50 ng / ml con los marcadores co-inyección estándar utilizando la línea germinal-8 técnicas de transformación: (p) ofm-1: : GFP 9 (25 ng / l), y (p) ODR 1-:: dsRed 10 (25 ng / l). El ODR-1 unidades promotor de expresión en el AWC y AWB amphid pares de neuronas, y el ofm-1 promotor de expresión en las unidades coelomocytes. </li><li> Someter a los animales transgénicos que expresan las matrices extracromosómicos detallado en el paso 1,2 para el ultravioleta (UV) / trimetilpsoraleno (TMP) protocolo de integración11. </li><li> Bleach sincronizar los 12 animales y cultivar en ellos el crecimiento de nematodos Medios (NGM) las placas que contenían E. OP50 coli a la etapa L4. Lave L4 animales en platos con NGM M9 tampón para eliminar E. OP50 bacterias coli. </li><li> Eliminar el tampón de M9 y añadir 50-100 l de 30 mg / ml de solución de trabajo TMP en el gusano pellet en el tubo de microcentrífuga de 1,5 ml. Envolver el tubo de microcentrífuga de 1,5 ml en papel de aluminio para bloquear la luz ambiental, y agitar suavemente en un agitador rotatorio durante 15 min en el embalaje flexible tubo de microcentrífuga de 1,5 ml. Transfiera los gusanos en la solución de TMP / gusano a una placa de NGM cabeza de serie grande. </li><li> Exponer a los gusanos en la placa de NGM a UV 350 μJ (X100) con un Stratalinker 2400. A continuación, añadir los gusanos a una placa que contiene NGM OP50 e incubar en la oscuridad durante 5 hr. </li><li> Elige 50 gusanos transgénicos para aproximadamente 10 placas sembradas (2-5 gusanos cada placa). Transferencia P0 es a las nuevas placas cada 24 horas durante 3 días. </li><li> Elige un 250 F1 clonalnimals (1 animal por placa). Clonar animales por 2-4 F2 de cada placa F1. Ver animales F3 para buscar una línea de transmisión 100% al ver la (p) ofm-1 :: patrón de expresión GFP bajo un microscopio de disección fluorescente. </li><li> Outcross la línea final integrado (s) con N2 animales de tipo salvaje 5 veces. Nos referiremos a la última línea integrada como pyIs500, y las buenas prácticas agrarias de etiquetado EGL-4 :: GFP molécula como EGL-4. </li></ol> 2. Cultivo y mantenimiento de animales para ensayos de translocación nuclear <ol><li> Cultivar los animales pyIs500 a 25 ° C en placas estándar de 10 cm NGM (ver más abajo para obtener la receta) se sembró con OP50 E. bacterias coli 13 (Figura 1). </li><li> En el Día 1, recoger cuatro y cincuenta y seis L4 pyIs500 animales de placas cultivadas a 25 ° C en cinco separados 10 cm OP50 E. coli sembraron placas e incubar a 25 ° C (es decir, L4 4-5 animales por placa). </li><li> El día 4, lavar adulto populations pyIs500 de animales de las placas de NGM grandes usando S-basal tampón (ver más abajo para obtener la receta). Usar pipetas desechables de vidrio para transferir los animales, ya que se pegará a la parte de puntas de pipeta de plástico. </li></ol> 3. Olor a largo plazo inducida por la translocación nuclear ensayos <ol><li> Lavar los animales pyIs500 3 veces en S-basal para eliminar las bacterias. No centrifugue los animales entre los mismos, sino que los animales puedan establecerse por gravedad en un tubo de microcentrífuga de 1,5 estacionario ml. Es crítico para asegurar que todas las bacterias se retira, y que los patés NGM están libres de contaminación, como las bacterias residuales afectará negativamente el resultado de la translocación de ensayo. Además, hemos encontrado que la esterilización en autoclave los tubos de 1,5 ml de microcentrífuga produce una &quot;pegajosa&quot; propiedad en las paredes interiores del tubo que hace que los animales se adhieren a los lados, y así utilizar no tratados en autoclave 1,5 ml tubos de microcentrífuga. </li><li> Mientras que los animales se están estableciendo entre los mismos, conforman tél adaptación solución. Añadir 100 ml de tampón de S-basal a un cilindro de 100 ml de graduación. Añadir la adaptación de olor a la S-basal y sellar el cilindro utilizando una banda de Parafilm ®. Si se realiza la adaptación benzaldehído añadir 7,5 l de benzaldehído a 100 ml de S-basal, para la adaptación butanona añadir 11 l de 2-butaone a 100 ml de S-basal. Invertir suavemente (no agitar) el cilindro graduado que contiene S-basales y olor 30 veces para crear una emulsión uniforme. </li><li> Después del lavado final en S-basal, que los animales puedan establecerse y eliminar todo el líquido. Etiquete un tubo &quot;+ ve&quot; o &quot;adaptar&quot; y el otro tubo &quot;-ve&quot; o &quot;unadapt&quot;. A continuación, añadir 1 ml de la mezcla de la adaptación al tubo de microcentrífuga de adaptación (+ ve), y se añade 1 ml de S-basal al tubo de microcentrífuga de control no adaptado (-ve). Trate de mantener el número de animales entre 100 y 200 en cada tubo. Con un poco de práctica, es posible estimar el número aproximado de animales en un sedimento por ojo. </li><li> Coloque un tubo de microcentrífuga de 1,5 ml tapón protectora cada tubo (impide que las tapas de hacer estallar abierto) y se colocan los tubos en un agitador rotatorio durante 80 min. Hemos encontrado que la exposición olor entre 60 min y 80 min produce resultados similares (ambos resultan en respuestas de adaptación a largo plazo). Sin embargo, 80 minutos de exposición proporciona resultados más consistentes en nuestras manos. Así, todos nuestros ensayos a largo plazo de adaptación están normalizados mediante el uso de una exposición min olor 80 (Figura 2). </li><li> Después de 80 minutos, lavar animales 3 veces en S-basal. Que los animales puedan establecerse por la gravedad entre cada lavado. </li></ol> 4. A corto plazo inducidos Odor-Ensayos de Translocación Nuclear <ol><li> En el caso de la adaptación olor a corto