Ces méthodes ont été développées par EEL pendant un congé de recherche dans le laboratoire du JT appui a été fourni par le Conseil de recherche et de l&#39;Université DePaul une NIH / NIDCR R01 accorder à JT (DE016678). Nous tenons à souligner les efforts de Hunter Caroline, technicien de soins aux animaux dans l&#39;établissement CMRC coeur de poisson zèbre et Paulina Pawluczuk, notre assistant de laboratoire de premier cycle.

Le poisson zèbre élevage Le poisson zèbre sont logés et élevés selon des méthodes standard 1. Passé la période de fin larvaire, le stade de développement d&#39;un poisson zèbre peut être mesurée par sa longueur standard (SL), ou la distance en ligne droite en millimètres du point plus antérieurs de la lèvre supérieure à la base de la nageoire caudale (la plaque posteriormost hypural) 2. A environ un mois après la fécondation (10-12 mm LS), les barbillons nasaux et maxillaires apparaissent comme transparents bourgeons épithéliaux près du puits olfactives et sur les coins postérieurs des maxillaires, respectivement. Comme le poisson zèbre mûrit, les barbillons s&#39;étendent dans étroite, moustache appendices semblables. Parce que le barbillon maxillaire est plus large (2-3 mm) et plus facile à manipuler, notre protocole ne s&#39;applique qu&#39;aux cet appendice particulier; des techniques similaires, cependant, pourrait être adapté aux plus petits le barbeau nasale. Clipping Barbel <ol><li> Anesthésier poisson zèbre adulte au stade désiré (p. ex., De 1,5-2,5 cm SL, ~ 3-6 mois après la fécondation) par immersion dans de 0,015% MS-222 (éthyl 3-aminobenzoate méthanesulfonate) tamponné à pH 7,0 dans de l&#39;eau du système. Observez les mouvements de natation jusqu&#39;à arrêter et ventilation des branchies devient lente et régulière. Selon la taille du poisson, une anesthésie complète prend environ 2-5 minutes. </li><li> Utiliser une résille d&#39;aquarium, le transfert de poissons 1-3 à la fois à un morceau plié de serviette en papier humide dans une boîte de Pétri. En utilisant une spatule, d&#39;orienter le poisson donc ils sont parallèles les uns aux autre côté et latéral gauche en place. Pliez une partie de la serviette en papier humide sur la partie caudale du poisson, couvrant tout le corps jusqu&#39;à l&#39;opercule (opercules) pour garder la peau et les branchies humides pendant la procédure. </li><li> Voir la plaque de Petri sous la loupe binoculaire, en utilisant la lumière incidente à angle faible pour éclairer la région de la tête. Focus sur la base de la gauche barbeau maxillaire, qui fait saillie de l&#39;angle ventral postérieur du maxillaire (Fig. 1). <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="Figure 1" /> Figure 1. </li><li> Étroitement saisir l&#39;arbre de l&#39;légèrement distale au bord du maxillaire barbeau. Élevez l&#39;arbre barbeau et insérez les mâchoires d&#39;une paire de ciseaux à ressort à pointe fine juste en amont de la pince. Les ciseaux peuvent être touchés à la pince pour la stabilité. Glissez les mâchoires de la paire de ciseaux le long de la tige du barbeau jusqu&#39;à la pointe se rapproche du bord du maxillaire. Fermer les ciseaux pour couper à travers l&#39;arbre barbeau. Le barbeau amputée, toujours serrée dans la pince, peut être enlevé pour la fixation ou observation. </li><li> Immédiatement transférer le poisson à un petit réservoir d&#39;eau potable du système (~ 500 ml) contenant une goutte de bleu de méthylène pour le contrôle superficiels infection fongique. Laisser le poisson à récupérer pendant la nuit. Le lendemain matin, retour le poisson pour le système d&#39;élevage. Barbel régénération est maximale après 2 semaines. </li></ol> Incorporation de gélose de paires appariées Barbel <ol><li> Recueillir les poissons par la technique d&#39;euthanasie appropriées et fixer dans un tube de 50 ml de 4% de paraformaldéhyde-tampon phosphate salin (PBS) avec agitation à 4 ° C pendant la nuit. Le volume fixateur doit être d&#39;au moins dix fois le volume du tissu. Après fixation, d&#39;éliminer les déchets paraformaldéhyde dans un contenant approuvé et rincer le tissu à fond dans plusieurs changements de PBS. </li><li> Préparer dans un flacon en verre de 250 ml 150 ml d&#39;agarose à 2% (T m ~ 37 ° C) dans de l&#39;eau distillée. Chauffer avec un micro-ondes ou plaque chauffante, en agitant périodiquement jusqu&#39;à dissolution