Ces études ont été soutenus par des subventions des Instituts nationaux de la santé, Institut national de l&#39;alcoolisme et l&#39;abus d&#39;alcool: R01AA016837 (JCB) et P20AA017828 (AGD et JCB).

1. Etapes à effectuer le jour avant la Assay <ol><li> Choisissez les vers de la scène à L4 milieu de croissance des nématodes frais (NGM) plaques ensemencées avec une pelouse de OP50 E. coli, et de la culture eux à 20 CO / N. Chaque condition d&#39;essai nécessite 10 vers; chercher un excès de vers pour permettre O / N perte de vers. <ol><li> Seuls les animaux dosage qui sont adultes premier jour; de nombreux mutants se développent à une vitesse plus lente que le type sauvage. Réglez le moment de la cueillette pour les souches qui ont des retards de développement, afin que tous les animaux testés sont adultes de la première jour. </li></ol></li></ol> 2. Etapes à effectuer sur le jour de l&#39;essai <ol><li> Préparation pour le test: <ol><li> Effectuer des essais sur 60 x 15 mm plaques NGM non ensemencés standard. Sécher toutes les plaques à utiliser à 37 ° C pendant 2 heures, avec des couvercles de congé. Pour chaque essai expérimental, utilisez quatre plaques NGM séchées; ce seront les 0 et 400 mm plaques de dosage de l&#39;éthanol et leurs plaques d&#39;acclimatation d&#39;accompagnement. NOTE:L&#39;utilisation de plaques NGM est important ici; différences dans la composition des plaques, en particulier leur osmolarité, peuvent affecter fortement la réponse à la dose d&#39;éthanol sur le comportement, ce est due, au moins en partie, à des changements dans la quantité d&#39;éthanol accumulé par les animaux 20. Agar NGM est de 160 mOsm. </li><li> Après séchage, peser chacune des plaques NGM non ensemencés pour être utilisé dans l&#39;essai et noter le poids. Déterminer le volume de milieu dans des plaques par rapport au poids d&#39;une plaque vide. Pour estimer le poids de conversion de la gélose au volume, supposons que le support pèse le même qu&#39;un volume égal d&#39;eau. NOTE: Nos résultats les plus constants ont été trouvés avec les médias NGM qui a suffisamment déshydratés qu&#39;un 10 volume d&#39;origine ml a un volume post-séchage entre 8.3 à 8.9 ml. Une alternative au temps de séchage de 2 heures est de sécher jusqu&#39;à ce que les plaques atteignent cette gamme de volume pour tenir compte des différences dans les incubateurs. </li><li> Faire fondre 4 anneaux de cuivre (diamètre interne de 1.6 cm) dans la surface de chacune des plaques d&#39;agir comme enclos pour les différents génotypes ou groupes de worms.Grasp chaque bague avec une pince, et le traitement thermique à flamme (une forte flamme d&#39;un brûleur Bunsen fonctionne bien) pour environ 3 sec. Placer immédiatement l&#39;anneau sur une plaque tout en maintenant la bague avec la pince pour l&#39;empêcher de «sauter». <ol><li> Assurez-vous que l&#39;anneau repose à plat sur la surface de la gélose en appuyant doucement avec la pince à plusieurs points sur l&#39;anneau. Lorsque vous placez l&#39;anneau, veillez à laisser de la place pour trois anneaux supplémentaires. </li><li> Faire fondre les trois anneaux de cuivre supplémentaires dans la surface de la plaque, en prenant soin de les placer aussi près que possible de sorte que tous les quatre seront dans le champ de vision de la caméra. NOTE: Faire une bonne étanchéité à l&#39;agar est essentiel de garder les vers dans les anneaux pendant l&#39;essai. Anneaux qui sautent autour sur la plaque sont peu susceptibles de faire l&#39;étanchéité correcte avec l&#39;agar etpeut cicatriser la gélose, qui permet aux vers de creuser et interfère avec la visualisation des vers. </li><li> Pour chaque plaque dosage préparer une plaque d&#39;accompagnement &quot;d&#39;acclimatation&quot;, qui devrait être séché et non ensemencées et recevra pas d&#39;éthanol. Placez quatre anneaux de cuivre sur ces plaques. </li></ol></li><li> Etiqueter le fond des assiettes à côté de l&#39;anneau avec la souche du ver à être utilisé dans le ring, en prenant soin de ne pas écrire dans le champ de vision de l&#39;anneau lui-même. Faites correspondre les étiquettes sur la plaque d&#39;essai avec sa plaque d&#39;acclimatation accompagnant. </li><li> Calculer la quantité de 100% d&#39;éthanol pour ajouter à chaque plaque de dosage de sorte que la concentration finale est de 0 mM ou 400 mM d&#39;éthanol (poids par volume). Pour chaque expérience (n = 1), il y aura une 0 mM et une plaque 400 mM d&#39;éthanol; plaques d&#39;acclimatation ne reçoivent pas l&#39;éthanol. Ajouter l&#39;éthanol, le pipetage autour de la surface de la plaque. Utilisez le film de laboratoire pour sceller la plaque et laisser se stabiliser sur la paillasse pendant 2 heures. </li><li> Lorsque 1,5 heure se est écoulée, commencer à l&#39;étape d&#39;acclimatation de l&#39;essai, l&#39;étape 2.2. </li></ol></li><li> Effectuer test de locomotion: plaques d&#39;essai <ol><li> Soigneusement transférer 10 vers de chaque groupe expérimental au centre d&#39;un anneau de cuivre sur la plaque d&#39;acclimatation. Enlevez tous les aliments qui est visible sur la gélose à ce point en grattant doucement avec le choix du ver. Variez l&#39;ordre dans lequel les