Ce travail a été soutenu par l&#39;Institut national pour la santé mentale, et la National Science Foundation. Un merci spécial à Dr. Jen Gutzman, le Dr Amanda Dickinson et d&#39;autres membres du laboratoire Sive pour de nombreuses discussions utiles et des critiques constructives, et à Olivier Paugois pour l&#39;élevage de poissons d&#39;experts.

1. Préparation Aiguilles microinjection cellulaire et Tram <ol><li> Remplissez Eppendorf CellTram huile microinjecteur appareil avec de l&#39;huile minérale selon les instructions du fabricant. </li><li> Préparer aiguilles de microinjection en tirant des tubes capillaires en utilisant extracteur Sutter aiguille instruments. </li><li> Monter l&#39;aiguille sur micromanipulateur connecté à Eppendorf CellTram. </li><li> Cassez délicatement la pointe de l&#39;aiguille. Pour la taille de la pointe uniforme, mesurer avec un micromètre, ou comparer à une aiguille de référence. </li><li> Remplissez la longueur de l&#39;aiguille avec de l&#39;huile, en utilisant le CellTram pour pousser l&#39;huile sur toute la longueur, en prenant soin d&#39;éviter les bulles. </li></ol> 2. Préparation des embryons <ol><li> Percez des trous dans un plat recouvert d&#39;agarose 1% avec une pointe de pipette 1-200 pi, et enlever les bouchons d&#39;agarose. Remplissez plat avec les médias embryon (faite selon Westerfield 25). </li><li> En utilisant des pinces, des embryons dechorionate sous stéréomicroscope. </li><li> Transfert d&#39;embryons dechorionateden agarose plat couché. </li><li> Pour anesthésier embryons, ajouter tricaïne (0,1 mg / ml) à plat agarose jusqu&#39;à ce que les embryons s&#39;immobilisent (faite en fonction Westerfield 25). </li></ol> 3. Vidange du ECSF <ol><li> Orienter les embryons dont la queue dans les trous de l&#39;agarose et leurs faces postérieures les plus proches du micromanipulateur, permettant la visualisation de la face dorsale du cerveau. </li><li> Placez l&#39;aiguille à la rhombomère 0/1 charnière-point ou point le plus large du cerveau postérieur du ventricule (figure 1A). </li><li> Soigneusement toit en tôle percer le cerveau postérieur du ventricule étant sûr de ne pas passer par la profondeur du cerveau dans le jaune. </li><li> Égoutter la FFPE utilisant le CellTram et collecter le fluide dans l&#39;aiguille de microinjection. Soyez prudent pour éviter toutes les cellules. </li></ol> 4. La collecte de la FFPE pour l&#39;analyse de la composition <ol><li> Une fois la FFPE est recueillie dans l&#39;aiguille, soigneusement arrêt d&#39;aspiration du CellTram. </li><li> Move le plat de dessous de l&#39;aiguille et l&#39;aiguille en y plaçant dans un tube contenant le tampon désiré. </li><li> En utilisant la CellTram, vidanger le ECSF sur l&#39;aiguille dans le tampon en essayant de ne pas contaminer la solution avec de l&#39;huile. </li><li> Afin d&#39;éliminer les cellules que vous avez aspiré avec la FFPE FFPE, tournent à 10.000 x g. Recueillir non contaminée ECSF (surnageant) et conserver à -80 ° C jusqu&#39;au moment de l&#39;analyse. </li></ol> 5. Réintroduction de certains facteurs <ol><li> Égoutter la FFPE chaque h 2 pendant l&#39;intervalle de temps souhaité, car le fluide est reconstitué dans ce délai. Entre temps les points de drainage, retirez l&#39;aiguille et de stocker les embryons à 28,5 ° C ou la température désirée. </li><li> Chargez aiguille micropipette deuxième facteur d&#39;essai. </li><li> Placez l &#39;aiguille dans micromanipulateur attaché à microinjecteur. </li><li> Ajuster le volume d&#39;injection de 1 nl par mesure de la taille des gouttes dans le temps d&#39;injection d&#39;huile et de réglage et pression. </li><li> Injecter 1-2 nl dans les ventricules du cerveau, comme décrit précédemment 26. </li></ol>

<ol>
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(26), e1218 (2009).</li><li>Parada, C., Gato, A., Bueno, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-trans+retinol+and+retinol-binding+protein+from+embryonic+cerebrospinal+fluid+exhibit+dynamic+behaviour+during+early+central+nervous+system+development.">All-trans retinol and retinol-binding protein from embryonic cerebrospinal fluid exhibit dynamic behaviour during early central nervous system development.</a> Neuroreport. 19, 945-950 (2008).</li><li>Parada, C., Escola-Gil, J. C., Bueno, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoproteins+from+embryonic+cerebrospinal+fluid+are+required+for+neural+differentiation.">Low-density lipoproteins from embryonic cerebrospinal fluid are required for neural differentiation.</a> J. Neurosci. Res. 86, 2674-2684 (2008).</li><li>Kramer-Zucker, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia-driven+fluid+flow+in+the+zebrafish+pronephros,+brain+and+Kupffer's+vesicle+is+required+for+normal+organogenesis.">Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis.</a> Development. 132, 1907-1921 (2005).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Lowery, L. A., De Rienzo, G., Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+classification+of+zebrafish+brain+morphology+mutants.">Characterization and classification of zebrafish brain morphology mutants.</a> Anat. Rec. (Hoboken). 292, 94-106 (2009).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

