Les auteurs tiennent à reconnaître que ce travail a été soutenu par le Conseil de recherches en génie du Canada de subvention (CRSNG) en sciences naturelles et.

<ol>
	<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>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>. <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+Sequencing+Consortium.+Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">C. elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Pennisi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worming+secrets+from+the+C.+elegans+genome.">Worming secrets from the C. elegans genome.</a> Science. 282, 1972-1974 (1998).</li><li>Tabara, H., 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+rde-1+gene,+RNA+interference,+and+transposon+silencing+in+C.+elegans.">The rde-1 gene, RNA interference, and transposon silencing in C. elegans.</a> Cell. 99, 123-132 (1999).</li><li>Chisholm, A. D., Hardin, 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> Epidermal morphogenesis. WormBook. The C. elegans Research Community. , 1-22 (2005).</li><li>Zhang, H., Gally, C., Labouesse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+morphogenesis:+how+multiple+cells+cooperate+to+generate+a+tissue.">Tissue morphogenesis: how multiple cells cooperate to generate a tissue.</a> Curr. Opin. Cell Biol. 22, 575-582 (2010).</li><li>Kiehart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing:+the+power+of+the+purse+string.">Wound healing: the power of the purse string.</a> Curr. Biol. 9, 602-605 (1999).</li><li>Ikegami, 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=Semaphorin+and+Eph+receptor+signaling+guide+a+series+of+cell+movements+for+ventral+enclosure+in+C.+elegans.">Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans.</a> Curr. Biol. 22, 1-11 (2012).</li><li>Chin-Sang, I. D., George, S. E., Ding, M., Moseley, S. L., Lynch, A. S., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ephrin+VAB-2/EFN-1+functions+in+neuronal+signaling+to+regulate+epidermal+morphogenesis+in+C.+elegans.">The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans.</a> C. elegans. Cell. 99, 781-790 (1999).</li><li>Wang, X., Roy, P. J., Holland, S. J., Zhang, L. W., Culotti, J. G., Pawson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Ephrins+Control+Cell+Organization+in+C.+elegans+Using+Kinase-Dependent+and+-Independent+Functions+of+the+VAB-1+Eph.">Multiple Ephrins Control Cell Organization in C. elegans Using Kinase-Dependent and -Independent Functions of the VAB-1 Eph.</a> Mol. Cell. 4, 903-913 (1999).</li><li>Chin-Sang, I. D., Moseley, S. L., Ding, M., Harrington, R. J., George, S. E., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+C.+elegans+ephrin+EFN-4+functions+in+embryonic+morphogenesis+in+a+pathway+independent+of+the+VAB-1+Eph+receptor.">The divergent C. elegans ephrin EFN-4 functions in embryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor.</a> Development. 129, 5499-5510 (2002).</li><li>Ghenea, S., Boudreau, J. R., Lague, N. P., Chin-Sang, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VAB-1+Eph+receptor+tyrosine+kinase+and+SAX-3/Robo+neuronal+receptors+function+together+during+C.+elegans+embryonic+morphogenesis.">The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis.</a> Development. 132, 3679-3690 (2005).</li><li>Bernadskaya, Y. Y., Wallace, A., Nguyen, J., Mohler, W. A., Soto, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNC-40/DCC,+SAX-3/Robo,+and+VAB-1/Eph+polarize+F-actin+during+embryonic+morphogenesis+by+regulating+the+WAVE/SCAR+actin+nucleation+complex.">UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex.</a> PLoS Genet. 8, (2012).