Estos métodos han sido desarrollados por EEL durante un permiso de investigación en el laboratorio de JT se prestó apoyo por la Universidad DePaul de Investigación del Consejo y un NIH / NIDCR R01 otorga a JT (DE016678). Agradecemos los esfuerzos de Hunter Caroline, técnico de cuidado de los animales en las instalaciones centrales CMRC pez cebra y Paulina Pawluczuk, nuestro asistente de laboratorio de grado.

Pez cebra cría Pez cebra son alojados y criados de acuerdo con los métodos estándar de 1. Pasado el período larval tarde, la etapa de desarrollo de un pez cebra puede ser medido por su longitud estándar (SL), o la distancia en línea recta en milímetros desde el punto más anterior del labio superior hasta la base de la aleta caudal (placa hipurales más posterior) 2. En aproximadamente un mes después de la fecundación (10-12 mm SL), las barbillas nasal y maxilar aparecen brotes epiteliales como transparente cerca de las fosas olfatorias y en las esquinas posteriores de los maxilares, respectivamente. Como el pez cebra madura, el barbo se extienden en estrecho, como barba-apéndices. Debido a que el maxilar barbos es más grande (2-3 mm) y más fácil de manipular, el protocolo sólo se aplica a este apéndice particular; técnicas similares, sin embargo, podría ser adaptado para los más pequeños barbos nasal. Barbo de recorte <ol><li> Anestesiar a adultos de pez cebra en la etapa deseada (por ejemplo., 1.5-2.5 cm SL, ~ 3-6 meses post-fecundación) por inmersión en el 0,015% de MS-222 (el 3-aminobenzoato metanosulfonato) tamponado a pH 7,0 en el agua del sistema. Observe los movimientos hasta que deje de nadar y ventilación de enmalle se vuelve más lenta y regular. Dependiendo del tamaño del pez, una anestesia completa tarda aproximadamente 2-5 minutos. </li><li> Usando una red de pesca del acuario, la transferencia de peces de 1-3 en un momento en que una hoja doblada de la toalla de papel húmeda en una placa de Petri. Usando una espátula roma, orientar a los peces por lo que son paralelos a cada lado lateral izquierdo y otra arriba. Parte de las veces la toalla de papel húmeda en la parte caudal de los peces, que cubre todo el cuerpo hasta el opérculo (branquias cubiertas) para mantener la piel y las branquias húmedas durante el procedimiento. </li><li> Ver la placa de Petri bajo el microscopio estereoscópico, con bajo ángulo de luz incidente para iluminar la zona de la cabeza. Se centran en la base de la izquierda del maxilar barbos, que sobresale de la parte posterior ventral del maxilar superior (Fig. 1). <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="Figura 1" /> Figura 1. </li><li> Comprender bien el eje de la ligeramente distal al borde del maxilar barbos. Elevar el eje de barbos e insertar las fauces de una tijera de primavera de punta fina justo por encima de las pinzas. Las tijeras se puede tocar a las pinzas para la estabilidad. Deslice las fauces de las tijeras a lo largo del eje de la barbilla hasta la punta se acerca al borde del maxilar superior. Cerca de las tijeras para cortar a través del eje barbos. El barbo amputada, que aún esté sujeta con las pinzas, se puede quitar para la fijación o la observación más. </li><li> Inmediatamente la transferencia de los peces a un pequeño tanque de agua limpia del sistema (~ 500 ml) que contiene una gota de azul de metileno para el control de infecciones fúngicas superficiales. Permiten a los peces a recuperarse durante la noche. A la mañana siguiente, vuelta a los peces en el sistema de crianza. Barbo de la regeneración es máxima después de 2 semanas. </li></ol> Agar incrustación de pares coincidentes barbo <ol><li> Recoger los peces por la técnica de eutanasia adecuados y fijar en un tubo de 50 ml de paraformaldehído al 4%-tampón fosfato salino (PBS) con agitación a 4 ° C durante la noche. El volumen de fijador debe ser por lo menos diez veces el volumen del tejido. Después de la fijación, la eliminación de residuos de paraformaldehído en un recipiente aprobado y enjuague el tejido a fondo en varios cambios de PBS. </li><li> Preparar en un frasco de vidrio de 250 ml 150 ml de agarosa al 2% (T m ~ 37 ° C) en agua destilada. Calor con un microondas o una placa caliente, agitando periódicamente