complète. Equilibrer la solution fondue dans un bain de 50-60 ° C eau. </li><li> Assemblez un gréement gel standard d&#39;électrophorèse en plaçant un moule gel de petits joints (10 x 10 cm; ~ volume de 100 ml de gel) fermement contre les parois de la chambre tampon. Ajouter 2-4 petit échantillon en dents de peigne (4 x 1,5 mm). </li><li> Sur une surface plane, verser la fondue d&#39;agarose dans le moule pour juste couvrir la base des peignes, formant des puits très peu profond. Immédiatement mis l&#39;agarose restes de nouveau dans le bain d&#39;eau chaude, ce qui sera utilisé plus tard pour intégrer le tissu (étapes 9-12). Mettez une longue pipette Pasteur en verre dans le ballon pour maintenir la pipette à la même température que l&#39;agarose. </li><li> Laisser le gel de durcir pendant 20-30 minutes. Retirer les peignes, puis retirez le moule du gel contenant le gel durci de la chambre tampon. Fermement bandes sur les côtés ouverts du moule avec du ruban de gel en laboratoire alors le gel ne glisse pas. Cette bande doit s&#39;étendre de quelques millimètres sur la surface de l&#39;agarose afin d&#39;agarose supplémentaires peuvent être ajoutés plus tard (étape 13). </li><li> Position le moule du gel sur la scène d&#39;un stéréomicroscope. Magnify un puits vide et rapprocher les bords au point. </li><li> Sur la surface de l&#39;adjacentes au puits d&#39;agarose, une pipette une goutte d&#39;eau ou de tampon contenant les échantillons fixés barbeau (par exemple, se régénérer et de contrôle). Using plié broches insectes, faites glisser les échantillons humides dans le puits et les pousser vers le bas. Orienter le barbeau arbres parallèles les uns aux autres et alignés contre les bords opposés long du bien. La tension de surface va contribuer à maintenir le tissu en position. </li><li> Lorsque vous êtes prêt à intégrer, utiliser une pipette fine ou au coin d&#39;un tissu de laboratoire pour enlever l&#39;excès de fluides dans le puits. Travaillez rapidement afin de ne pas laisser les tissus se dessèchent. </li><li> Utilisation de la pipette Pasteur en verre réchauffé doucement pipette propre agarose fondu dans et autour des spécimens pour sceller le tissu barbeaux en place. Ne remplissez pas trop. Avant l&#39;agarose durcit, faire ajustements de dernière minute avec les broches d&#39;insectes. </li><li> Placez une étiquette en papier numéroté spécimen à côté du puits et le fixer avec une petite goutte d&#39;agarose. </li><li> Répétez les étapes 6 à 10 pour chacun des puits d&#39;échantillons. </li><li> Si vous le souhaitez pour le calibrage d&#39;image, insérez un morceau de papier avec une échelle micrométrique imprimés (par exemple, <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> ) dans le fond d&#39;un puits vide. Aplatir le papier au fond du puits et recouvrez chaude d&#39;agarose. </li><li> Après tous les spécimens sont intégrés, la surface du gel sera irrégulière, avec des gouttes durcies d&#39;agarose. Placer le gel sur une surface plane et verser doucement supplémentaires fondue agarose jusqu&#39;à la surface supérieure est uniforme. Utilisez la plus petite quantité d&#39;agarose nécessaire pour obtenir une surface lisse, trop d&#39;agarose sera obscure diascopie photographie. Permettre que cette couche supérieure de durcir. </li><li> Le gel peut désormais être photographiés sur la scène d&#39;un stéréomicroscope pour enregistrer la morphologie brute pour chaque paire de barbeau. Calibration des images est obtenue en photographiant l&#39;échelle micrométrique embarqués. </li><li> Le gel complet peut être stocké enveloppé dans des serviettes en papier humide et scellé dans un sac en plastique à 4 ° pendant plusieurs semaines. </li><li> Pour une analyse plus poussée, des blocs de gélose contenant paire de barbillons peuvent être découpées dans du gel avec une lame de scalpel ou un rasoir et traitées pour l&#39;histologie de paraffine ou cryosectioning par des méthodes standard 3,4. Alternativement, barbeaux individuel peut être disséquée de l&#39;agarose avec de fines aiguilles, rincés à l&#39;eau, et traitées à d&#39;autres applications en aval (par exemple, l&#39;ensemble de montage immunohistochimie ou la microscopie confocale). </li></ol>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, 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=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