</li><li>Piekny, A. J., Werner, M., Glotzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis:+welcome+to+the+Rho+zone.">Cytokinesis: welcome to the Rho zone.</a> Trends Cell Biol. 15, 651-658 (2005).</li><li>Piekny, A. J., Maddox, A. 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+myriad+roles+of+Anillin+during+cytokinesis.">The myriad roles of Anillin during cytokinesis.</a> Sem. Cell Dev. Biol. 21, 881-891 (2010).</li><li>Zhao, W. M., Fang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+is+a+substrate+of+anaphase-promoting+complex/cyclosome+(APC/C)+that+controls+spatial+contractility+of+myosin+during+late+cytokinesis.">Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis.</a> J. Biol. Chem. 280, 33516-33524 (2005).</li><li>Piekny, A. J., Glotzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+is+a+scaffold+protein+that+links+RhoA,+actin+and+myosin+during+cytokinesis.">Anillin is a scaffold protein that links RhoA, actin and myosin during cytokinesis.</a> Curr. Biol. 18, 30-36 (2008).</li><li>Hickson, G. R., O&#8217;Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+control+of+anillin+behavior+during+cytokinesis.">Rho-dependent control of anillin behavior during cytokinesis.</a> J. Cell Biol. 180, 285-294 (2008).</li><li>Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Kamath, R. S., 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=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry. WormBook, The C. elegans Research Community. , 1-61 (2006).</li><li>Maddox, A. S., Habermann, B., Desai, A., Oegema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+two+C.+elegans+anillins+in+the+gonad+and+early+embryo.">Distinct roles for two C. elegans anillins in the gonad and early embryo.</a> Development. 132, 2837-2848 (2005).</li><li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Dev. Biol. 100, 64-119 (1983).</li><li>Chin-Sang, I. D., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+of+the+worm:+genetics+of+epidermal+morphogenesis+in+C.+elegans.">Form of the worm: genetics of epidermal morphogenesis in C. elegans.</a> Trends Genet. 16, 544-551 (2000).</li><li>Zhang, H., Labouesse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+mechanical+inputs+&#8211;+a+coordinated+process.">Signaling through mechanical inputs &#8211; a coordinated process.</a> J. Cell Sci. 125, 3039-3049 (2012).</li><li>Guenther, C., Garriga, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+distribution+of+the+C.+elegans+HAM-1+protein+in+neuroblasts+enables+daughter+cells+to+adopt+distinct+fates.">Asymmetric distribution of the C. elegans HAM-1 protein in neuroblasts enables daughter cells to adopt distinct fates.</a> Development. 122, 3509-3518 (1996).</li><li>Audhya, A., 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+complex+containing+the+Sm+protein+CAR-1+and+the+RNA+helicase+CGH-1+is+required+for+embryonic+cytokinesis+in+Caenorhabditis+elegans.">A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.</a> J. Cell Biol. 171, 267-279 (2005).</li><li>Maddox, A. S., Lewellyn , L., Desai, A., Oegema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+and+the+septins+promote+asymmetric+ingression+of+the+cytokinetic+furrow.">Anillin and the septins promote asymmetric ingression of the cytokinetic furrow.</a> Dev. Cell. 12, 827-835 (2007).</li><li>Dorn, J. F., 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=Actomyosin+tube+formation+in+polar+body+cytokinesis+requires+Anillin+in+C.+elegans.">Actomyosin tube formation in polar body cytokinesis requires Anillin in C. elegans.</a> Curr. Biol. 20, 2046-2051 (2010).</li><li>Calixto, A., Ma, C., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+neuronal+RNAi+in+C.+elegans+using+SID-1.">Enhanced neuronal RNAi in C. elegans using SID-1.</a> Nat. Methods. 7, 554-559 (2010).</li></ol>

