Ce travail a été financé par des subventions des Instituts nationaux de la santé: EY015748, EY017964 (MET), et EY019517 (ASV). Ce travail a également été soutenue par la recherche pour prévenir la cécité sans restriction Grant au département d&#39;ophtalmologie et les Lions de New York Central. Nous tenons également à remercier notre technicien animalier, Matthew Mellini, pour son excellent soin des animaux et pour le renforcement de la vedette dans cette vidéo.

Protection des animaux Le têtards de Xenopus laevis utilisées dans cette étude ont été cultivés, élevés et traités conformément aux procédures approuvées par Upstate Medical University IACUC et le Guide pour les soins et l&#39;utilisation des animaux de laboratoire. . 1 Configuration Pré-comportement: têtards <ol><li> Obtenir des embryons de xénope fécondés provenant d&#39;une source disponible dans le commerce ou in vitro féconder les ovocytes, comme décrit précédemment 6. </li><li> Placez blastula des embryons de neurones de la scène de la plaque à 18 ˚ C en 60 mm boîtes de Pétri contenant 0,1 X MMR [10 modifié (ROR) solution mère de Ringer X Marc (10 mM MgCl 2, 20 mM de KCl, 20 mM de CaCl2; HEPES 50 mM; NaCl 1 M; ajusté à pH 7,5, passé à l&#39;autoclave à température ambiante et stockées)] avec 50 ug / ml de l&#39;antibiotique gentamicine. Changez 0,1 X MMR chaque jour et éliminer les embryons morts. </li><li> Une fois les embryons atteignent Nieuwkoop et Faber stade ~ 27 au 30 juillet, passer èmee embryons dans 0,1 X MMR sans antibiotique pour permettre la croissance bactérienne de l&#39;intestin. </li><li> Cultiver les têtards jusqu&#39;à ce qu&#39;ils atteignent le stade d&#39;alimentation (étape 45) et déplacez-les dans des boîtes de Pétri de 100 mm remplis de 0,1 X MMR. Les nourrir ortie poudre surnageant chaque jour pendant une semaine, en changeant le 0,1 X MMR le jour intervenir. </li><li> Après une semaine dans le plat 100 mm Petri, déplacer les animaux de la moitié des réservoirs de gallons remplis d&#39;eau de grenouille [0,5 g / L océan instantanée et 2 mM de phosphate de sodium dibasique (Na 2 HPO 4: mw 141,96) pH 6,8]. Nourrissez-les comme dans l&#39;étape 2.4, mais changer l&#39;eau seulement 2-3 fois par semaine lorsque l&#39;eau est trouble. Maintenir les animaux dans un cycle lumière obscurité de 12 h de lumière/12 avec les lumières allumer à 6 heures. </li><li> Placez le réservoir demi-gallon avec les sujets de test sur une surface blanche, comme un sous-tapis de paillasse, au moins 12 heures O / N, avant le test. </li><li> têtards d&#39;essai aux stades 45 à 50 comme décrit dans l&#39;étape 3 ci-dessous. </li></ol> . 2 Configuration Pré-comportement: Equipment <ol><li> Mettez de côté une zone de laboratoire pour l&#39;analyse du comportement. Il devrait être dans un quartier calme à faible trafic avec l&#39;éclairage fluorescent standard. </li><li> Préparer les bacs de test pour les animaux. Les bacs de test, un demi-gallon imbriqués sont composées de deux parties: un réservoir interne qui contiendra l&#39;eau et têtards; et un réservoir externe avec le stimulus visuel. <ol><li> Pour le réservoir interne, faire une petite marque sur les coins extérieurs avec un feutre, 5 cm du fond; il s&#39;agit de la ligne d&#39;eau (figure 1A, la ligne pointillée verte). </li><li> Aussi, pour le réservoir intérieur, remplir les divets dans le réservoir (dans les coins et au centre) avec un composé inerte, comme Sylgard élastomère. NOTE: Les têtards s&#39;attarder dans ces domaines si elles ne sont pas remplis po </li><li> Pour le réservoir externe, couvrir exactement la moitié de l&#39;extérieur d&#39;un réservoir avec du ruban électrique noir et l&#39;autre moitié avec le tissu blanc papier Figure 1B. </li></ol></li><li> Placez les réservoirs sur leinoculation platine. </li><li> Configuration de la webcam / trépied afin qu&#39;il soit au-dessus des bacs de test. Régler la caméra pour permettre la visualisation de la zone d&#39;essai, comme le montre la figure 1C. </li><li> Connectez la webcam à un ordinateur avec le logiciel QuickTime Player installé 5. Allumez l&#39;appareil photo. Dans QuickTime Player, sous Fichier, sélectionnez &quot;Nouvel enregistrement de séquence. La configuration de test de comportement sera visible sur l&#39;ordinateur tout de suite. </li><li> Drapé d&#39;un chiffon de coton léger sur l&#39;ensemble de la configuration pour réduire indices externes qui pourraient influer sur le comportement des têtards, ainsi que, de réduire la lumière réfléchie sur la surface de l&#39;eau. </li><li> Veiller