Diese Methoden wurden von EEL während eines Forschungsaufenthaltes verlassen im Labor von JT-Unterstützung durch die DePaul University Research Council und ein NIH / NIDCR R01 Zuschuss an JT (DE016678) zur Verfügung gestellt wurde entwickelt. Wir danken für die Bemühungen von Caroline Hunter, Tierpflege-Techniker in der CMRC Zebrafisch Core Facility und Paulina Pawluczuk, unsere Studenten Laborantin.

Zebrafisch Tierhaltung Zebrafische sind untergebracht und nach der Norm Methoden 1 aufgezogen. Vorbei am späten Larvenstadien Frist kann der Entwicklungsstand eines Zebrafisch durch seine Standard-Länge (SL), oder die gerade Linie in Millimetern von der vordersten Punkt der Oberlippe an der Basis der Schwanzflosse (posteriormost hypural Platte) gemessen werden 2. Bei ca. 1 Monat nach der Befruchtung (10-12 mm SL), erscheinen die Nasen-und Kieferhöhle Barben als transparent epithelialen Knospen in der Nähe des Riechgruben und auf den hinteren Ecken des Oberkiefers bzw.. Da der Zebrafisch reift, erstrecken sich die Barben in schmale, whisker Fortsätzen. Da die oberen Barbe ist größer (2-3 mm) und leichter zu manipulieren, gilt unser Protokoll nur für diese besondere Anhängsel; ähnliche Techniken konnte jedoch zu den kleineren nasalen Barben angepasst werden. Barbel Clipping <ol><li> Betäuben erwachsenen Zebrafisch auf die gewünschte Stufe (z. B.., 1,5-2,5 cm SL, ~ 3-6 Monate nach der Befruchtung) durch Eintauchen in 0,015% MS-222 (Ethyl-3-Aminobenzoesäure Methansulfonat) gepuffert auf pH 7,0 im System Wasser. Beachten Sie, bis schwimmen Bewegungen zu stoppen und gill Belüftung wird langsam und regelmäßig. Je nach Größe der Fische, nimmt Vollnarkose ca. 2-5 Minuten. </li><li> Mit einem Aquarium Fischnetz, Transfer 1-3 Fische auf einmal zu einem gefalteten Stück nassen Papiertuch in einer Petrischale. Mit einem stumpfen Spatel, orientieren die Fische, so dass sie parallel zueinander und linken lateralen Seite bis sind. Falten Sie ein Teil der nassen Papiertuch über den hinteren Teil der Fische, die den gesamten Körper bis zum Operculum (Kiemendeckel), um die Haut und Kiemen feucht während des Verfahrens. </li><li> Sehen Sie sich die Petrischale unter dem Stereomikroskop, mit geringem Winkel einfallende Licht auf den Kopf zu beleuchten. Konzentrieren Sie sich auf der Basis der linken Oberkiefer-Barbe, die von der hinteren ventralen Ecke des Oberkiefers (Abb. 1) ragt. <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="Abbildung 1" /> Abbildung 1. </li><li> Dicht erfassen die Welle der Barbe leicht distal an den Rand des Oberkiefers. Elevate die Barbe Welle und legen Sie die Backen einer spitzen Feder Schere genau proximal der Pinzette. Die Schere kann die Zange für die Stabilität berührt werden. Schieben Sie die Backen der Schere entlang der Welle der Barbe, bis die Schneide nähert sich der Rand des Oberkiefers. Schließen Sie die Schere durch die Barbe Welle geschnitten. Die amputiert Barbe, noch griff in die Zange, kann zur Fixierung oder weitere Beobachtung entfernt werden. </li><li> Sofort überweisen Sie den Fisch auf einem kleinen Tank mit sauberem Wasser System (~ 500 ml) enthält einen Tropfen Methylenblau zu oberflächliche Pilzinfektion zu kontrollieren. Lassen Sie den Fisch über Nacht erholen. Am nächsten Morgen kehren die Fische, die Aufzucht-System. Barbel Regeneration nach 2 Wochen maximal. </li></ol> Agar Einbettung von Matched Pairs Barbel <ol><li> Sammeln Sie Fische durch geeignete Euthanasie Technik und fix in einem 50 ml-Röhrchen mit 4% Paraformaldehyd-Phosphat-gepufferter Kochsalzlösung (PBS) unter Rühren bei 4 ° C über Nacht. Die Fixierlösung sollte mindestens das Zehnfache des Volumens des Gewebes werden. Nach der Fixierung von Paraformaldehyd Abfälle in einer zugelassenen Behälter und spülen Sie das Gewebe gründlich in mehrere Änderungen der PBS. </li><li> Bereiten Sie sich in einem 250 ml-Glasflasche 150 ml 2% Agarose (T m ~ 37 ° C) in destilliertem Wasser. Hitze mit einer Mikrowelle oder Heizplatte, Rühren in regelmäßigen Abständen, bis sie vollständig