Les auteurs n&#39;ont rien à révéler.

Ce protocole décrit l&#39;utilisation de divers types de microscopie à des divisions cellulaires de l&#39;image au cours de l&#39;embryogenèse milieu. En particulier, ce protocole met en évidence comment l&#39;image de la division des neuroblastes, des cellules qui peuvent faciliter la morphogenèse épidermique. La communication cellulaire est important pour la formation des tissus au cours du développement de métazoaires et C. elegans est un excellent modèle pour étudier la formation du tissu in v...

Bien qu&#39;il existe des protocoles décrivant la manière de la division cellulaire de l&#39;image au début C. elegans embryon, ce protocole décrit comment la division cellulaire de l&#39;image dans un tissu à la mi embryogenèse. L&#39;un des principaux défis dans les organismes d&#39;imagerie au cours du développement a été leur sensibilité à la phototoxicité. Cependant, l&#39;augmentation de l&#39;accessibilité à la filature microscopes confocaux disque ou microscopes champ balayé a permis des applications d&#39;imagerie les plus répandues. Les deux systèmes utilisent des lasers à l&#39;état solide et la lumière diffusée, ce qui limite les niveaux de rayons UV que les organismes sont exposés. Toutefois, les peuplements Widefield peuvent encore être utilisés pour l&#39;imagerie in vivo, en particulier s&#39;ils sont équipés de caméras à haute sensibilité (par exemple EMCCD), le contrôle de l&#39;ouverture et contrôle de la lumière (par exemple des LED ou des ampoules de mercure réglables). Ce protocole décrit comment utiliser soit un système confocal base ou un système à grand champ de la division cellulaire de l&#39;image dans le développement C. elegans &lt;/ Em&gt; embryons. A titre d&#39;exemple, nous décrivons comment l&#39;image de la division cellulaire des neuroblastes. Neuroblastes peuvent faciliter la morphogenèse épidermique en fournissant des signaux chimiques ou mécaniques pour les cellules de l&#39;épiderme sus-jacentes, et de fournir un excellent exemple de l&#39;importance de la communication intercellulaire dans la formation des tissus. Caenorhabditis elegans est un organisme modèle idéal pour les études fondées microscopie en raison de sa transparence et de l&#39;organisation simple tissu 1. Par ailleurs, C. elegans se prête à des méthodes génétiques et RNAi, et puisque plusieurs de ses gènes ont des homologues humains, il peut être utilisé pour identifier les mécanismes conservées pour la formation de tissu 2-5. Dans C. elegans, la formation de l&#39;épiderme survient pendant l&#39;embryogenèse milieu, quand l&#39;embryon a&gt; 300 cellules. Morphogenèse épidermique se compose de plusieurs phases principales, au cours de laquelle l&#39;embryon est enfermé dans une couche de cellules épidermiques qui compriment et s&#39;étendent à transformer le embryo d&#39;une forme ovoïde dans la forme allongée d&#39;un ver 6. Décrit une enceinte ventrale de ces événements morphogénétiques, lorsque les cellules épidermiques ventrales migrent vers la ligne médiane ventrale à couvrir la surface ventrale de l&#39;embryon (figure 1). Tout d&#39;abord, deux paires de cellules de premier plan de bord antérieure située migrent vers la ligne médiane ventrale, où elles adhèrent et fusionnent avec leurs voisins controlatérales 6. Elle est suivie par la migration des cellules de poche postérieure situé, qui forment coin comme des formes en créant une poche ventrale 6-7. Le mécanisme qui ferme la poche n&#39;est pas bien comprise. Une possibilité est que la structure de la myosine contractile actine supracellular lie les cellules de poche ensemble dans une chaîne de sac à main comme la mode, semblable à la cicatrisation des plaies 8. Fait intéressant, la migration d&#39;une partie des cellules de la poche est médiée par des sous-ensembles spécifiques de sous-jacents 9 neuroblastes (précurseurs neuronaux qui se trouvent en dessous de la epidermis, figure 1B). Des études antérieures ont montré que les neuroblastes régulent la migration ventrale des cellules épidermiques et de l&#39;enceinte ventral. VAB-1 (récepteur Ephrin) et (ligand Ephrin) VAB-2 est fortement exprimé dans les neuroblastes et faciliter le tri des antérieur et neuroblastes postérieures