hasta que se disuelva. Equilibrar la solución fundida en un baño de 50-60 ° C el agua. </li><li> Montar un equipo de electroforesis en gel estándar mediante la colocación de un pequeño molde de gel de juntas (10 x 10 cm, aproximadamente 100 ml de volumen de gel) con firmeza contra los lados de la cámara de amortiguación. Añadir 2-4 dientes pequeños peines de la muestra (4 x 1,5 mm). </li><li> Sobre una superficie plana, verter la agarosa derretida en el molde para sólo cubrir la base de los panales, formando pozos muy poco profundos. Inmediatamente los restos de agarosa de nuevo en el baño de agua caliente, lo que se utilizará posteriormente para integrar el tejido (Pasos 9-12). Ponga una larga pipeta Pasteur de vidrio en el matraz para mantener la pipeta a la misma temperatura que la agarosa. </li><li> Deje que el gel para endurecer durante 20-30 minutos. Quitar los panales, luego retire el molde del gel que contiene el gel endurecido de la cámara de amortiguación. Firmemente la cinta de los lados abiertos del molde con cinta de gel de laboratorio por lo que el gel no se desliza. Esta cinta debe extenderse varios milímetros sobre la superficie de la agarosa, para que más de agarosa se puede agregar más adelante (Paso 13). </li><li> Coloque el molde del gel en el escenario de un microscopio estereoscópico. Ampliar un pozo vacío y llevar a los bordes en el foco. </li><li> Sobre la superficie del lado del bien de agarosa, una pipeta una gota de agua o un tampón que contiene las muestras de barbo fijo (por ejemplo, regenerar y control). Using pines doblados insectos, arrastre las muestras húmedas en el pozo y los empuja hacia el fondo. Orientar el barbo ejes paralelos entre sí y alineados en contra de lo contrario los bordes largos del pozo. Tensión de la superficie ayudará a mantener el tejido en su posición. </li><li> Cuando esté listo para insertar, utilice una pipeta de punta fina o la punta de un pañuelo de papel de laboratorio para eliminar el exceso de líquido del pozo. Trabaje con rapidez para no dejar que los tejidos se sequen. </li><li> Utilizando la pipeta Pasteur de vidrio caliente, con cuidado pipeta nueva agarosa fundida en y alrededor de las muestras para sellar el tejido de barbos en el lugar. No llene en exceso. Antes de la agarosa se endurece, hacer ajustes de última hora con las patas de insectos. </li><li> Coloque una etiqueta de papel ejemplar numerado al lado del bien y seguro que con una pequeña gota de agarosa. </li><li> Repita los pasos 6 a 10 para cada pozo de las muestras. </li><li> Si se desea para la calibración de la imagen, insertar una hoja de papel con una escala micrométrica impresos (por ejemplo, <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> ) en el fondo de un pozo vacío. Alise el papel en el fondo del pozo y se cubre con agua tibia de agarosa. </li><li> Después de todas las muestras se insertan, la superficie del gel será irregular, con caídas endurecido de agarosa. Colocar el gel en una superficie plana y suavemente vierta adicionales agarosa derretida hasta que la superficie superior es uniforme. Use la menor cantidad de agarosa necesaria para obtener una superficie lisa, demasiado oscuro agarosa se transmite la luz de la fotografía. Permitir que esta capa superior se endurezca. </li><li> El gel puede ahora ser fotografiados en el escenario de un microscopio estereoscópico para registrar la morfología bruto para cada par de barbos. Calibración de la imagen se obtiene mediante la fotografía la escala del micrómetro incorporado. </li><li> El gel se puede almacenar todo envuelto en toallas de papel húmedas y selladas en una bolsa de plástico a 4 º durante varias semanas. </li><li> Para su posterior análisis, los bloques de agar que contiene par emparejado de barbos se puede cortar el gel con un bisturí o una navaja y procesados ​​para histología de parafina o muestras criostáticas por métodos estándar 3,4. Por otra parte, barbos individuales pueden ser diseccionados de la agarosa con agujas muy finas, se enjuagan en agua, y se procesaron para otras aplicaciones posteriores (por ejemplo, todo el montaje inmunohistoquímica o microscopía confocal). </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>