à ce que la luminance au titre des mesures de tissu entre 35-50 cd / m 2. </li></ol> 3. Comportement Assay <ol><li> Remplir le réservoir de test interne pour la marque de 5 cm d&#39;eau fait dans l&#39;étape 1.2.1 avec de l&#39;eau de grenouille. </li><li> L&#39;utilisation d&#39;un petit filet, déplacez doucement l&#39;animal dans le réservoir de test interne. </li><li> Ouvrir QuickTime logiciel sur ordinateur et, à l&#39;aide du réservoir externe comme un guide, assurez-vous que l&#39;appareil est en mesure de visualiser et d&#39;enregistrer la zone de test. </li><li> Écrivez le nom de l&#39;animal, la date et l&#39;heure sur un morceau de papier et d&#39;enregistrer avec la caméra. </li><li> Placez le réservoir de test dans le réservoir extérieur, le côté noir du réservoir vers la droite Figure 2. </li><li> Commencer à enregistrer le film immédiatement après l&#39;organisation du réservoir et réglez la minuterie pendant 2 min. </li><li> Quand le minuteur sonne, retirer le réservoir de test interne, tourner le réservoir externe de 180 ° et placez le réservoir de test interne à l&#39;intérieur du réservoir externe. Démarrer la minuterie. Remarque: le côté noir devrait maintenant être de l&#39;autre côté. </li><li> Répétez l&#39;étape 3.7, huit fois plus pour un total de dix essais. Utilisez la figure 2 comme un guide, en cochant chaque essai. </li><li> Répétez le test de comportement sur deux jours différents. </li></ol> 4. Rétinienne axotomie <ol><li> Placez l&#39;animal dans 0,02% tricaine jusqu&#39;à ce qu&#39;ils ne répondent pasà une pincée de queue avec un # 3 forceps. </li><li> Faire fondre 1% d&#39;agarose dans 0,1 X MMR puis ajouter à une boîte de Pétri de 60 mm. Une fois refroidie, une petite motte de terre rectangulaire dans l&#39;agarose et ajouter le têtard de la motte de terre avec un peu de tricaïne 0,02%, de sorte que l&#39;animal est partiellement immergé dans le liquide. </li><li> Pierce la peau à un angle de 45 degrés derrière la région dorsale de l&#39;oeil avec une aiguille 25 G, tout en s&#39;agrippant de l&#39;animal contre la pince sur le côté opposé. </li><li> Prendre soin de ne pas couper la veine, qui se trouve à côté du nerf optique, atteindre soigneusement dans le trou avec # 5 pinces, couper le nerf optique et le retourner à l&#39;écart. Si il ya une hémorragie sévère en raison de la biopsie accidentellement l&#39;artère, puis placer rapidement l&#39;animal dans 2% tricaïne à euthanasier. ATTENTION: solution de tricaine 2% peut provoquer un engourdissement dans les humains. Cette solution doit être manipulé avec des gants. </li><li> Pour récupérer l&#39;animal après la chirurgie, placer l&#39;animal dans 100 mm boîte de Petri avec 0.7X ROR et 50 pg / ml de gentamicine pendant 20 min. Next, transfert têtard à la cuve de récupération d&#39;eau de grenouille. Laisser reposer animale O / N. </li><li> Le lendemain matin, tester la fonction visuelle comme décrit ci-dessus (étapes 3.1 à 3.9). </li></ol> 5. Analyser les résultats <ol><li> Après les essais sont terminés, mesurer la quantité de temps le têtard reste sur le côté noir de la cuve. <ol><li> Voir manuellement les vidéos en utilisant un logiciel qui affiche l&#39;heure et permet des pauses fréquentes. Regardez le temps d&#39;affichage de la vidéo. Notez le début du procès de deux minutes, ce qui est quand le réservoir interne niche avec succès à l&#39;intérieur du réservoir externe et est carré en vue de la caméra. </li><li> Utilisez la position des yeux du têtard à définir de quel côté de la cuve les animaux nagent sur. Définir la traversée d&#39;un côté de la cuve à l&#39;autre que lorsque les deux yeux ont franchi la ligne noir / blanc. </li></ol></li><li> Notez le début et la fin de chaque intervalle (en secondes) que l&#39;animal passe sur le côté noir. Faites une pause et reenrouler la vidéo pour assurer l&#39;exactitude. Additionner le sec dans chaque essai. </li><li> Entrée ces nombres dans la feuille de calcul du comportement joint. La feuille de calcul calcule le rapport entre le temps que l&#39;animal a passé sur le côté blanc en soustrayant le temps passé sur le côté noir de la cuve de l&#39;essai sec 120 au total et le diviser par le temps total en secondes [(120 - nombre total de secondes consacrées à le côté noir) / 120 sec]. Les moyennes de feuille de calcul de ce ratio sur l&#39;ensemble des 10 essais. </li><li> Utilisez le T-test de Student, jumelé, distribution bilatérale afin de déterminer si les essais entre les jours sont significatives (P ≤ 0,05). </li></ol>