les unes des autres, et mutations dans VAB-1 ou VAB-2 provoquent enceinte ventrale phénotypes 10-13. Cependant, les expériences de sauvetage de promoteurs ont montré que VAB-1 est également nécessaire dans les cellules épidermiques ventrales jacentes et des récepteurs pour d&#39;autres signaux de guidage sécrétées par les neuroblastes sont exprimés dans les cellules de l&#39;épiderme ventral 9. Bien que des mutations dans l&#39;un de ces récepteurs provoquent phénotypes de l&#39;enceinte ventrales, il n&#39;est pas clair si les défauts se produisent en raison de problèmes de positionnement des neuroblastes ou en raison de l&#39;échec des cellules épidermiques ventrales pour répondre aux signaux de guidage 14. Modification de la répartition des neuroblastes wiThoout affectant leur capacité à sécréter des signaux de guidage pourrait faire la lumière sur le rôle des neuroblastes et leur capacité à apporter une contribution mécanique au cours de la morphogenèse épidermique. Récemment, il a été constaté qu&#39;un gène de la division cellulaire, ani-1 (anillin) est fortement exprimé dans les neuroblastes (figure 2A) et son appauvrissement provoque neuroblastes défauts de division. Fait intéressant, ces embryons présentent ventrales phénotypes de l&#39;enceinte (Fotopoulos, Wernike et Piekny, observations non publiées). Anillin est requis pour la division cellulaire, et en particulier pour la cytokinèse, qui décrit le processus par lequel une cellule mère sépare physiquement en deux cellules filles. Cytocinèse est entraîné par la formation d&#39;un anneau contractile actomyosine, qui doit être étroitement contrôlée dans l&#39;espace et le temps pour s&#39;assurer qu&#39;il est correctement couplée avec la sœur de chromatides ségrégation. Le régulateur principal de la cytokinèse métazoaires est RhoA (RHO-1 chez C. elegans), une petite GTPase qui est unctive dans sa forme liée au GTP. La FEM Ect2/ECT-2 RhoA active, après quoi interagit RhoA-GTP avec des effecteurs en aval, qui forment l&#39;anneau contractile et la médiation de son l&#39;admission 15. Anillin est une protéine de domaine qui se lie à plusieurs RhoA via son extrémité C-terminale et à l&#39;actine et de la myosine par l&#39;intermédiaire de son extrémité N-terminale. Anillin est nécessaire pour stabiliser la position de l&#39;anneau contractile dans des cellules de mammifères ou de Drosophila S2 16. Anillin épuisement provoque des anneaux contractiles à des oscillations latérales, et la cytokinèse échoue finalement former des cellules multinucléées 17-19. Fait intéressant, bien que C. elegans ani-1 coordonne actomyosin contractilité dans l&#39;embryon précoce, il n&#39;est pas essentiel pour la cytokinèse. Cependant, comme décrit ci-dessus, ani-1 est requise pour la cytokinèse neuroblastes durant l&#39;embryogenèse (mi-Fotopoulos, Wernike et Piekny, observations non publiées). l&#39;échec de la cytokinèse modifierait le nombre et la position des neuroblastes et pourrait affecter la loction de signaux de guidage chimiques, ou il peut modifier les propriétés mécaniques du tissu. Les deux modèles mettent en évidence le rôle non autonome de neuroblastes pour enceinte ventrale, et l&#39;importance de la communication tissus tissus au cours du développement embryonnaire. Ce protocole expérimental décrit comment la division cellulaire de l&#39;image pendant C. elegans mi embryogenèse en utilisant la microscopie de fluorescence. La majorité des expériences qui étudient les mécanismes de la division cellulaire ont été réalisées dans des cellules individuelles dans des boîtes de culture (par exemple. Des cellules HeLa ou S2) ou dans les embryons précoces avec un nombre limité de cellules (par exemple C. elegans embryon d&#39;une cellule, Xenopus, ou échinoderme embryons). Cependant, il est également important d&#39;étudier la division cellulaire dans les tissus, car il existe des indices externes qui peuvent influer sur la synchronisation et la mise en place du plan de division. En outre, les cellules peuvent fournir des indices chimiques ou mécaniques d&#39;influencer le développement du tissu voisins, et il est important de comprendre comment la communication intercellulaire aide tissus pour former au cours du développement.

