Este trabajo fue apoyado por el Instituto Nacional de Salud Mental, y la National Science Foundation. Un agradecimiento especial a la Dra. Jen Gutzman, la Dra. Amanda Dickinson y otros miembros del laboratorio Sive para muchas discusiones útiles y críticas constructivas, y Olivier Paugois para la cría de peces experto.

1. Preparación de agujas de microinyección y tranvía Celular <ol><li> Llenar Eppendorf CellTram aceite aparato microinyector con aceite mineral de acuerdo con las instrucciones del fabricante. </li><li> Preparar agujas de microinyección tirando tubos capilares utilizando Sutter extractor aguja instrumentos. </li><li> Montar la aguja en micromanipulador Eppendorf CellTram conectado a. </li><li> Corte cuidadosamente la punta de la aguja. Para el tamaño de punta uniforme, medir con un micrómetro, o comparar a una aguja de referencia. </li><li> Llene la longitud de la aguja con aceite, usando la CellTram para empujar el aceite hacia abajo de la longitud, teniendo cuidado de evitar cualquier burbuja. </li></ol> 2. Preparación de los embriones <ol><li> Hacer agujeros en un 1% de agarosa al plato cubierto con una punta de pipeta 1-200 l, y eliminar bloques de agarosa. Rellene plato con los medios de embriones (hechas de acuerdo con Westerfield 25). </li><li> Utilizando pinzas, embriones dechorionate bajo microscopio estereoscópico. </li><li> Transferencia de embriones dechorionateden la fuente de agarosa recubierta. </li><li> Para anestesiar embriones, añadir tricaína (0,1 mg / ml) a plato de agarosa hasta que dejan de moverse embriones (hecho de acuerdo con Westerfield 25). </li></ol> 3. Vaciado del ECSF <ol><li> Orientar los embriones con sus colas en los agujeros de la agarosa y sus lados posteriores más cercanos a la micromanipulador, permitiendo la visualización de la cara dorsal del cerebro. </li><li> Colocar la aguja en el rhombomere 0/1 bisagra de punto o el punto más ancho de hindbrain ventrículo (Figura 1A). </li><li> Con cuidado, perfore placa del techo del ventrículo cerebro posterior asegurándose de no pasar por la profundidad del cerebro en la yema. </li><li> Vacíe el ECSF utilizando el CellTram y recoger el líquido en la aguja de microinyección. Tenga cuidado de evitar cualquier célula. </li></ol> 4. La recolección de la ECSF para el Análisis de Composición <ol><li> Una vez que el ECSF se recoge en la aguja, cuidadosamente detener la succión de la CellTram. </li><li> MoVE el plato de debajo de la aguja y colocar la aguja cuidadosamente en un tubo que contiene el tampón deseado. </li><li> Uso de la CellTram, drenar el ECSF de la aguja en el tampón tratando de evitar la contaminación de la solución con aceite. </li><li> Con el fin de eliminar las células que se han aspirado junto con el ECSF ECSF, centrifugado a 10.000 x g. Recoger no contaminada ECSF (sobrenadante) y se almacena a -80 ° C hasta que esté listo para su posterior análisis. </li></ol> 5. La reintroducción de factores seleccionados <ol><li> Vacíe el ECSF cada 2 h durante el intervalo de tiempo deseado, ya que el líquido se repone dentro de ese período de tiempo. Entre los puntos temporales de drenaje, retire la aguja y almacenar los embriones a 28,5 ° C o temperatura deseada. </li><li> Coloque la aguja micropipeta segundo factor de la prueba. </li><li> Coloque la aguja en micromanipulador unido a microinyector. </li><li> Ajustar el volumen de inyección de 1 nl midiendo el tamaño de gota en el tiempo de inyección de aceite y regulación y presión. </li><li> Inyectar 1-2 nl en los ventrículos del cerebro como se ha descrito previamente 26. </li></ol>

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No hay conflictos de interés declarado.