Le poisson zèbre barbillon maxillaire est un système de tissu sous-utilisés pour l&#39;étude de la croissance, l&#39;entretien et la régénération de plusieurs types cellulaires chez le poisson zèbre. Bien que l&#39;appendice n&#39;a pas le barbeau analogue humain, les types de cellules qu&#39;il contient sont hautement conservées, ce qui rend possible d&#39;étudier la peau, les glandes, les mélanocytes vaisseaux circulatoires, et les nerfs dans une structure optiquement clair et anatomiquement cylindrique simple. Semblable à la nageoire caudale bien étudiés, le barbeau tissus peut être induite par l&#39;amputation de se régénérer. Utilisation de la frontière du maxillaire comme un repère anatomique, le plan de l&#39;amputation peut être placé précisément, à faciliter la mesure des barbeaux repousse. Pour un opérateur expérimenté, chaque opération ne prend que quelques secondes. La récupération est rapide, et nous avons jusqu&#39;ici détecté aucun effet à court ou à long terme sur le comportement des poissons. Le poisson zèbre avec un barbeau nager maxillaires, manger et se reproduisent aussi efficacement que non-chirurgical des contrôles, et ont de survie comparable à l&#39;époque de la collecte de tissus, jusqu&#39;à 6 mois après la chirurgie. L&#39;impact physiologique de cette chirurgie prédit d&#39;être minime, car 1) les papilles extra réalisé sur le barbeau se retrouvent également sur ​​de nombreuses autres parties de l&#39;épithélium de poissons, y compris les lèvres, les joues et la tête 5, et 2) les papilles de différenciation apparaissent sur le barbeau régénération dans les 72 heures (Leclair et al., données non publiées). Cela rend le barbillon maxillaire un système minimalement invasives pour étudier la cicatrisation des plaies, la revascularisation et une réinnervation dans le cadre d&#39;un vertébré adulte. Après l&#39;induction chirurgicale de la régénération, barbillons peuvent être prélevés à intervalles de mesure morphométrique et / ou l&#39;analyse microscopique des tissus fixés. Idéalement, le barbillon maxillaire est d&#39;environ la longueur (2-3 mm) et diamètre (100-200 mm) d&#39;un embryon de poisson zèbre, de faciliter l&#39;application de plusieurs protocoles standard, y compris histologie paraffine, cryosectioning, toute monture immunohistochimie, et in situ hybridation. Pris ensemble, ces caractéristiques font du barbillon maxillaire hautement réalisable modèle in vivo pour l&#39;étude de la réparation des tissus et la régénération.