aufgelöst. Äquilibrieren das geschmolzene Lösung in einem 50-60 ° C Wasserbad. </li><li> Bauen Sie ein Standard-Gel-Elektrophorese rig, indem Sie einen kleinen gedichtete Gelform (10 x 10 cm; ~ 100 mL Gelvolumen) fest gegen die Seiten der Pufferkammer. Fügen Sie 2-4 kleinen Zähnen Probe Kämme (4 x 1,5 mm). </li><li> Auf einer ebenen Fläche, gießen Sie die geschmolzene Agarose in die Form gerade bedeckt die Basis der Waben bilden sehr seichten Brunnen. Sofort setzen die übrig gebliebenen Agarose zurück in den warmen Wasserbad, das wird später verwendet werden, um das Gewebe (Schritte 9-12) einbinden. Legen Sie ein langes Glas Pasteur Pipette in den Kolben der Pipette auf die gleiche Temperatur wie die Agarose zu halten. </li><li> Lassen Sie das Gel zu härten für 20-30 Minuten. Entfernen Sie die Kämme, entfernen Sie dann das Gel enthaltende Form der gehärteten Gel aus der Pufferkammer. Fest Band die offenen Seiten des Gels Form mit Labor-Band so das Gel nicht rutscht aus. Diese Band sollte einige Millimeter über der Oberfläche der Agarose zu verlängern, so dass zusätzliche Agarose später hinzugefügt werden können (Schritt 13). </li><li> Positionieren Sie die Gelform auf der Bühne ein Stereomikroskop. Magnify einen leeren Brunnen und bringen die Kanten in den Fokus. </li><li> Auf die Oberfläche der Agarose neben dem gut, einen Tropfen von Wasser oder Puffer, der den festen Barben Proben (zB, regenerieren und Kontrolle). Using gebogen Insektennadeln, ziehen Sie den feuchten Proben in den Brunnen und schieben Sie sie nach unten. Orient die Barbe Wellen parallel zueinander ausgerichtet und an gegenüberliegenden langen Kanten des Brunnens. Die Oberflächenspannung wird dazu beitragen, halten das Gewebe in Position. </li><li> Wenn Sie bereit sind zu verankern, mit einem feinen Pipettenspitze oder die Ecke eines Labors Gewebe, um überschüssige Flüssigkeit aus dem Brunnen zu entfernen. Arbeiten Sie schnell, um nicht zu lassen, das Gewebe austrocknen. </li><li> Mit der erwärmten Glas Pasteur Pipette vorsichtig Pipette frisch geschmolzene Agarose in und um die Proben zu den Barben Gewebe an Stelle abzudichten. Nicht überfüllen. Bevor die Agarose erhärtet, machen Last-Minute-Korrekturen mit dem Insekt Pins. </li><li> Legen Sie eine nummerierte Papier Probe Etikett neben dem Brunnen und sichern Sie sie mit einem kleinen Tropfen Agarose. </li><li> Wiederholen Sie die Schritte 6 bis 10 für jede Vertiefung der Proben. </li><li> Wenn für die Bild-Kalibrierung gewünscht, legen Sie ein Blatt Papier mit einem gedruckten Mikrometerbereich (zB <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> ) in den Boden eines leeren Brunnen. Streichen Sie das Papier auf den Boden des Brunnens und decken Sie es mit warmen Agarose. </li><li> Nachdem alle Proben eingebettet sind, wird die Oberfläche des Gels werden unregelmäßig, mit gehärteter Tropfen Agarose. Legen Sie das Gel auf eine ebene Fläche und sanft in zusätzliche geschmolzener Agarose pour bis die Oberfläche gleichmäßig ist. Verwenden Sie die kleinste Menge an Agarose notwendig, um eine glatte Oberfläche zu erhalten, zu viel Agarose wird Durchlicht-Fotografie im Dunkeln. Lassen Sie diese oberste Schicht zu härten. </li><li> Das Gel kann nun auf der Bühne ein Stereomikroskop auf grobe Morphologie für jede Barbe Paar Rekord fotografiert werden. Bild Kalibrierung durch Fotografieren der embedded Mikrometerbereich erreicht. </li><li> Das ganze Gel gespeichert eingewickelt in feuchte Papiertücher und versiegelt in einem Plastikbeutel bei 4 ° C für mehrere Wochen. </li><li> Zur weiteren Analyse können Agar Blöcke mit zwei gepaarten Barben aus dem Gel geschnitten werden mit einem Skalpell oder einer Rasierklinge und verarbeitet Paraffin Histologie oder Kryoschneiden nach Standardmethoden 3,4. Alternativ können einzelne Barben aus der Agarose seziert werden mit feinen Nadeln, in Wasser gespült, und verarbeitet für andere Downstream-Anwendungen (zB ganze-mount Immunhistochemie oder die konfokale Mikroskopie). </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>