<ol>
	<li>Maurer, C. M., Huang, Y. Y., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zebrafish+oculomotor+behavior+to+model+human+disorders.">Application of zebrafish oculomotor behavior to model human disorders.</a> Rev Neurosci. 22 (1), 5-16 (2011).</li><li>Solessio, E., Scheraga, D., Engbretson, G. A., Knox, B. E., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+temporal+properties+of+the+rod+pathway+in+larval+Xenopus.">Circadian modulation of temporal properties of the rod pathway in larval Xenopus.</a> J Neurophysiol. 92 (5), 2672-2684 (2004).</li><li>Moriya, T., Kito, K., Miyashita, Y., Asami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+background+color+of+the+Xenopus+laevis+tadpole.">Preference for background color of the Xenopus laevis tadpole.</a> J Exp Zool. 276 (5), 335-344 (1996).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7 (8), (2009).</li><li>Choi, R. Y., 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=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 52 (1), 364-373 (2011).</li><li>Viczian, A. S., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+determination+using+the+animal+cap+transplant+(ACT)+assay+in+Xenopus+laevis.">Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.</a> J Vis Exp. 16 (39), (2010).</li><li>Nieuwkoop, P. D., Faber, 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> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second-generation+device+for+automated+training+and+quantitative+behavior+analyses+of+molecularly-tractable+model+organisms.">A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.</a> PLoS One. 5 (12), (2010).</li><li>Rosemberg, D. B., 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=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS One. 6 (5), (2011).</li><li>Dong, W., 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=Visual+Avoidance+in+Xenopus+Tadpoles+is+Correlated+With+the+Maturation+of+Visual+Responses+in+the+Optic+Tectum.">Visual Avoidance in Xenopus Tadpoles is Correlated With the Maturation of Visual Responses in the Optic Tectum.</a> J Neurophysiol. 101 (2), 803-815 (2009).</li><li>Lan, L., 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=Noggin+Elicits+Retinal+Fate+In+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate In Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. 27 (9), 2146-2152 (2009).</li><li>De Robertis, E. M., Kuroda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+and+neural+induction+in+Xenopus+embryos.">Dorsal-ventral patterning and neural induction in Xenopus embryos.</a> Annu Rev Cell Dev Biol. 20, 285-308 (2004).</li></ol>

The authors have nothing to disclose.