El uso de esta técnica para drenar manualmente ECSF desde los ventrículos del cerebro del pez cebra será útil para determinar el requisito de ECSF durante el desarrollo. Además, esta técnica permitirá descripción del perfil de proteínas ECSF en el curso del desarrollo embrionario. Identificación de proteínas diferentes durante este tiempo permitirá una mayor investigación en la función del CSF y su posible papel en el desarrollo cerebral. En amniotas, algunos factores identificados en ECSF (IGF2, FGF2, ácido retinoico, y apolipoproteínas) tienen un papel en la supervivencia demostró neuroepitelial celular, la proliferación y la neurogénesis 13,17,20,23,27,28. Sin embargo, parece que hay cientos de proteínas no caracterizadas en la ECSF, que se obtuvo después de la formación del plexo coroideo, más tarde que los puntos de tiempo se ha demostrado aquí. Además, nuestro laboratorio y otros han identificado mutantes de pez cebra con el tamaño anormal del ventrículo cerebral o defectos cerebrales 29-31, y VHA puedeanormalidades en la composición e ECSF y función. Este método permite fácilmente para aislar y probar ECSF anormal putativo de mutantes o bajo diferentes condiciones ambientales. Una ventaja importante que el sistema de pez cebra es reemplazar factores seleccionados a través de la inyección en los ventrículos del cerebro después de la eliminación de ECSF, con lo que prueba su función durante el desarrollo del cerebro. Las pequeñas moléculas, proteínas y ECSF obtenidos después de la perturbación genética o química pueden ser probados. Reintroducción de ECSF de otras especies permitirá la comparación de factores y funciones entre especies y en diferentes condiciones patológicas, como la hidrocefalia, que complementa y su interconexión con los estudios de mamíferos. En resumen, el uso del pez cebra para eliminar ECSF, analizar su composición y reintroducir ECSF o factores específicos en los ventrículos cerebrales, aumentará significativamente la comprensión de la función y la regulación de la función ECSF y la del sistema ventricular del cerebro.

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Eppendorf CellTram Petróleo </td><td> Eppendorf </td><td> 516 000.025 </td></tr><tr><td> Aceite mineral </td><td> Sigma </td><td> M8410 </td></tr><tr><td> Tricaína polvo </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Los tubos capilares </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

manual drainage of the zebrafish embryonic brain ventricles

El líquido cefalorraquídeo (LCR) es un líquido rico en proteínas contenidas dentro de los ventrículos cerebrales. Está presente durante el desarrollo embrionario temprano de vertebrados y persiste durante toda la vida. Adulto CSF se piensa para amortiguar el cerebro, eliminar los desechos, y llevar a 1,2 secretada moléculas. En el embrión y adulto mayor, la mayoría de los CSF se hace por el plexo coroideo, una serie de regiones altamente vascularizados secretoras situados adyacentes al cerebro ventrículos 3-5. En el pez cebra, el plexo coroideo está completamente formado a 144 horas después de la fertilización (hpf) 6. Antes de esto, en embriones de vertebrados tanto pez cebra y otros incluyendo el ratón, una cantidad significativa de embrionario CSF ​​(ECSF) está presente. Estos datos y los estudios sugieren que el polluelo en el neuroepitelio es secretora temprana en el desarrollo y puede ser la fuente principal de ECSF antes de plexo coroideo desarrollo 7. ECSF contiene aproximadamente tres veces más proteínas tHan adulto CSF, sugiriendo que podría tener un papel importante durante el desarrollo 8,9. Los estudios en pollo y ratón demuestran que los factores secretados en el ECSF, la presión del fluido, o una combinación de éstos, son importantes para la neurogénesis, la expresión de genes de supervivencia, la proliferación celular y de la célula en el neuroepitelio 10-20. Análisis proteómico de rata humana, el ratón y el pollo ECSF han identificado muchas proteínas que pueden ser necesarios para la función LCR. Estos incluyen componentes de la matriz extracelular, apolipoproteínas, proteínas que regulan la presión osmótica, y proteínas implicadas en la muerte celular y la proliferación 21-24. Sin embargo, las funciones complejas de la ECSF son en gran medida desconocidos. Hemos desarrollado un método para eliminar ECSF desde los ventrículos del cerebro del pez cebra, permitiendo así la identificación de los componentes ECSF y para el análisis de la exigencia ECSF durante el desarrollo. Aunque más ECSF se pueden obtener de otra w sistemas de vertebradosith embriones más grandes, ECSF se pueden recoger de las primeras etapas del desarrollo del pez cebra, y bajo condiciones genéticas o ambientales que conducen a volumen anormal ventrículo cerebral o en la morfología. La eliminación y recogida de ECSF permite el análisis de espectrometría de masas investigación, de la función ECSF, y la reintroducción de los factores seleccionados en los ventrículos para ensayar su función. Por lo tanto la accesibilidad del embrión de pez cebra temprana permite el análisis detallado de la función ECSF durante el desarrollo.