groupes expérimentaux sont mis sur les plaques à travers des essais expérimentaux de sorte que les mêmes souches ne sont pas mis sur les plaques dans le même ordre entre les essais. NOTE: L&#39;objectif est de transférer les animaux à la plaque avec des quantités minimales de nourriture, parce que si la nourriture sur la plaque d&#39;acclimatation est transférée à la plaque de dosage, les vers vont se agréger autour de la nourriture et de l&#39;effet du médicament sur la locomotion sera obscurci. </li><li> Veillez à ne pas briser la surface de la gélose à la reprise, car cela va permettre aux vers de creuser et de perturber le test. Incuber les vers à température ambiante pendant 30 min. </li><li> Assurez-vous que il ya un intervalle approprié entre les initiations de chaque plaque. Un expérimentateur expérimenté peut déplacer 40 animaux, 10 / ring pour quatre anneaux dans &lt;1,5 min, mais ne importe quel intervalle jusqu&#39;à 2 min est acceptable. Le test standard a l&#39;enregistrement de films à 10-12 min et 30-32 min d&#39;exposition à la fois pour 0 mM et de l&#39;éthanol 400 mM. </li><li> Initier la plaque d&#39;acclimatation pour l&#39;exposition 0 mM et la plaque d&#39;acclimatation pour l&#39;exposition de 400 mm environ 2 min et 30 sec en dehors pour permettre économie du premier fichier de film avant le deuxième film doit commencer. </li><li> Après la période d&#39;acclimatation de 30 minutes, les transférer vers des plaques d&#39;acclimatation aux plaques de dosage. Transférer les vers aux plaques d&#39;essai (0 mm ou 400 mm) éthanol dans le même ordre qu&#39;ils ont été ajoutés à la plaque d&#39;acclimatation, garder la trace de la synchronisation entre les finitions de chaque plaque. Sceller la plaque avec un film de laboratoire pour minimiser la perte d&#39;éthanol à la vaporisation. </li><li> USE mouvement d&#39;écopage avec un ver choix mince tranchant aplati pour recueillir vers le haut de la reprise aplatie. Effectuer le transfert de vers de la plaque d&#39;acclimatation non ensemencée à la plaque de dosage, sans l&#39;utilisation de bactéries, qui est couramment utilisé dans le transfert vers car il contribue à coller au ver de la reprise. NOTE: La vitesse à laquelle les animaux sont transférés à cette étape est très important parce que les premiers vers sur la plaque seront exposés à l&#39;éthanol plus que les derniers vers ajoutés à la plaque, et des points de temps antérieurs peut montrer certains effets dépendant du temps. Ce est la principale raison pour faire tourner les souches sont placés sur une première plaque travers répétitions expérimentales. </li></ol></li><li> Effectuer Locomotion Assay: Tournage <ol><li> Utiliser une combinaison microscope / de caméra qui permet de imagerie simultanée de tous les quatre anneaux dans le champ de vision (environ 42x42 mm 2 carré), tel qu&#39;un objectif de microscope 0,5x, 0,8x grossissement et une caméra avec un 2048x2048 7,481; m pixel CCD. </li><li> Utilisez un éclairage uniforme à travers le champ de vision, ce qui facilite la reconnaissance d&#39;objets à l&#39;étape de seuil d&#39;intensité (voir ci-dessous). A 3 &quot;x3&quot; rétro-éclairage fonctionne bien. </li><li> Image les vers sur une plaque positionnée médias vers le haut (couvercle vers le bas), ce qui génère contraste qui est perdue lorsque le rétroéclairage par rapport au microscope traditionnelle transmise source de lumière. </li><li> Préparer le logiciel d&#39;analyse d&#39;image pour capturer des films en accéléré des vers mobiles. Réglez le logiciel pour capturer des images 12 bits de gris toutes les 1 sec, comme un 2-min (120 images) film. Pour réduire la taille du fichier de sortie, tout en conservant une résolution d&#39;image suffisante, utiliser un mode de regroupement de 2x2 pour capturer des images de 1024x1024 pixels. </li><li> Enregistrer les films. Commencer l&#39;enregistrement d&#39;un film de 120 châssis de la première plaque (0 d&#39;éthanol mM) 10 min après la dernière des vers ont été placés sur cette plaque. Enregistrez le fichier de film. </li><li> Enregistrez la deuxième plaque (éthanol 400 mM). Répétez ce processus pourles deux plaques commençant 30 min après la dernière vers ont été placés sur la première plaque (0 mM d&#39;éthanol) pour capturer les points de temps de 30 à 32 min pour chaque exposition. NOTE: Prévoyez suffisamment de temps pour enregistrer les fichiers image après chaque session de capture avant de commencer l&#39;enregistrement de la plaque suivante à analyser. </li><li> Pour l&#39;avenir épreuvage des films archivés et pour permettre à ces films d&#39;être ouverts et analysés par d&#39;autres logiciels d&#39;imagerie open source dans le domaine public, tels que ImageJ 28, convertir une copie de chaque film à 8 bits 256 fichiers TIFF d&#39;échelle de gris. REMARQUE: Le logiciel d&#39;analyse d&#39;image décrit ici utilise un format de fichier propriétaire. </li></ol></li><li> Analyse des films en utilisant le logiciel ImagePro. <ol><li> Une fois capturé, analyser les films en utilisant plus le logiciel Image-Pro ou son équivalent. REMARQUE: Une variété d&#39;autres logiciels de suivi d&#39;objet est disponible qui a été