plazo, utilizar el mismo protocolo como a largo plazo por los ensayos de translocación inducida olor-(Pasos 3.1-3.4), sin embargo en lugar de incubar los pyIs500 animales en la adaptación de olor para 80 min, utilizar una exposición min 30 . La cuantificación de la inducción de olor ensayos de translocación nuclear se describen a continuación (Etapa 5) es la misma a largo plazo y corto-term exposiciones. </li></ol> 5. Calificación del Evento olor inducida por la translocación nuclear <ol><li> Hacer 2% de agarosa que contienen pastillas de 5 mM de azida sódica (NaN 3) por disolución de electroforesis en gel de agarosa en dH 2 0. Coloque una sola gota de agarosa fundida que contiene NaN 3 sobre un portaobjetos de microscopio de vidrio. Inmediatamente colocar otro portaobjetos de microscopio de vidrio en la primera diapositiva para aplanar la gota de agarosa fundida. Dejar durante 30 segundos, y luego suavemente separar el portaobjetos dejando un portaobjetos de microscopio con una almohadilla plana de agarosa de ~ 1 mm. </li><li> Después del lavado final en S-basal, permita que los gusanos a instalarse en el tubo de microcentrífuga de 1,5 ml y eliminar todo el líquido. A continuación, agregue los animales sabios de abandono en las almohadillas de agarosa de preparaciones microscópicas varios. Es importante mantener el número de animales de cada diapositiva para entre 50 y 100. Demasiados animales por diapositiva complicará puntuación y con frecuencia crean un resplandor después de la dispersión de la luz azul de excitación. El exceso de líquido puedeser malos de la almohadilla de agarosa utilizando un pequeño Kim-wipe. Poner un vaso de 0,5 mm cubreobjetos sobre la almohadilla y se deja reposar durante 2 minutos para permitir la NaN 3 para inmovilizar a los animales. Nota, los animales pueden ser recuperados de las diapositivas después de la puntuación. Los animales se recuperan de la anestesia NaN 3 después de ~ 1 hora si se transfiere a una placa sembrada NGM en una gota de buffer M9. </li><li> En este punto es imprescindible que cada experimentador idear una clave para rastrear qué diapositiva es el experimental (+ ve) diapositiva y diapositiva que es el control (-ve) diapositivas, las diapositivas y pasar a otra persona que ciegamente marcará el GFP: EGL-4 patrón de localización. Esto controlará cualquier sesgo experimentador. </li><li> Puntuación entre 50 y 100 animales por diapositiva utilizando un microscopio fluorescente vertical con los objetivos de aumento de 10X, 40X y 63X y filtros GFP / RFP. En primer lugar, localizar animales de menos de 10 aumentos con iluminación de campo brillante. En segundo lugar, localizar la neurona AWC apagando el bright-campo de iluminación y utilizando el filtro de RFP. (P) ODR-1 :: unidades DsRed expresión en las neuronas y AWB AWC. Las neuronas tienen AWC distintivos de células en forma de cuerpos ovalados, en comparación con los cuerpos celulares AWB que son más pequeños y de forma circular (Figura 2). </li><li> Una vez que el cuerpo de la célula AWC ha sido localizado, cambiar el filtro GFP y registrar la localización de las buenas prácticas agrarias: EGL-4 como la nuclear o citoplasmática. El uso de un contador permite al experimentador que seguir mirando por el ocular y anotó la diapositiva. Nota: 5.1-5.5 pasos se repiten al menos 3 veces en días diferentes para producir datos estadísticamente significativos (Figuras 1-2). </li></ol> 6. La supervisión, GFP-etiquetados EGL-4 durante la recuperación de la adaptación Olor a largo plazo <ol><li> Después del paso 3,5, dividir las poblaciones de pyIs500 animales en múltiples alícuotas y la puntuación para GFP nuclear versus citoplásmica :: EGL-4 en la AWC punto en el tiempo cero (es decir, después de la exposición min 80) y multiple puntos de tiempo posteriores (Figura 3). </li><li> Transferir tanto el control (-ve) y animales experimentales (+ ve) usando pipetas desechables de vidrio en placas de NGM. </li><li> Permitir que los animales se recuperen durante varios períodos de tiempo y volver a examinar la localización de GFP :: EGL-4in AWC en cada punto de tiempo como en los pasos desde 5,1 hasta 5,5. </li></ol> 7. Materiales Placas de NGM: Por 1 L añadir a 1 L ddH 2 0: 21 g de Bacto-Agar; 3 g Cloruro sódico (NaCl); 2,5 g de Bacto-peptona. Autoclave Media. Después del autoclave, enfriar el agar hasta que la temperatura se lee 54 ° C. Añadir las siguientes soluciones: 25 ml de 1 M tampón fosfato de potasio; 1 ml 1 M sulfato de magnesio (MgSO 4); 1 ml cloruro de calcio 1 M (CaCl 2); 1 ml de colesterol en etanol (5 mg / ml stock). 1 M dibásico K 2 HPO 4 174.2 g / mol: Por 500 ml: En 400 ml ddH 2 O, se disuelven 87,1 g Dibasic K 2 HPO 4. Ajustar el volumen a 500 L con ddH 2 O. Esterilizar por filtración utilizando un filtro de 500 ml Unidad estéril. Monobásico 1 M KH 2 PO 4 136.1 g / mol: Para 1 L: En 800 ml ddH 2 O, se disuelven 136,1 g monobásico KH 2 PO 4. Ajustar el volumen a 1 l con ddH 2 O. Esterilizar por filtración utilizando un filtro de 500 ml Unidad estéril. 1 M de fosfato de potasio (K 3 PO 4) pH 6,0: Para 1 L: En un filtro de 1 L Unidad estéril, añadir 868 ml de 1 M KH 2 PO 4 monobásico. Deje que la unidad se filtre, a continuación, añadir 132 ml de 1 M dibásico K 2 HPO 4. Deje que la unidad de filtrar todo el líquido. Retirar la unidad de filtro y ajustar el pH a 6,0. Almacenar a temperatura ambiente. S-basal: Para 1 L: añadir 50 ml de 1M K 3 PO 4 Buffer, pH ~ 6,0, y 20 ml de 5 M NaCl ac. Llene hasta 1 L conddH 2 O. Autoclave de soluciones. M9: Para 1 litro: 3 g de KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl, 1 ml 1 M MgSO 4, H 2 O. Llenar hasta 1 L con ddH 2 0. Esterilizar en autoclave.