Matériel Microscope Un microscope stéréoscopique à disséquer à transmettre et l&#39;incident éclairage par fibre optique est nécessaire pour écrêtage barbeau et intégrant agar. Clipping Barbel <ul><li> Poissons d&#39;eau du système </li><li> 2 réservoirs de poissons zèbres traversant </li><li> MS-222 (éthyl 3-aminobenzoate méthanesulfonate; Sigma Chemical # E10521-10G) </li><li> stériles de 120 mm boîte de Pétri </li><li> serviettes en papier humide </li><li> spatule en métal émoussée </li><li> Dumont # 55 Bio Inox forceps (Outils Fine Science # 11255-20) </li><li> Des ciseaux à ressort Mini-Vannas (Outils Fine Science # 15000-00) </li><li> agent bleu de méthylène antifongique (Drs Foster &amp; Smith # CD-905781) </li><li> petits aquariums résille </li></ul> Collection de tissus et d&#39;incorporation d&#39;agar <ul><li> 4% en paraformaldéhyde 1x tampon phosphate salin (PBS-PF), jusqu&#39;à aliquotés dans 50 ml tubes coniques et conservés congelés à -20 ° C </li><li> 1x tampon phosphate salin (pH de 7,27 à 6) </li><li> 250 ml flacon en verre </li><li> Agarose à 2% dans de l&#39;eau distillée </li><li> standard de petites agarose rig électrophorèse sur gel (taille de gel = ~ 10 cm carrés) </li><li> petites dents peigne échantillon d&#39;électrophorèse (taille peigne 4 x 1,5 mm) </li><li> bain d&#39;eau tiède (50-60 ° C) </li><li> bandes de laboratoire </li><li> noir émaillé broches d&#39;insectes, de taille 000 (Fine Science Tools # 26000-25) </li><li> 1-2 détenteurs broches (outils Fine Science # 26016-12) </li><li> 9-inch pipette Pasteur en verre </li><li> poire ou la pompe </li><li> tissulaire en laboratoire </li><li> étiquettes en papier petits </li><li> (Optionnel) échelle d&#39;étalonnage imprimée </li><li> serviettes en papier humide </li><li> sac plastique zip de fermeture </li><li> bistouri / lame de rasoir (pour découper des blocs de gélose) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbillons sont phanères sensorielle trouve dans les poissons, les reptiles et les amphibiens. Le poisson zèbre, Danio rerio, développe deux paires de barbillons-une paire de courte nasale et une paire plus maxillaires. Barbel tissus contient des cellules d&#39;origine ectodermique, mésodermique et l&#39;origine de la crête neurale, y compris les cellules de peau, les glandes, les papilles, les mélanocytes, les navires circulatoires et des nerfs sensoriels. Contrairement à la plupart des tissus adultes, le barbillon maxillaire est optiquement clair, nous permettant de visualiser le développement et la maintenance de ces types de tissus tout au long du cycle de vie. Cette vidéo montre le développement précoce de l&#39;barbillon maxillaire (en commençant un mois environ après la fécondation), et démontre un protocole chirurgical pour favoriser la régénération de l&#39;appendice pour adultes (&gt; 3 mois après la fécondation). Brièvement, le barbeau maxillaire gauche d&#39;un poisson anesthésié est élevée avec une pince stérile juste distal à la face caudale du maxillaire. Une amende, des ciseaux à ressort stérile est positionné contre la pince pour couper l&#39;arbre le barbeau à ce niveau, l&#39;établissement d&#39;un repère anatomique pour le plan de l&#39;amputation. Une croissance régénérative peut être mesurée par rapport à ce plan, et en comparaison à l&#39;controlatéral barbeau. Tissus Barbel régénère rapidement, atteignant la repousse maximale dans les 2 semaines de blessure. Techniques d&#39;analyse de la régénération barbeau comprennent la dissection et l&#39;inclusion des paires de barbillons (se régénérer et de contrôle) dans les puits d&#39;un gel d&#39;électrophorèse d&#39;ADN standard. Spécimens embarqués sont commodément photographié sous un stéréomicroscope pour la morphologie et la morphométrie brute, et peut être stocké pendant des semaines avant d&#39;applications en aval tels que l&#39;histologie de paraffine, cryosectioning, et / ou immunohistochimie monter ensemble. Ces méthodes d&#39;établir le barbeau maxillaires comme un roman dans le système de tissus in vivo pour l&#39;étude de la capacité régénératrice de multiples types de cellules dans le contexte génétique de poisson zèbre.

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

Le poisson zèbre barbillon maxillaire est un organe des sens tégumentaires contenant ectodermique, mésodermique et dérivés de la crête neurale. Surtout, le barbeau adulte peut se régénérer après une amputation proximale. Cette vidéo présente le développement maxillaire barbeau et démontre un protocole chirurgical pour induire la régénération, suivie par imagerie de collecte, l&#39;intégration et en aval de spécimens barbeau.

Méthodes pour l&#39;étude de l&#39;Barbel Zebrafish maxillaire

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Research

JoVE Journal

Biology

12.4K vues.  DePaul University. Le poisson zèbre barbillon maxillaire est un organe des sens tégumentaires contenant ectodermique, mésodermique et dérivés de la crête neurale. Surtout, le barbeau adulte peut se régénérer après une amputation proximale. Cette vidéo présente le développement maxillaire barbeau et démontre un protocole chirurgical pour induire la régénération, suivie par imagerie de collecte, l'intégration et en aval de spécimens barbeau.

Vidéo: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injection intrapéritonéale en Zebrafish adultes

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Laser Ablation de l&#39;pronéphros Zebrafish d&#39;étude rénale régénération épithéliale

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En électroporation in vivo de morpholinos dans la régénération des adultes Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavage poisson zèbre pour adultes

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Méthodes d&#39;extraction des endosymbionts de la mouche blanche<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...