Nous rapportons ici un test de comportement guidé par la vision simple qui peut être facilement réalisée en moins d&#39;une semaine par le personnel de laboratoire une formation minimale. Alors que d&#39;autres tests nécessitent un équipement spécialisé et de l&#39;expertise en comportement animal, ce test permet un test rapide pour déterminer la fonction visuelle. Un autre essai de comportement, le test d&#39;évitement visuel, a été développé pour déterminer comment le tectum contribue à la perception v...

Xenopus laevis ont été utilisés comme un organisme modèle pour étudier la formation des yeux. Les yeux se développent rapidement, de plus en plus de maturité dans moins d&#39;une semaine des gènes ou des voies d&#39;essai qui ont un effet sur le développement et la fonction visuelle. Pour tester la fonction visuelle, la réponse optocinétique et optomoteur a été utilisée dans le poisson-zèbre et les têtards de Xenopus, respectivement 1,2. Parce que les yeux de têtards de Xenopus sont relativement plus petit que le poisson-zèbre, ce test nécessite l&#39;utilisation d&#39;équipement spécialisé et de personnel qualifié pour détecter le comportement subtil queue-flip et mouvements oculaires chez Xenopus. Un comportement plus robuste chez Xenopus est la préférence pour la natation dans un réservoir avec un fond blanc, ce qui est décrit ici 3. Lorsque vous placez un têtard dans un réservoir à moitié noir / moitié blanc, le têtard pré-métamorphique nage rapidement sur le côté blanc de la cuve. Nous avons déjà utilisé ce test pour déterminer si pluripotentes dérivé des cellulesyeux étaient fonctionnels 4. Nous rapportons ici une version détaillée de ce test, qui peut être utilisé pour tester la fonction visuelle des têtards de Xenopus prémétamorphique. Ce test est plus simple que le test de réponse optomoteur, car il ne nécessite un caméscope monté numérique, logiciel d&#39;appareil photo numérique et un équipement standard dans la plupart des laboratoires de Xenopus. En outre, la réponse enregistrée ne nécessite aucune formation particulière aux résultats de pointage. Nos résultats représentatifs montrent que le même groupe de têtards, ayant subi deux axotomy rétinienne, nagent aléatoirement autour du réservoir. Nous avons également inclus les résultats d&#39;analyse du comportement d&#39;un têtard représentant, en montrant comment un oeil peut être testé pour la réponse visuelle. Une feuille de calcul a été inclus afin que les numéros acquises au cours de l&#39;essai peuvent être insérés et analysés. Cette feuille peut être utilisée pour déterminer si les têtards testés ont une réponse visuelle.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1/2-gal. Flex-Tank with Cover</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">SB19271M</td>
			<td style="width:225px;">size: 5-3/8&#34; x 7&#34; x 3-3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black electrical tape</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White tissue paper</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large Inoculating Turntable</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">50809-022</td>
			<td>size: Dia 114.3 x&#160; H 76.2 mm (4 1/2&#160; x&#160; 3&#34;)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Durasorb Underpad</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">82004-836</td>
			<td>size: 43.2 x 60.1 cm (17 x 24&#34;)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Kim wipe</td>
			<td style="width:95px;">Krackeler Scientific, Inc.</td>
			<td style="width:119px;">1945-34155-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Tripod</td>
			<td style="width:95px;"></td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iSight camera or webcam</td>
			<td style="width:95px;">Apple</td>
			<td style="width:119px;">M8817LL/A</td>
			<td>Good for larger tadpoles but small ones are difficult to see</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable computer</td>
			<td style="width:95px;">Apple/PC</td>
			<td style="width:119px;">various</td>
			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiniDV Handycam Camcorder</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCR-HC42</td>
			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Handycam Station</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCRA-C121</td>
			<td>This can be used for connecting firewire to camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuickTime Player software</td>
			<td style="width:95px;">Quicktime</td>
			<td style="width:119px;">Version 10.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">26 Gauge Needle (5/8&#34; length)</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">BD305115</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Dumont #5 Forceps Biology</td>
			<td style="width:95px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Disposables</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamicin Sulfate [50 mg/ml]</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">17-528Z</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sylgard 184 Silicone Elastomer</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Instant Ocean</td>
			<td style="width:95px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td>Stock solution = 100 g/L stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">S0876</td>
			<td>Stock solution = 0.4 M stored at room temperature</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tricaine (Ethyl-3-aminobenzoate methanesulfonate salt)</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td>Stock solution = 10% stored at -20&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">LM00490MX</td>
			<td>100 embryos/unit</td>
		</tr>
	</tbody>
</table>