L&#39;utilisation de cette technique pour purger manuellement ECSF à partir des ventricules cérébraux poisson zèbre sera utile pour déterminer l&#39;exigence de ECSF au cours du développement. En outre, cette technique permettra description du profil protéique ECSF au cours du développement embryonnaire. L&#39;identification des différentes protéines au cours de cette période permettra enquête plus approfondie sur la fonction de la LCR et son rôle potentiel au cours du développement du cerveau. En amniotes...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Eppendorf CellTram huile </td><td> Eppendorf </td><td> 516 000.025 </td></tr><tr><td> Huile minérale </td><td> Sigma </td><td> M8410 </td></tr><tr><td> Tricaïne poudre </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Tubes capillaires </td><td> FHC Inc </td><td> 30-30-1 </td></tr></tbody></table>

manual drainage of the zebrafish embryonic brain ventricles

Liquide céphalo-rachidien (LCR) est un liquide riche en protéine contenue dans les ventricules du cerveau. Il est présent pendant le développement embryonnaire précoce des vertébrés et persiste toute la vie. Adulte CSF est pensé pour amortir le cerveau, éliminer les déchets, et effectuer sécrétée 1,2 molécules. Dans l&#39;embryon adultes et plus, la majorité des CSF est faite par la choroïde, du plexus une série de régions hautement vascularisées sécrétrices situées à côté du cerveau ventricules 3-5. Chez le poisson zèbre, le plexus choroïde est entièrement formé à 144 après la fécondation heures (hpf) 6. Avant cela, dans les deux embryons de vertébrés poisson zèbre et autres, y compris la souris, une quantité importante de CSF embryonnaire (FFPE) est présent. Ces données et d&#39;études de poussin suggèrent que la neuro-épithélium sécrétoire est tôt dans le développement et peut-être la principale source de ECSF avant plexus choroïde développement 7. ECSF contient environ trois fois plus de protéines than adulte CSF, suggérant qu&#39;il peut jouer un rôle important au cours du développement 8,9. Studies in poussin et de la souris démontrent que les facteurs sécrétés dans la FFPE, la pression de fluide, ou une combinaison de ceux-ci, sont importantes pour la neurogenèse, la survie expression des gènes, la prolifération cellulaire et de cellules dans le neuroépithélium 10-20. Analyses protéomiques de l&#39;homme, le rat, la souris et poussin FFPE ont identifié de nombreuses protéines qui peuvent être nécessaires pour la fonction LCR. Ils comprennent des composants de la matrice extracellulaire, les apolipoprotéines, des protéines de régulation de la pression osmotique, et des protéines impliquées dans la mort cellulaire et la prolifération 21-24. Cependant, les fonctions complexes de la FFPE sont largement inconnues. Nous avons développé un procédé pour éliminer ECSF à partir des ventricules cérébraux poisson zèbre, permettant ainsi l&#39;identification des composants ECSF et pour l&#39;analyse de l&#39;exigence ECSF au cours du développement. Bien que plus ECSF peuvent être collectées à partir d&#39;autres systèmes avec des vertébrésvec embryons plus grands, ECSF peuvent être recueillies dès les premiers stades de développement du poisson zèbre, et dans des conditions génétiques ou environnementaux qui conduisent à volume cérébral anormal du ventricule ou de la morphologie. L&#39;enlèvement et la collecte de ECSF permet d&#39;analyse de masse, spectrométrie enquête de la fonction ECSF, et la réintroduction des facteurs de sélection dans les ventricules pour doser leur fonction. Ainsi, l&#39;accessibilité de l&#39;embryon du poisson zèbre précoce permet une analyse détaillée de la fonction ECSF au cours du développement.

Un exemple d&#39;un ventricule cérébral drainée est illustré à la figure 1B-C. Ventricules cérébraux sont effondrés car ils n&#39;ont pas ECSF (figure 1B vs C). Comme on le voit dans les images dorsales (figure 1B-C et la Figure 2A-D) neuroépithélium rhombencéphale ne conserve sa morphologie caractéristique et semble être ouverte malgré l&#39;absence de ECSF probablement en raison de robustes charnières points. Cependant, les vues latéra...

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

Nous présentons une méthode pour collecter le fluide céphalo-rachidien (LCR) et de créer un système qui manque de LCR dans le système ventriculaire du cerveau du poisson zèbre embryonnaire. Cela permet un examen plus approfondi de la composition du LCR et son exigence au cours du développement embryonnaire du cerveau.

Drainage Manuel des ventricules cérébraux embryonnaires de poisson-zèbre

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

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Neuroscience

9.9K vues.  Massachusetts Institute of Technology. Nous présentons une méthode pour collecter le fluide céphalo-rachidien (LCR) et de créer un système qui manque de LCR dans le système ventriculaire du cerveau du poisson zèbre embryonnaire. Cela permet un examen plus approfondi de la composition du LCR et son exigence au cours du développement embryonnaire du cerveau.

Vidéo: 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 test de perméabilité du neuro-épithélium embryonnaire poisson zèbre

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

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Neurosciences

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.

Micromanipulation de l&#39;expression génique dans le cerveau de poisson zèbre adulte en utilisant microinjection Cerebroventricular d&#39;oligonucléotides 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 enregistrements de cellules embryonnaires de poisson zèbre 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 protocole complet pour la segmentation manuelle des structures du lobe temporal médial

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.

Isolement et culture de cellules endothéliales de la embryonnaire du cerveau antérieur

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.

Enregistrement électrophysiologique dans le cerveau d&#39;Intact poisson zèbre adulte

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.

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

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.

Transsynaptique génétique conditionnelle traçage dans le cerveau embryonnaire de souris

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.

Imagerie en direct de la épendymaire Cilia dans les ventricules latéraux du cerveau de souris

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.

L&#39;exposition du porc du système nerveux central pour l&#39;analyse anatomopathologique: Un manuel pour Décapitation, Crâne d&#39;ouverture et cerveau suppression