utilisé pour suivre avec succès multiples C. elegans à un moment 29 et telslogiciel pourrait raisonnablement se substituer à celle décrite ici comme les étapes d&#39;identification de l&#39;objet sont fondées sur des principes similaires. La méthode décrite se rapporte aux versions Image-Pro Plus logiciels de 6,0 à 6,3 et 7,0, de nouvelles versions de Image-Pro Plus logiciels peut avoir des différences mineures. </li><li> Analyser les films dans les segments 2 min (120 images). En premier lieu, appliquer un filtre aux images pour aplatir le fond et à améliorer le contraste des objets à vis sans fin. Sélectionnez le filtre comme suit: Menu&gt; Processus&gt; Filtres&gt; Amélioration&gt; Aplatir: Paramètres: background = dark; Feature Largeur = 20 Pix. </li><li> Re-enregistrer les films après l&#39;étape de filtrage mais toujours garder le film original dans sa forme non filtré. <ol><li> Analyser le locomotion des animaux dans chaque cycle séparément avec une zone circulaire d&#39;intérêt (ROI) qui est placé et dimensionné pour recouvrir l&#39;anneau de cuivre. Identifier et suivre les vers avec le menu&gt; Mesure&gt; suivre les objets ... commande; cela soulève Tracking Dfenêtre de la table de ata. Le bouton Options de suivi permet pistes spécifiques à exclure et de limiter les artefacts expérimentaux. </li></ol></li><li> Sous l&#39;onglet Suivi automatique, utiliser les paramètres suivants: Paramètres Piste: limite de vitesse (recherche de rayon) = 400 um / cadre, Accélération limite = Auto, la longueur minimale d&#39;une plage totale = 400 um, le type de mouvement prédominante = chaotique; Objets dans les paramètres de pistes: Permettre aux pistes partielles = yes, longueur min = 21 cadres, suivi profondeur de prédiction = 1 cadre. </li><li> Pour lancer le processus de suivi, cliquez sur le Trouver toutes les pistes automatiquement touche fonction pour faire apparaître le / boîte de dialogue Options de taille de comte et la boîte de dialogue de suivi. Sélectionnez l&#39;option Manuel pour la sélection de gamme d&#39;intensité dans la boîte de dialogue comte / Taille, qui fournit l&#39;étape de seuil importante qui utilise l&#39;intensité d&#39;échelle de gris, le ver pixels de mettre en évidence un objet de ver pour analyse et d&#39;ignorer les légers fond pixels. <ol><li> Réglez le seuil d&#39;intensité sliDER (une pour définir la limite supérieure, une pour définir la limite inférieure) pour créer une gamme inclusive qui met en évidence tous les objets sombres. Une gamme de 0-1,500 sur une échelle de 0-4,095 est un bon point de départ pour le réglage plus fin. </li><li> Appliquer un filtre de taille pour exclure les objets qui sont plus grandes et plus petit qu&#39;un objet ver unique, comme l&#39;anneau de cuivre et tous les débris sur les plaques. Placer les deux paramètres de taille pour ce faire que couvrent tailles pour chaque ver dans une variété de postures avec la mesure&gt; sélectionner les mesures élément de menu sur la boîte de dialogue des options comte / Taille. (Gamme Area: 28,000-120,000 um 2 et la gamme Périmètre: 600-2,500 um). </li><li> Si une souche mutante est nettement plus petit ou plus grand que les autres animaux sur la plaque pour être analysé, élargir la gamme des paramètres de filtre pour la reconnaissance d&#39;objet pour accueillir les différentes tailles des souches avant de suivre les animaux afin que les paramètres ne ont pas besoin d&#39;être changé mi-analyse. </li></ol> &lt;/ Li&gt; <li> Achever le processus de suivi en cliquant sur Continuer dans la boîte de dialogue de suivi. Comparer visuellement les pistes de sortie avec les progrès de chaque ver dans le film afin de se assurer que chaque ver est représenté moins qu&#39;il y ait une raison valable pour exclure sur la base des réglages automatiques de filtrage. Supprimez manuellement les pistes qui ont été produites par la présence d&#39;objets non-ver confirmés qui répondaient aux critères de filtrage de taille. </li><li> Utilisez le logiciel pour calculer la vitesse de chaque ver entre chaque cadre (distance parcourue pour le centre de gravité d&#39;un objet par une sec entre les cadres) et afficher la vitesse moyenne pour chaque piste et la vitesse moyenne sur toutes les pistes pour la population de vers dans le anneau de cuivre. Considérez cette moyenne finale soit n = 1 en termes d&#39;essais expérimentaux. Exporter les données vers un tableur pour les analyses statistiques et l&#39;archivage des données. </li><li> Une fois que les données ont été enregistrées pour le premier anneau sur la plaque, déplacer le ROI à l&#39;anneau suivant etrépéter le processus de suivi, sans modifier les paramètres. </li><li> Calculer la vitesse relative de la locomotion à travers: </li><li> La vitesse relative (%) = traité (400 mM) de vitesse / non traitée (0 mm) Vitesse x 100. REMARQUE: les manipulations génétiques différentes modifient souvent le taux de la locomotion de base des animaux. Afin de déterminer l&#39;effet de l&#39;éthanol sur la vitesse de locomotion des animaux et d&#39;être en mesure de comparer ces effets à travers différents génotypes ou conditions, calculer une vitesse relative. </li></ol></li></ol>