<ol>
	<li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14 (4), 803 (1995).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+modulate+olfactory+acuity,+discrimination,+and+memory+in+Caenorhabditis+elegans.">Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.</a> Learning and Memory. 4 (2), 179 (1997).</li><li>Bargmann, C. I., 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=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Neuron. 74 (3), 515 (1993).</li><li>L'Etoile, N. D., 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+cyclic+GMP-dependent+protein+kinase+EGL-4+regulates+olfactory+adaptation+in+C.+elegans.">The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans.</a> Neuron. 36, 1079-10 (2002).</li><li>Lee, J. I., 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=Nuclear+entry+of+a+cGMP-dependent+kinase+converts+transient+into+long-lasting+olfactory+adaptation.">Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.</a> PNAS. 107 (13), 6016 (2010).</li><li>O'Halloran, D. M., 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=Regulators+of+AWC-mediated+olfactory+plasticity+in+Caenorhabditis+elegans.">Regulators of AWC-mediated olfactory plasticity in Caenorhabditis elegans.</a> PLoS Genetics. 5 (12), e1000761 (2009).</li><li>O'Halloran, D. M., 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=Changes+in+cGMP+levels+affect+the+localization+of+EGL-4+in+AWC+in+Caenorhabditis+elegans.">Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans.</a> PLoS ONE. 7 (2), e31614 (2012).</li><li>Mello, C. C., 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=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO Journal. 10, 3959-39 (1991).</li><li>Miyabayashi, T., 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=Expression+and+function+of+members+of+a+divergent+nuclear+receptor+family+in+Caenorhabditis+elegans.">Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans.</a> Dev. Biol. 215, 314 (1999).</li><li>L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+and+odor+discrimination+are+mediated+by+the+C.+elegans+guanylyl+cyclase+ODR-1.">Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1.</a> Neuron. 25 (3), 575 (2000).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451 (1995).</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. , (2006).</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 (1), 71 (1974).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <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+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+Dopamine+Neuron+Degeneration+Assay+for+the+Validation+of+Potential+Parkinson's+Disease+Genes.">Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes.</a> J. Vis. Exp. (17), e835 (2008).</li><li>Pittenger, C., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+general+mechanisms+for+long-lasting+plasticity:+Aplysia+and+the+hippocampus.">In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358 (1432), 757 (2003).</li></ol>

