Nos cortésmente las gracias al Dr. John Hayes para los scripts, los Dres. Eric Bradley y Christopher Del Negro para obtener ayuda con la microscopía confocal, de Drew Hughes, Laura Odorizzi, Garafalo Alex, Rebecca Lowden, y Liz MacMurray por su trabajo en el desarrollo del proyecto y proporcionar datos preliminares, el Dr. Greg Smith por sus útiles sugerencias sobre el análisis estadístico. Este trabajo fue apoyado por una subvención del NIH (NINDS IR15N5067566-01) para el SMS y el Howard Hughes Medical Institute Science Education Grant al Colegio de William y Mary.

No hay conflictos de interés declarado.

Con sus tipos celulares bien caracterizadas que se conservan en todos los vertebrados, la retina proporciona un modelo útil para el estudio de los procesos moleculares-celulares que regulan la especificación del tipo de célula y la diferenciación. Cultivo celular primario proporciona un método potente para la investigación de una gran variedad de procesos que incluyen la expresión génica, la dinámica de proteínas y el calcio y la actividad eléctrica en el nivel de resolución única célula. A continuación l...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td></td></tr><tr><td></td><td></td><td></td><td> Para las disecciones y Cultivo </td></tr><tr><td> BD Falcon Easy Grip placas de cultivo tisular de 35 mm, </td><td> Pescador </td><td> 08-772A </td><td></td></tr><tr><td> Platos desechables de poliestireno Petri, 60 x 15 mm </td><td> Pescador </td><td> 0875713A </td><td></td></tr><tr><td> 35 mm Nunclon superficie Petri Dishes (con Airvent) </td><td> Pescador </td><td> 12-565-91 </td><td></td></tr><tr><td> Dumont Fine Pinzas (Dumostar # 55) </td><td> Pescador </td><td> NC9341917 </td><td></td></tr><tr><td> Cellattice Micro-dictaminó cubreobjetos de plástico, 25 mm </td><td> Pescador </td><td> 50-313-17 </td><td></td></tr><tr><td> Etil-m-aminobenzoato de metanosulfonato de sal (MS-222) </td><td> MP Biomedicals, LLC </td><td> 103106 </td><td></td></tr><tr><td> Sulfato de Gentamicina </td><td> Enzo Ciencias de la Vida </td><td> 380-003-G025 </td><td></td></tr><tr><td> Gibco tripsina (1:250) en polvo </td><td> Life Technologies </td><td> 27250-018 </td><td></td></tr><tr><td> Colagenasa B de Clostridium histolyticum </td><td> Roche </td><td> 11088831001 </td><td></td></tr><tr><td> Penicilina-Estreptomicina </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> Nilo Azul Sulfato (opcional) </td><td> Pfaltz &amp; Bauer </td><td> N05550 </td><td></td></tr><tr><td> 500 ml de vacío Filtro / almacenamiento de botellas del sistema, 0,22 micras de poro 33,2 cm 2 de membrana CN </td><td> Corning </td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ Td&gt; <td></td><td> Para obtener imágenes de calcio </td></tr><tr><td> Fluo-4 AM 1 mM en solución de DMSO, Cell Permanente </td><td> Life Technologies </td><td> F-14217 </td><td></td></tr><tr><td> Pluronic F-127 10% de solución en agua </td><td> Life Technologies </td><td> P-6866 </td><td></td></tr><tr><td> LSM 510 sistema de microscopio confocal </td><td> Zeiss </td><td> Modelo Descatalogado </td><td></td></tr><tr><td> Reactivo bloqueador </td><td> Roche </td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> Por hibridación fluorescente in situ (FISH) </td></tr><tr><td> Anti-digoxigenina-Pod, fragmentos Fab </td><td> Roche </td><td> 11207733910 </td><td></td></tr><tr><td> Anti-DNP-HRP Anticuerpo </td><td> Perkin-Elmer </td><td> NEL747A </td><td></td></tr><tr><td> Cy3Éster de NHS </td><td> GE Healthcare </td><td> PA13101 </td><td></td></tr><tr><td> NHS-fluoresceína </td><td> Thermo Scientific </td><td> 46409 </td><td></td></tr><tr><td> Formamida desionizada, </td><td> Amresco </td><td> 0606-950ml </td><td></td></tr><tr><td> Torula ARN, Tipo IX </td><td> Sigma-Aldrich </td><td> R3629 </td><td></td></tr><tr><td> Sal sódica de heparina, a partir de mucosa intestinal porcina </td><td> Sigma-Aldrich </td><td> H3393-250KU </td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich </td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 2. Reactivos y equipos específicos. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Solución </td><td> Referencia </td><td> Contenido </td></tr><tr><td> Medio de cultivo celular </td><td> Chang y Spitzer, 2009 54 </td><td> 116 mM NaCl 0,67 mM KCl 2 mM CaCl 2 1,31 mM MgSO 4 4,6 mM Tris 1% (v: v) Penicilina / Estreptomicina Ajustar el pH a 7,8 con HCl. Esterilizar por filtración mediante el paso a través de un filtro de 0,22 micras CN. Almacenar a 4 ° C. </td></tr><tr><td> Calcio-Magnesio medio libre (CMF) </td><td> Gu et al, 1994 42;. Gu y Spitzer, 1995 55 </td><td> 116 mM NaCl 0,67 mM KCl 4,6 mM Tris 0,4 mM EDTA 1% (v: v) Penicilina / Estreptomicina Ajustar el pH a 7,8 con HCl. Esterilizar por filtración mediante el paso a través de un filtro de 0,22 micras CN. Almacenar a 4 ° C. </td></tr><tr><td> Tampón de ácido maleico (MAB) </td><td> Sive y col., 2000 53 </td><td> 100 mM de ácido maleico 150 mM NaCl (pH 7,5). </td></tr><tr><td> Marc Modificado Ringers (MMR), 10 veces más </td><td> Sive y col., 2000 53 </td><td> 100 mM NaCl mM KCl 1 mM MgSO 4 2 mM CaCl 2 5 mM de HEPES pH ajustado a 7,4 con NaOH 0,1 X MMR también contiene 50 mg / ml de sulfato de gentamicina y el pH se ajustó a 7,4 con NaOH. </td></tr><tr><td> Solución MEMFA, 10X </td><td> Sive y col., 2000 53 </td><td> 0,1 M MOPS (pH 7,4) 2 mM EGTA 1 mM MgSO 4 3,7% de formaldehído Una solución de 10 veces, sin formaldehído, se puede almacenar a 4 ° C. El formaldehído se añadió fresco (1/10 de volumen de un patrón madre 37%). </td></tr><tr><td> En tampón de hibridación in situ </td><td> Sive et al., 2000 53 </td><td> 50% de formamida 5X SSC 1 mg / ml de ARN Torula 100 mg / ml de heparina Solución Denhart 1X de 0,1% de Tween 20 CHAPS 0,1% 10 mM EDTA. </td></tr></tbody></table> Soluciones. * Gentamicina, un antibiótico, se usa en nuestras soluciones MMR mientras penicilina y estreptomicina se utilizan en los medios de nuestra cultura. </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