Der Zebrafisch Oberkiefer Barbe ist eine nicht ausgelastete Gewebe für die Untersuchung des Wachstums, Wartung und Erneuerung von mehreren Zelltypen im Zebrafisch. Obwohl die Barbe Anhängsel hat kein Mensch analog, sind die Zelltypen enthält hoch konserviert, die es ermöglichen, Haut, Drüsen, Melanozyten, Kreislauf-Gefäße und Nerven in eine optisch klare und anatomisch einfachen zylindrischen Struktur zu studieren. Ähnlich wie bei den gut untersuchten Schwanzflosse kann Barben Gewebe induziert durch Amputation zu regenerieren. Mit der Grenze des Ober-als anatomische Landmarke, kann die Amputation Flugzeug genau platziert werden, die Messung der Barbe Nachwachsen. Für einen erfahrenen Operateur nimmt jeder Operation nur ein paar Sekunden. Recovery ist schnell, und wir haben bisher keine kurz-oder langfristige Auswirkungen auf Fische Verhalten erkannt. Zebrafisch mit einem Ober-Barben schwimmen, essen und Rasse so effektiv wie nicht-chirurgischen Kontrollen und haben vergleichbare Überlebensrate bis zum Zeitpunkt der Gewebe Sammlung, bis zu 6 Monate nach der Operation. Die physiologische Wirkung dieser Operation voraussichtlich minimal sein, weil 1) die extraorale Geschmacksknospen auf der Barbe durchgeführt auch auf vielen anderen Teilen der Fisch Epithel, einschließlich der Lippen, Wangen und Kopf 5 gefunden werden, und 2) unterscheiden Gaumen erscheinen auf die regenerierende Barben innerhalb von 72 Stunden (LeClair et al., unveröffentlichte Daten). Das macht die Ober-Barbe ein minimal-invasives System zur Untersuchung der Wundheilung, Revaskularisation und Reinnervation im Rahmen eines erwachsenen Wirbeltieren. Nach der chirurgischen Induktion von Regeneration, kann Barben in Abständen für morphometrische Messung und / oder mikroskopische Analyse von fixiertem Gewebe gesammelt werden. Praktischerweise ist der Oberkiefer Barben etwa die Länge (2-3 mm) und Durchmesser (100-200 mm) eines Zebrafisch-Embryos, die Erleichterung der Anwendung von vielen Standard-Protokollen, einschließlich Paraffin Histologie, Kryoschneiden, ganze-mount Immunhistochemie und in situ Hybridisierung. Zusammen genommen machen diese Features die oberen Barben ein sehr machbar in vivo Modell für die Untersuchung die Reparatur von Gewebe und Regeneration.