El pez cebra maxilar barbos es un sistema de tejido subutilizadas para estudiar el crecimiento, mantenimiento y regeneración de varios tipos de células en el pez cebra. Aunque el apéndice barbo no tiene analogía humana, los tipos de células que contiene son altamente conservadas, lo que hace posible el estudio de la piel, las glándulas, los melanocitos, los vasos circulatorios y los nervios en una estructura cilíndrica ópticamente transparente y simple anatomía. Similar a la aleta caudal bien estudiado, el tejido barbos se puede inducir la regeneración de la amputación. Utilizando la frontera del maxilar como un hito anatómico, el avión amputación puede ser colocado con precisión, lo que facilita la medición de los barbos rebrote. Para un operador con experiencia, cada cirugía es sólo unos pocos segundos. La recuperación es rápida, y hasta ahora hemos detectado ningún efecto a corto o largo plazo en el comportamiento de los peces. Pez cebra con un maxilar barbos nadar, comer y reproducirse tan eficazmente como no quirúrgicos de control, y tienen una supervivencia comparable a la época de la recogida de tejidos, hasta 6 meses después de la cirugía. El impacto psicológico de esta cirugía prevé que sea mínimo porque 1) las papilas gustativas extraoral realizado en el barbo también se encuentran en muchas otras partes del epitelio de los peces, incluyendo los labios, las mejillas y la cabeza 5, y 2) diferenciar las papilas gustativas aparecen en el barbo regeneración dentro de las 72 horas (LeClair et al., datos no publicados). Esto hace que el maxilar barbos un sistema mínimamente invasivo para el estudio de la cicatrización de heridas, la revascularización y la reinervación en el contexto de un vertebrado adulto. Después de la inducción quirúrgica de la regeneración, barbos se pueden recoger en los intervalos de medición morfométrica y / o el análisis microscópico de tejido fijado. Convenientemente, el maxilar barbos es aproximadamente de la longitud (2-3 mm) y diámetro (100-200 mm) de un embrión de pez cebra, para facilitar la aplicación de muchos de los protocolos estándar, incluyendo la histología de parafina, muestras criostáticas, inmunohistoquímica todo el montaje, e in situ hibridación. En conjunto, estas características hacen que el maxilar barbos una muy factible en un modelo in vivo para el estudio de la reparación de tejidos y la regeneración.