El proceso por el que la región anterior de la placa neural da lugar a la retina de vertebrados sigue siendo un foco importante de investigación tanto clínica como básica. Además de la evidente importancia médica para la comprensión y el tratamiento de enfermedades de la retina, el desarrollo de la retina de vertebrados continúa sirviendo como un importante sistema modelo y elegante para la comprensión neuronal determinación de tipo celular y la diferenciación 1-16. La retina neural consta de seis tipos de células discretas (ganglio, fotorreceptores amacrinas, horizontal,, células bipolares, y las células gliales de Müller) dispuestas en capas estereotipadas, un patrón que es muy conservada entre todos los vertebrados 12,14-18. Si bien el estudio de la retina en el embrión en desarrollo está intacto claramente necesaria para la comprensión de cómo este órgano complejo se desarrolla a partir de una protuberancia del cerebro anterior en una estructura en capas, hay muchas preguntas que benefician from empleando métodos que utilizan cultivos celulares primarios de presuntas células de la retina 7,19-23. Por ejemplo, el análisis de las células de los tejidos y se disocia en diferentes etapas permite discernir el estado de la especificación de las células individuales en diferentes etapas de desarrollo, es decir, el destino de las células en la ausencia de interacciones con los tejidos vecinos 8,19-22 ,24-33. Cultivo de células primarias también permite al investigador para tratar el cultivo con reactivos específicos y analizar los resultados en un nivel de célula única 5,8,21,24,27-30,33-39. Xenopus laevis, un sistema modelo para el estudio clásico de desarrollo neural temprano 19,27,29,31-32,40-42, sirve como un sistema particularmente adecuado para el cultivo de células primarias de retina 10,38,43-45. Tejido de la retina presuntiva se puede acceder desde las primeras etapas de desarrollo, inmediatamente después de la inducción neural 25,38,43. Además, dado then cada célula en el embrión contiene un suministro de yema de huevo, células de la retina pueden ser cultivadas en un medio de comunicación muy simples definidos que consisten en una solución salina tamponada, eliminando así los efectos de confusión de incubación o de otros sueros de los productos basados ​​en 10,24,44-45 . Sin embargo, el aislamiento del tejido de la retina de los tejidos circundantes y el posterior procesamiento es un reto. Aquí, se presenta un método para la disección y disociación de las células de la retina en Xenopus laevis que se usa para preparar cultivos de células primarias que, a su vez, se analizó la actividad de calcio y la expresión génica en la resolución de las células individuales. Si bien el tema presentado en este documento es el análisis de los transitorios de calcio espontáneos, la técnica es ampliamente aplicable a una amplia gama de temas de investigación y enfoques (Figura 1).