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La entrada nuclear inducida por el olor de un GFP-etiquetados EGL-4 molécula descrito aquí proporciona una lectura sólida molecular de la adaptación olfativa en C. elegans. Los ensayos nucleares inducidas olor-translocación son sencillas y requieren sólo unos pocos días de tiempo de preparación. El animal pyIs500 que hemos construido para estos ensayos, expresa un marcador que ilumina la neurona AWC, así como la expresión de la GFP-etiquetados EGL-4 proteínas. Por lo tanto, creemos que un ex...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Bacto Agar </td><td> Difco </td><td> DF0140-07-4 </td><td> NGM placas </td></tr><tr><td> Cloruro de Sodio </td><td> Fisher Chemical </td><td> S671-10 </td><td> NGM placas </td></tr><tr><td> Bacto Peptona </td><td> Difco </td><td> DF0118-07-2 </td><td> NGM placas </td></tr><tr><td> Fosfato de potasio dibásico </td><td> Fisher Chemical </td><td> S375-500 </td><td> S-Basal tampón y NGM placas </td></tr><tr><td> Fosfato de potasio monobásico </td><td> Fisher Chemical </td><td> P285-500 </td><td> S-Basal tampón y NGM placas </td></tr><tr><td> Kimwipes - Pequeño </td><td> Kimberly-Clark </td><td> LS2770 </td><td></td></tr><tr><td> Etanol 100% </td><td> Gold Shield QuímicaCo. </td><td> 43196-115 </td><td> olores de dilución para los ensayos de quimiotaxis </td></tr><tr><td> Cloruro de calcio </td><td> Sigma-Aldrich </td><td> C8106-500G </td><td> NGM placas </td></tr><tr><td> Sulfato de magnesio </td><td> MP Biomedicals </td><td> 150136-500G </td><td> NGM placas </td></tr><tr><td> De azida de sodio 99% </td><td> Fisher Scientific </td><td> ICN10289180 </td><td> Anestésico </td></tr><tr><td> Agarosa - UltraPure </td><td> Invitrogen </td><td> 16500-500 </td><td> La agarosa almohadillas </td></tr><tr><td> Benzaldehído </td><td> Sigma-Aldrich </td><td> B1334-100G </td><td> Olor AWC </td></tr><tr><td> Butanona, ACS Grado </td><td> Sigma-Aldrich </td><td> 360473-500ML </td><td> Olor AWC </td></tr><tr><td> Tubos de microcentrífuga de 1,5 ml de colores - </td><td> Denville </td><td> LS8147 </td><td></td></tr><tr><td> Pasteur 5-3/4 pipeta de vidrio desechable &quot; </td><td> Fisher Scientific </td&gt; <td> 13-678-20B </td><td></td></tr><tr><td> Stratalinker </td><td> Stratagene </td><td> Stratalinker 2400 </td><td> Integración UV </td></tr><tr><td> Botella de vacío del filtro - 500 ml </td><td> Nalgene </td><td> 09-740-25B </td><td></td></tr></tbody></table>