<table border="0" cellpadding="0" cellspacing="0" width="1056">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
			<td style="width:185px;">#AGR001.1</td>
			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Bio Basic Inc.</td>
			<td>#9002-18-0</td>
			<td>For making bacteria LB agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>BioShop Canada Inc.</td>
			<td>#AGA001.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse Alexa 488 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse anti-GFP antibody</td>
			<td>Roche Applied Science</td>
			<td>#11814460001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit Alexa 568 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>BioShop Canada Inc.</td>
			<td>#AMP201.5</td>
			<td>Store powder at 4 &#176;C and dissolved ampicillin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bactopetone&#160; (peptone-A)</td>
			<td>Bio Basic Inc.</td>
			<td>#G213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (calcium chloride)</td>
			<td>BioShop Canada Inc.</td>
			<td>#C302.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>BioShop Canada Inc.</td>
			<td>#CHL380.25</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#D9542</td>
			<td>Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>BioShop Canada Inc.</td>
			<td>#GLY001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG (isopropylthio-&#946;-galactoside)</td>
			<td>Bio Basic Inc.</td>
			<td>#367-93-1</td>
			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

Ce protocole décrit l&#39;utilisation de la microscopie de fluorescence à l&#39;image à l&#39;intérieur de la division des cellules en développement Caenorhabditis elegans embryons. En particulier, ce protocole met l&#39;accent sur la façon de l&#39;image divisant neuroblastes, qui se trouvent sous les cellules de l&#39;épiderme et peuvent être importants pour la morphogenèse épidermique. formation de tissus est cruciale pour le développement de métazoaires et s&#39;appuie sur des indices externes de tissus voisins. C. elegans est un excellent organisme modèle pour étudier la morphogenèse des tissus in vivo en raison de sa transparence et de l&#39;organisation simple, faire ses tissus faciles à étudier par microscopie. Enceinte ventrale est le processus où la surface ventrale de l&#39;embryon est recouvert d&#39;une seule couche de cellules épithéliales. Cet événement est pensé pour être facilitée par les neuroblastes sous-jacents, qui fournissent orientation chimique indices de médiation migration des cellules épithéliales recouvrant. Toutefois, les neuroblastes sont très proliférante et peuvent également agircomme substrat mécanique pour les cellules épidermiques ventrales. Les études utilisant ce protocole expérimental pourraient découvrir l&#39;importance de la communication intercellulaire pendant la formation du tissu, et pourraient être utilisées pour révéler les rôles des gènes impliqués dans la division cellulaire dans les tissus en développement.

Ce protocole expérimental décrit la façon de la division cellulaire de l&#39;image en C. elegans embryons en moyenne embryogenèse. En particulier, il décrit comment les neuroblastes de l&#39;image, ce qui peut faciliter la morphogenèse épidermique. Épidermique morphogenèse est due à une combinaison de changements de forme des cellules épidermiques, la migration et l&#39;adhérence, mais aussi s&#39;appuie sur des indices chimiques ou mécaniques des neuroblastes sous-jacents (figure 1B).</str...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Ce protocole décrit comment l&#39;image des cellules en division dans un tissu en Caenorhabditis elegans embryons. Bien que de nombreux protocoles décrivent comment la division cellulaire de l&#39;image dans l&#39;embryon précoce, ce protocole décrit comment l&#39;image de la division cellulaire dans un tissu en développement au cours de l&#39;embryogenèse tardive milieu.

Visualisation neuroblaste Cytocinèse cours<em&gt; C. elegans</em&gt; Embryogenèse

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

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.8K vues.  Concordia University.  Ce protocole décrit comment l'image des cellules en division dans un tissu en Caenorhabditis elegans embryons. Bien que de nombreux protocoles décrivent comment la division cellulaire de l'image dans l'embryon précoce, ce protocole décrit comment l'image de la division cellulaire dans un tissu en développement au cours de l'embryogenèse tardive milieu. 

Vidéo: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

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

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

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

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Dosage β-amyloïde toxicité utilisant un transgéniques C. elegans</emModèle>

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

Recherche

Neurosciences

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 à l&#39;apprentissage butanone, à court terme et à long terme Tests mémoire associative

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

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.

In vivo laser axotomie 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.

Quantification automatisée de la fluorescence dans 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 Suivi et évaluation comportementale

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

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

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

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

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

Un indicateur moléculaire de l&#39;adaptation à long terme olfactive dans<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 ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

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

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

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

La micro-injection d&#39;insuline de l&#39;hippocampe et<em&gt; In vivo</emMicrodialyse&gt; Test de la mémoire spatiale cours

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

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.

En imagerie calcique in vivo des neurones 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, ...

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

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

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

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

Imagerie in vivo de Dauer spécifique neuronale Rénovation 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 ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un test pour mesurer les effets de l&#39;éthanol sur la vitesse de Locomotion<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...