Mikroskop Ausstattung Ein Stereo-Binokular mit Durchlicht und Vorfall Glasfaser-Beleuchtung ist für Barben Clipping und Agar Einbettung erforderlich. Barbel Clipping <ul><li> Fisch System Wasser </li><li> 2 Zebrafisch Kreuzung Tanks </li><li> MS-222 (Ethyl-3-Aminobenzoesäure Methansulfonat; Sigma Chemical # E10521-10G) </li><li> sterile 120-mm Petrischale </li><li> nasse Papiertücher </li><li> stumpfen Spatel </li><li> Dumont Nr. 55 Bio Inox Pinzette (Fine Science Tools # 11255-20) </li><li> Mini-Vannas Frühjahr Schere (Fine Science Tools # 15000-00) </li><li> Methylenblau Antimykotikum (Drs. Foster &amp; Smith # CD-905781) </li><li> kleines Aquarium fishnet </li></ul> Tissue-Sammlung und Agar Einbettung <ul><li> 4% Paraformaldehyd in 1x Phosphat-gepufferter Kochsalzlösung (PF-PBS), aliquotiert in 50 ml konische Röhrchen und bei -20 ° C bis zur Verwendung </li><li> 1x Phosphat-gepufferter Kochsalzlösung (pH 7,27 bis 6) </li><li> 250 ml Glasflasche </li><li> 2% Agarose in destilliertem Wasser </li><li> Standard klein Agarosegelelektrophorese rig (Gel size = ~ 10 cm im Quadrat) </li><li> kleinen Zähnen Elektrophorese-Kamm (Kamm Größe 4 x 1,5 mm) </li><li> warmen Wasserbad (50-60 ° C) </li><li> Labor-Band </li><li> schwarz emailliert Insektennadeln, Größe 000 (Fein Science Tools # 26000-25) </li><li> 1-2 Pin-Halter (Fine Science Tools # 26016-12) </li><li> 9-Zoll-Glas Pasteurpipette </li><li> Peleusball oder Pumpe </li><li> Laborwischtuch </li><li> kleine Papier-Etiketten </li><li> (Optional) gedruckt Kalibrierungsskala </li><li> nasse Papiertücher </li><li> zip-Schließung Plastiktüte </li><li> Skalpell / Rasierklinge (zum Ausschneiden Agar-Blöcke) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barben sind hautsensorischen Anhängsel bei Fischen, Reptilien und Amphibien gefunden. Der Zebrafisch, Danio rerio, entwickelt zwei Paar Barteln-eine kurze Nase Paar und einen längeren Oberkiefer Paar. Barbel Gewebe enthält Zellen ektodermalen, mesodermalen und Neuralleiste Ursprungs, einschließlich Hautzellen, Drüsen, Gaumen, Melanozyten, Kreislauf-Gefäße und sensorischen Nerven. Anders als die meisten adulten Gewebe ist die Ober-Barbe optisch klar, so dass wir die Entwicklung und Wartung dieser Gewebearten während des gesamten Lebenszyklus zu visualisieren. Dieses Video zeigt frühe Entwicklung des Oberkiefers Barbe (Beginn etwa einen Monat nach der Befruchtung) und zeigt eine chirurgische Protokoll, um die Regeneration im adulten Anhängsel (&gt; 3 Monate nach der Befruchtung) zu induzieren. Kurz gesagt, ist die linke Oberkiefer Barben eines betäubten Fische mit einer sterilen Pinzette gerade distal bis zum kaudalen Rand des Oberkiefers erhöht. Eine feine, sterile Schere Frühjahr gegen die Zange positioniert, um die Barbe Welle auf dieser Ebene geschnitten, Gründung einer anatomischen Landmarke für die Amputation Ebene. Regenerative Wachstum kann in Bezug auf diese Ebene gemessen werden, und im Vergleich zu der kontralateralen Barben. Barbel Gewebe regeneriert sich schnell und erreichte maximal Nachwachsen innerhalb von 2 Wochen nach der Verletzung. Techniken für die Analyse der regeneriert Barben gehören sezieren und Einbettung paarweise Barben (regenerieren und Kontrolle) in die Vertiefungen einer Standard-DNA-Elektrophorese-Gel. Embedded Proben sind bequem unter einem Stereomikroskop für grobe Morphologie und Morphometrie fotografiert, und kann für mehrere Wochen aufbewahrt vor Downstream-Anwendungen wie Paraffin Histologie werden, Kryoschneiden und / oder ganze Berg Immunhistochemie. Diese Methoden zu etablieren Oberkiefer Barbe als neuartige In-vivo-Gewebe-System für die Erforschung der Regenerationsfähigkeit der verschiedenen Zelltypen innerhalb des genetischen Zusammenhang mit der Zebrafisch.

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

Der Zebrafisch Oberkiefer Barbe ist ein integumentary Sinnesorgan mit ektodermalen, mesodermalen und Neuralleiste Derivate. Wichtig ist, dass die erwachsenen Barben nach proximal Amputation zu regenerieren. Dieses Video stellt Oberkiefer Barben Entwicklung und zeigt eine chirurgische Protokoll, um die Regeneration zu induzieren, durch Sammlung, Einbett-und nachgelagerten Bildgebung von Barben Exemplare folgten.

Methoden zur Untersuchung des Zebrafisch Maxillary Barbel

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 Aufrufe.  DePaul University. Der Zebrafisch Oberkiefer Barbe ist ein integumentary Sinnesorgan mit ektodermalen, mesodermalen und Neuralleiste Derivate. Wichtig ist, dass die erwachsenen Barben nach proximal Amputation zu regenerieren. Dieses Video stellt Oberkiefer Barben Entwicklung und zeigt eine chirurgische Protokoll, um die Regeneration zu induzieren, durch Sammlung, Einbett-und nachgelagerten Bildgebung von Barben Exemplare folgten.

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

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-Tiermodell Stimuli

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.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

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.

Intraperitoneale Injektion in Adult Zebrafisch

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 des Zebrafisch Vorniere auf Study Renal Epithelregeneration

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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.

In-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

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 Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

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.

Gavaging Adult Zebrafisch

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.

Methoden zur Extraktion von Endosymbionten aus der Whitefly<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 ...