Ejemplos de éxito disecados vesículas ópticas (etapa 25) y retinas (etapa 35) se muestran en las Figuras 2E y 2J. Aunque este protocolo se puede utilizar en varias etapas de desarrollo, es crítico para obtener sólo tejido de la retina para asegurar la precisión para experimentos adicionales. Retire con cuidado la epidermis en todo momento y asegurarse de que las pinzas no perforar el tejido de la retina. En la etapa 35 años o más, la lente puede ser visto como una capa transparente en la parte s...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

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

Xenopus laevis proporciona un sistema modelo ideal para el estudio de la especificación del destino celular y la función fisiológica de las células individuales de la retina en cultivo de células primarias. Aquí se presenta una técnica para la disección de tejidos retinales y la generación de cultivos de células primarias que se toman imágenes de la actividad del calcio y se analizaron mediante hibridación in situ.

Disección, Cultura y Análisis de<em&gt; Xenopus laevis</em&gt; Tejido de la retina embrionaria

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

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

Research

JoVE Journal

Neuroscience

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

13.8K vistas.  College of William and Mary. Xenopus laevis proporciona un sistema modelo ideal para el estudio de la especificación del destino celular y la función fisiológica de las células individuales de la retina en cultivo de células primarias. Aquí se presenta una técnica para la disección de tejidos retinales y la generación de cultivos de células primarias que se toman imágenes de la actividad del calcio y se analizaron mediante hibridación in situ.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection y cultivos primarios de neuronas del hipocampo de embriones de ratón y corticales

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Disección de un ojo del ratón de un monte entero del epitelio pigmentario de la retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina basado en Aislamiento y cultivo de motoneuronas embrionarias de ratón

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Aislamiento y cultivo de células neuronales de ratas embrionarias: un protocolo rápido

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neural Explant Cultures from Xenopus laevis

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

Neural Explant Cultures from Xenopus laevis

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

Los cultivos neuronales de explantes de<em&gt; Xenopus laevis</em

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

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

La disección y de montaje lateral de embriones de pez cebra: Análisis para el Desarrollo de la médula espinal

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Un ensayo de comportamiento simple para Pruebas de función visual en<em&gt; Xenopus laevis</em

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

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

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

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

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

Grabación de la actividad neuronal inducida por temperatura a través de la supervisión de cambios de calcio en el bulbo olfatorio de<em&gt; Xenopus laevis</em

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

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