a simple behavioral assay for testing visual function in xenopus laevis

Mesure de la fonction visuelle chez les têtards de la grenouille Xenopus laevis, permet le dépistage de la cécité chez les animaux vivants. La réponse optocinétique est un comportement réfléchi basé sur la vision qui a été observée chez tous les vertébrés testés. Tadpole yeux sont petits, la réponse queue de bascule a été utilisé comme mesure de rechange, ce qui nécessite un technicien qualifié pour enregistrer la réponse subtile. Nous avons développé un test de comportement alternatif basé sur le fait que les têtards préfèrent nager sur le côté blanc d&#39;un réservoir lorsqu&#39;il est placé dans une cuve avec des côtés à la fois en noir et blanc. L&#39;analyse présentée ici est une alternative simple peu coûteux qui crée une réponse qui est facilement mesurable. L&#39;installation se compose d&#39;un trépied, d&#39;une webcam et bacs de test imbriqués, facilement disponibles dans la plupart des laboratoires de Xenopus. Cet article comprend un film montrant le comportement des têtards, avant et après le sectionnement du nerf optique. Afin de tester la fonction d&#39;un oeil, on inclut également reprérésultats représentatifs d&#39;un têtard dans lequel chaque œil a subi axotomy rétinienne sur plusieurs jours consécutifs. Les études futures pourraient développer une version automatisée de ce test pour tester la vision de nombreux têtards à la fois.

Les rapports précédents ont montré que les têtards de Xenopus prémétamorphique préfèrent nager sur le côté blanc d&#39;un réservoir noir / blanc et a appelé cet essai, l&#39;arrière-plan de couleur préférée de dosage 3 Nous avons changé ce test afin de tester la fonction visuelle des deux yeux de têtards dans moins d&#39;une semaine. De cette manière, les yeux peuvent être collectées pour un examen histologique. Nous montrons ici comment la réponse e...

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Têtards de Xenopus laevis préfèrent nager sur le côté blanc d&#39;un réservoir noir / blanc. Ce comportement est guidé par la vision. Sur la base de ce comportement, nous présentons un test simple pour tester la fonction visuelle des têtards.

Un test comportemental simple pour tester la fonction visuelle dans<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

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Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.7K vues.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  Têtards de Xenopus laevis préfèrent nager sur le côté blanc d'un réservoir noir / blanc. Ce comportement est guidé par la vision. Sur la base de ce comportement, nous présentons un test simple pour tester la fonction visuelle des têtards. 

Vidéo: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Les vermicelles et tests de manipulation Capellini: Simple mesures quantitatives de la fonction patte habile dans les rats et les souris

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Recherche

Neurosciences

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Une méthode générale pour l&#39;évaluation d&#39;incubation du désir saccharose chez les rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Des cultures d&#39;explants neuraux de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, de la culture, et de l&#39;analyse des<em&gt; Xenopus laevis</em&gt; Tissus embryonnaires rétine

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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 ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Un léger Casque-based System pour manipuler Rétroaction sonore chez les oiseaux chanteurs

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultané<em&gt; Ex vivo</em&gt; Tests fonctionnels de deux rétines par<em&gt; In vivo</em&gt; Système Électrorétinogramme

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

A Behavioral Essai pour mécanosensibilité de marcm-fondé clones dans<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Enregistrement Neuronal activité induite de Température-travers le contrôle des modifications de calcium dans le bulbe olfactif de<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Préparations et protocoles pour toute cellule Patch Clamp enregistrement des neurones Tectal Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...