Un ejemplo de un ventrículo cerebral escurrido se muestra en la figura 1B-C. Ventrículos cerebrales se derrumbó ya que carecen de ECSF (Figura 1B vs C). Como se ve en las imágenes dorsales (Figura 1B-C, y la Figura 2A-D) del neuroepitelio hindbrain retiene su morfología característica y parece estar abierto a pesar de la falta de ECSF probable debido a las robustas de bisagras puntos. Sin embargo, las vistas laterales (Figura 2A&#39;-D &#39;) demuestran que el ventrículo hindbrain ha sido drenado, consistente con la presencia de un delgado epitelio flexible de la placa de techo que se derrumba ventralmente. En los embriones de tipo salvaje, ECSF se produce continuamente y rellena el cerebro ventrículos 2-3 h después de drenaje (Figura 2). Por lo tanto, CSF debe ser continuamente empobrecido sobre el tiempo de interés. Mientras que la eliminación del fluido a las 24 HPF no interrumpa la morfología gruesa o el desarrollo, la eliminación de ECSF durante un largo per<html> iod de desarrollo puede tener efectos adversos en el desarrollo de todo el cuerpo. Esto debe ser tenido en cuenta al determinar la eficacia de los factores inyectados en los ventrículos del cerebro. Además, para determinar si los defectos de desarrollo son un resultado de la pérdida de ECSF o debido a una aguja perfore el cerebro, un control importante es la inserción de una aguja en el cerebro sin quitar ECSF. Hemos demostrado que ECSF tiene un perfil de proteína diferente de extracto de embrión de conjunto, y el análisis en un gel de proteína de SDS-PAGE demuestra que una cantidad detectable de proteína puede ser obtenida de pez cebra ECSF (Figura 3). Esto demuestra que neuroepiteliales contaminantes celulares son a niveles inferiores a proteínas específicas ECSF, y, además, que una cantidad sustancial de proteína puede ser recogido que es suficiente para el análisis de espectrometría de masas. 3/4243fig1.jpg &quot;/&gt; Figura 1. Manualmente drenados ventrículos cerebrales. (A) Diseño Experimental. Lateral (izquierda) y dorsal (derecha) ve de dónde insertar la aguja de microinyección. Aguja (triángulo negro) se inserta en la placa de techo del ventrículo cerebro posterior en el punto más ancho de la parte posterior del cerebro. La aguja se inserta más en el cerebro medio y ventrículos del cerebro anterior según lo señalado por las flechas rojas. (BC) Ejemplos de 24 HPF manualmente drena embrión. El embrión misma imagen antes de drenaje (B) o después de desagüe (C). (B &#39;C&#39;) Trazos de BC. Línea gris indica la morfología presente pero no visible. F = cerebro anterior, cerebro medio = M, H = rombencéfalo. Las barras de escala = 50 micras. <img alt="Figura 2" src="/files/ftp_upload/4243/4243fig2.jpg" /> Figura 2. ECSF recargas de los ventrículos cerebrales con el tiempo. (A) 24 hpf embrión antes de drenar (B) inmediatamente después de t = 0 (C) 15, (D) 60, (E) 120, (F) 180, (G) 240, y (h) 300 min (m) después de drenar. (AH) y dorsal (A&#39;-H &#39;) imágenes laterales luminoso con anterior a la izquierda. F = cerebro anterior, cerebro medio = M, H = rombencéfalo. Las barras de escala = 50 micras. <img alt="Figura 3" src="/files/ftp_upload/4243/4243fig3.jpg" /> Perfil de la figura 3. Proteína varía entre 24 hpf todo embrión de pez cebra (WE) extracto (0,5 g de proteína total) y 24 hpf ECSF de 50 embriones. Tampón de carga SDS añadido al recogieron ECSF para desnaturalizar las proteínas de la muestra, se ejecutan en un gel PAGE 8% de Tris-HCl, y se detectaron con Sypro Ruby. * Indican bandas únicas para ECSF.

Watch this Scientific Journal Video about Manual Drainage of the Zebrafish Embryonic Brain Ventricles at JoVE.com

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Se presenta un método para recoger líquido cefalorraquídeo (LCR) y para crear un sistema que carece de CSF dentro del sistema ventricular del cerebro embrionario de pez cebra. Esto permite un examen más detenido de la composición de CSF y sus requerimientos durante el desarrollo embrionario del cerebro.

Drenaje Manual de los ventrículos del cerebro embrionario de pez cebra

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

manual-drainage-of-the-zebrafish-embryonic-brain-ventricles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.9K vistas.  Massachusetts Institute of Technology. Se presenta un método para recoger líquido cefalorraquídeo (LCR) y para crear un sistema que carece de CSF dentro del sistema ventricular del cerebro embrionario de pez cebra. Esto permite un examen más detenido de la composición de CSF y sus requerimientos durante el desarrollo embrionario del cerebro.

Video: Manual Drainage of the Zebrafish Embryonic Brain Ventricles

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Un ensayo para la permeabilidad del neuroepitelio embrionario de pez cebra

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

Investigación

Neurociencias

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Micromanipulación de la expresión génica en el cerebro de pez cebra adulto Uso Microinyección cerebroventricular de oligonucleótidos Morpholino

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Las grabaciones de las células embrionarias de pez cebra Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

Un protocolo integral para la segmentación manual de las estructuras mediales Temporal Lobe

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

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 ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Registro electrofisiológico en el cerebro del pez cebra adultos Intacto

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condicional transsynaptic genética Seguimiento en el cerebro de embriones de ratón

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.