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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+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, 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=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., 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=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

La neurobiologie simple et des outils génétiques disponibles en C. elegans faire le ver un excellent modèle pour étudier les bases moléculaires des effets de l&#39;éthanol sur le comportement. Ici, nous décrivons un test qui a été utilisé pour identifier plusieurs médiateurs moléculaires et environnementaux de la réponse comportementale aiguë en éthanol 8,10,20,24,25. Cette méthode permet la différenciation et l&#39;examen simultané des deux phénotypes de comportement de réponse d...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

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.

Les données représentatives (figure 1) de plusieurs génotypes différents et leurs contrôles appariés sont présentés 8,24; données ont été spécifiquement choisis que les différences de mettre en évidence chez les animaux testés. Le degré d&#39;effet à 10 min de l&#39;exposition est considérée comme la sensibilité initiale d&#39;une souche, qui est représentée sur les axes de gauche de la figure 1B-G. Les souches mutantes avec une vitesse relative plus gra...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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 ...

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Neuroscience

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

8.5K vues.  Virginia Commonwealth University. 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. 

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

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialyse d&#39;éthanol au cours de l&#39;éthanol détermination opérant auto-administration et d&#39;éthanol par chromatographie en phase gazeuse

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Recherche

Neurosciences

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Un test de perméabilité du neuro-épithélium embryonnaire poisson zèbre

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Caenorhabditis elegans Model System for Amylopathy Study

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

A Caenorhabditis elegans Model System for Amylopathy Study

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

A<em&gt; Caenorhabditis elegans</em&gt; Système modèle pour l&#39;étude de Amylopathy

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

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Méthodes pour étudier les mécanismes d&#39;action des antipsychotiques dans<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Un, test de comportement évolutives et économiques pour mesurer l&#39;éthanol sédation sensibilité et la tolérance rapide dans<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

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.

Microchambres agarose à long terme de l&#39;imagerie calcique<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 ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Novel Metrics à Caractériser Embryonic Allongement du nématode<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Méthode électrophysiologique de réponses d&#39;enregistrement intracellulaire tension de<em&gt; Drosophila</em&gt; Photorécepteurs et Interneurones à Light Stimuli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UNE<em&gt; Caenorhabditis elegans</em&gt; Ensemble d&#39;aversion en cuivre à base d&#39;état nutritionnel

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagerie ratiométrique Calcium des neurones individuels en comportement Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...