a molecular readout of long-term olfactory adaptation in c. elegans

Durante la estimulación sostenida neuronas sensoriales más adaptarán su respuesta al disminuir su sensibilidad a la señal. La respuesta de adaptación ayuda a la atención de forma y también protege las células de la sobre-estimulación. La adaptación en el circuito olfatorio de C. elegans fue descrita por primera vez por Colbert y Bargmann 1,2. Aquí, los autores definieron los parámetros del paradigma de la adaptación olfativa, que se utiliza para diseñar una pantalla genética para aislar mutantes defectuosos en su capacidad para adaptarse a los olores volátiles detectados por las células amphid Ala tipo C (AWC) neuronas sensoriales. Cuando wildtype C. elegans animales se exponen a un atractivo AWC-detectados olor 3 de 30 min se adaptarán su capacidad de respuesta al olor y entonces ignorar el olor en la adaptación de un ensayo de quimiotaxis de comportamiento para ~ 1 hr. Cuando wildtype C. elegans animales se exponen a un atractivo AWC-detectados olor de ~ 1 hr luego de ignorar el olor en la adaptación de achemotaxis comportamiento ensayo para ~ 3 h. Estas dos fases de adaptación olfativa en C. elegans fueron descritos como de corto plazo adaptación olfativa (inducido después de 30 min de exposición olor), y adaptación a largo plazo olfativo (inducido después de 60 min de exposición olor). Un trabajo posterior de L&#39;Etoile et al., 4 descubierto una proteína kinasa G (PKG) llamado EGL-4 que se requiere tanto para la adaptación olfativa a corto plazo ya largo plazo en las neuronas de AWC. El EGL-4 proteína contiene una secuencia de localización nuclear que es necesaria para la respuesta a largo plazo de adaptación olfativas pero prescindible para respuestas a corto plazo de adaptación olfativas en el AWC 4. Mediante el etiquetado de EGL-4 con una proteína fluorescente verde, que era posible visualizar la localización de EGL-4 en la AWC durante la exposición prolongada olor. El uso de este completamente funcional GFP-etiquetados EGL-4 (GFP :: EGL-4) molécula hemos sido capaces de desarrollar una lectura molecular de adaptación a largo plazo olfativo en la AWC 5. El uso de este molecular lectura de la adaptación olfativa hemos sido capaces de realizar ambas pantallas genéticas de avance y retroceso para identificar animales mutantes que exhiben defectuosos patrones de localización subcelular de GFP :: EGL-4 en la AWC 6,7. Aquí se describe: 1) la construcción de GFP :: EGL-4 animales que expresan; 2) el protocolo para el cultivo de los animales para los ensayos a largo plazo inducida por la translocación olor nucleares, y 3) la puntuación de la largo plazo olor inducida evento translocación nuclear y la recuperación (re-sensibilización) de la GFP nuclear :: EGL-4 Estado.

Un ejemplo del patrón de localización de GFP :: EGL-4in la AWC antes y después de la exposición prolongada olor se muestra en la Figura 2. Antes de la exposición prolongada olor, GFP :: EGL-4 se localiza en el citosol de la AWC (Figura 2B), y después de 80 min de exposición GFP olor :: EGL-4 se localiza en el núcleo de la AWC (Figura 2D). En el nivel conductual, los animales con citosólicas GFP :: EGL-4 en AWC son atraídos a una fuente puntual de olor ...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

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

Aquí describimos una lectura molecular de adaptación a largo plazo olfativo en<em&gt; Caenorhabditis elegans</em&gt;. La proteína quinasa G, EGL-4, es necesario para las respuestas de adaptación estables en el par de neurona sensorial primaria llamada AWC. Durante la exposición prolongados olor EGL-4 transloca del citosol al núcleo de la 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 ...

a-molecular-readout-of-long-term-olfactory-adaptation-in-c-elegans

Research

JoVE Journal

Neuroscience

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

10.7K vistas.  George Washington University. Aquí describimos una lectura molecular de adaptación a largo plazo olfativo en Caenorhabditis elegans. La proteína quinasa G, EGL-4, es necesario para las respuestas de adaptación estables en el par de neurona sensorial primaria llamada AWC. Durante la exposición prolongados olor EGL-4 transloca del citosol al núcleo de la AWC.

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

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans de aprendizaje positivo butanona, a corto plazo y largo plazo Ensayos de la memoria asociativa

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

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 ...

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, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Seguimiento del comportamiento a largo plazo de Libremente Natación débilmente eléctrico Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

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 ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

El análisis automatizado de<em&gt; C. elegans</em&gt; Comportamiento de la nadada El uso de software Célest

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Imunocoloración Cuádruple del Bulbo Olfativo para la Visualización de Códigos de Identidad Molecular del Axón Sensorial Olfativo

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Usando un Chip de microfluidos adaptado olfativo para la proyección de imagen de la actividad Neuronal en respuesta a las feromonas en las neuronas de cabeza de macho de C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...