Microscopio de Equipo Un microscopio estéreo con disección de transmisión y la iluminación incidente de fibra óptica se requiere para recortar el barbo y la incrustación de agar. Barbo de recorte <ul><li> Peces de agua del sistema </li><li> 2 tanques de pez cebra cruce </li><li> MS-222 (el 3-aminobenzoato metanosulfonato, Sigma Chemical # E10521-10G) </li><li> estéril de 120 mm placa de Petri </li><li> toallas de papel mojadas </li><li> espátula de metal contundente </li><li> Dumont # 55 Bio Inox Pinzas (Herramientas Artes Ciencia # 11255-20) </li><li> Mini-Vannas primavera tijeras (Herramientas Artes Ciencia # 15000-00) </li><li> azul de metileno agente antifúngico (Drs. Foster &amp; Smith # CD-905781) </li><li> pequeña red de pesca del acuario </li></ul> Tejido de recogida e incorporación de agar <ul><li> 4% de paraformaldehído en 1x tampón fosfato salino (PBS-PF), hasta alícuotas en tubos cónicos de 50 ml y congelar a -20 ° C </li><li> 1x tampón fosfato salino (pH 7,27 a 6) </li><li> 250 ml frasco de vidrio </li><li> Agarosa al 2% en agua destilada </li><li> estándar pequeña en gel de agarosa equipo de electroforesis (tamaño de gel = ~ 10 cm cuadrados) </li><li> pequeño peine de dientes electroforesis de la muestra (el tamaño de un peine de 4 x 1,5 mm) </li><li> baño de agua caliente (50-60 ° C) </li><li> laboratorio de la cinta </li><li> negro esmaltado alfileres de insectos, de tamaño 000 (Fina Ciencia Herramientas # 26000 a 25) </li><li> 02.01 titulares de pin (herramientas de Bellas Ciencia # 26.016-12) </li><li> De 9 pulgadas pipeta Pasteur de vidrio </li><li> pipeta de bulbo o la bomba </li><li> laboratorio de los tejidos </li><li> pequeñas etiquetas de papel </li><li> (Opcional) de calibración impresa escala </li><li> toallas de papel mojadas </li><li> zip de cierre de las bolsas de plástico </li><li> bisturí / hoja de afeitar (para cortar bloques de agar) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbos son apéndices de la piel sensoriales que se encuentran en los peces, reptiles y anfibios. El pez cebra, Danio rerio, desarrolla dos pares de barbillas, un par de cortos nasal y un par más largo del maxilar superior. Barbos tejido contiene células del ectodermo, mesodermo y el origen de la cresta neural, incluyendo células de la piel, las glándulas, las papilas gustativas, los melanocitos, los vasos circulatorios y los nervios sensoriales. A diferencia de la mayoría de los tejidos adultos, el maxilar barbos es ópticamente transparente, que nos permite visualizar el desarrollo y mantenimiento de estos tipos de tejidos en todo el ciclo de vida. Este video muestra el desarrollo temprano del maxilar barbos (a partir de aproximadamente un mes después de la fertilización) y demuestra un protocolo quirúrgico para inducir la regeneración en el apéndice de adultos (&gt; 3 meses después de la fertilización). En pocas palabras, el barbo superior izquierdo de un pez anestesiado se eleva con una pinza estéril, distal hasta el extremo caudal del maxilar superior. Un par de tijeras finas, primavera estéril se coloca en contra de las pinzas para cortar el eje de barbos en este nivel, estableciendo un punto de referencia anatómicos para el plano de la amputación. Crecimiento regenerador puede ser medido con respecto a este plano, y en comparación con el contralateral barbos. El tejido se regenera barbos rápidamente, alcanzando su máximo en el nuevo crecimiento de 2 semanas de la lesión. Técnicas de análisis del regenerado barbos incluyen la disección y la incrustación de pares de barbos (regenerarse y control) en los pocillos de un gel de electroforesis de ADN estándar. Especímenes incrustados están convenientemente fotografiados bajo un microscopio estereoscópico para la morfología y la morfometría bruto, y se puede almacenar durante varias semanas antes de que las aplicaciones posteriores como la histología de parafina, muestras criostáticas, y / o inmunohistoquímica montar todo. Estos métodos de establecer el barbo maxilar como una novela en el sistema de tejido vivo para el estudio de la capacidad regenerativa de los múltiples tipos de células en el contexto genética del pez cebra.

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

El pez cebra maxilar barbos es un órgano sensorial que contiene tegumentario ectodérmica, mesodérmica y derivados de la cresta neural. Es importante destacar que, el barbo adultos pueden regenerarse después de una amputación proximal. Este video presenta el desarrollo del maxilar barbos y demuestra un protocolo quirúrgico para inducir la regeneración, seguida por la imagen recogida, incorporación y posterior de las muestras barbos.

Métodos para el Estudio de la barbo pez cebra maxilar

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 vistas.  DePaul University. El pez cebra maxilar barbos es un órgano sensorial que contiene tegumentario ectodérmica, mesodérmica y derivados de la cresta neural. Es importante destacar que, el barbo adultos pueden regenerarse después de una amputación proximal. Este video presenta el desarrollo del maxilar barbos y demuestra un protocolo quirúrgico para inducir la regeneración, seguida por la imagen recogida, incorporación y posterior de las muestras barbos.

Video: 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.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

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.

Los estímulos generados por ordenador Modelo 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.

Usando la respuesta de optocinético para estudiar la función visual del pez cebra

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.

Inyección intraperitoneal en adultos de pez cebra

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.

La ablación con láser de la pronefros pez cebra para el Estudio de la regeneración epitelial renal

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 electroporación in vivo de Morpholinos en la retina de pez cebra adultos

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 electroporación in vivo de Morpholinos en la regeneración de adultos de pez cebra Aleta de cola

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 de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

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 adultos de pez cebra

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.

Métodos para la extracción de endosimbiontes de la mosca blanca<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 ...