Diese Arbeit wurde von der National Institute for Mental Health und der National Science Foundation unterstützt. Besonderer Dank geht an Dr. Jen Gutzman, Dr. Amanda Dickinson und anderen Sive lab Mitglieder für viele hilfreiche Diskussionen und konstruktive Kritik, und Olivier Paugois für Experten Fische Tierhaltung.

Ein. Vorbereiten Mikroinjektion Needles and Cell Tram <ol><li> Füllen Eppendorf CellTram Öl Mikroinjektor Apparat mit Mineralöl nach den Anweisungen des Herstellers. </li><li> Bereiten Mikroinjektion Nadeln durch Ziehen Kapillarrohre mit Sutter Instrumente Nadel abziehen. </li><li> Montieren Nadel auf Mikromanipulator an Eppendorf CellTram. </li><li> Vorsichtig brechen die Spitze der Nadel. Für eine gleichmäßige Größe der Spitze, mit einem Mikrometer messen oder vergleichen, um eine Referenz Nadel. </li><li> Füllen Sie die Länge der Nadel mit Öl, mit dem CellTram Öl über die gesamte Länge zu schieben, dabei darauf achten, keine Blasen zu vermeiden. </li></ol> 2. Vorbereitung der Embryonen <ol><li> Stoßen Löcher in einem 1% Agarose-beschichteten Schale mit einem 1-200 ul Pipettenspitze und entfernen Agarose Steckern. Füllen Schale mit Embryo Medien (hergestellt nach Westerfield 25). </li><li> Mit Pinzette dechorionate Embryonen unter Stereomikroskop. </li><li> Übertragen dechorionated Embryonenin Agarose-beschichteten Gericht. </li><li> Embryonen zu betäuben, fügen Tricaine (0,1 mg / ml) an Agarose Antenne, bis Embryonen bewegten Anschlag (hergestellt nach Westerfield 25). </li></ol> 3. Entleeren des ECSF <ol><li> Orient die Embryonen mit ihren Endstücken in den Löchern der Agarose und ihre hinteren Seiten am nächsten zu dem Mikromanipulator, so dass für die Visualisierung der dorsalen Seite des Gehirns. </li><li> Positionieren Sie die Nadel am Rhombomer 0/1 Scharnier-Punkt oder der breitesten Stelle Hinterhirn Ventrikel (Abbildung 1A). </li><li> Sorgfältig durchbohren Dachblech Rautenhirn Ventrikel sicher zu sein, um nicht durch die Tiefe des Gehirns in dem Eigelb zu gehen. </li><li> Lassen Sie das ECSF mit dem CellTram sammeln und Flüssigkeit in Mikroinjektionsnadel. Seien Sie vorsichtig, um alle Zellen zu vermeiden. </li></ol> 4. Das Sammeln der ECSF für Composition Analysis <ol><li> Sobald die ECSF in der Nadel gesammelt wird, zu stoppen sorgfältig Absaugung aus dem CellTram. </li><li> Move die Schale aus unter der Nadel und die Nadel genau positionieren zu einem Schlauch mit dem gewünschten Puffer. </li><li> Mit dem CellTram, lassen Sie das ECSF aus der Nadel in den Puffer zu vermeiden versucht Verunreinigung der Lösung mit Öl. </li><li> Um Zellen, die Sie haben zusammen mit dem ECSF, Spin ECSF bei 10.000 x g abgesaugt entfernen. Unberührten ECSF (Überstand) und bei -80 ° C bis zur weiteren Analyse zu sammeln. </li></ol> 5. Wiedereinführung der ausgewählten Faktoren <ol><li> Lassen Sie das ECSF alle 2 Stunden während der gewünschte Zeitintervall, da die Flüssigkeit in diesem Zeitraum wieder aufgefüllt wird. Zwischen Entwässerungszeit-Punkte, entfernen Sie die Nadel und bewahren die Embryonen bei 28,5 ° C oder Temperatur gewünscht wird. </li><li> Legen zweiten Mikropipette Nadel mit Test-Faktor. </li><li> Ort Nadel in den Mikromanipulator Mikroinjektorkopfs befestigt. </li><li> Passen Injektionsvolumen bis 1 nl durch Messung der Tropfengröße in Öl und Einstellen Einspritzzeit und pressure. </li><li> Inject 1-2 nl in Hirnkammern wie zuvor beschrieben 26. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+in+cell+proliferation+in+the+germinal+epithelium+of+the+HTx+rats.">Failure in cell proliferation in the germinal epithelium of the HTx rats.</a> Eur. J. Pediatr. Surg. 11, S57-S59 (2001).</li><li>Lehtinen, M. K., 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=The+cerebrospinal+fluid+provides+a+proliferative+niche+for+neural+progenitor+cells.">The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.</a> Neuron. 69, 893-905 (2011).</li><li>Zappaterra, M. D., 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=A+comparative+proteomic+analysis+of+human+and+rat+embryonic+cerebrospinal+fluid.">A comparative proteomic analysis of human and rat embryonic cerebrospinal fluid.</a> J. Proteome. Res. 6, 3537-3548 (2007).</li><li>Parvas, M., Parada, 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=A+blood-CSF+barrier+function+controls+embryonic+CSF+protein+composition+and+homeostasis+during+early+CNS+development.">A blood-CSF barrier function controls embryonic CSF protein composition and homeostasis during early CNS development.</a> Dev. Biol. 321, 51-63 (2008).</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=Mammalian+embryonic+cerebrospinal+fluid+proteome+has+greater+apolipoprotein+and+enzyme+pattern+complexity+than+the+avian+proteome.">Mammalian embryonic cerebrospinal fluid proteome has greater apolipoprotein and enzyme pattern complexity than the avian proteome.</a> J. Proteome Res. 4, 2420-2428 (2005).</li><li>Gato, A., 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=Analysis+of+cerebro-spinal+fluid+protein+composition+in+early+developmental+stages+in+chick+embryos.">Analysis of cerebro-spinal fluid protein composition in early developmental stages in chick embryos.</a> J. Exp. Zool. A Comp. Exp. Biol. 301, 280-289 (2004).</li><li>Westerfield, M., Sprague, J., Doerry, E., Douglas, S., Grp, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Information+Network+(ZFIN):+a+resource+for+genetic,+genomic+and+developmental+research.">The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.</a> Nucleic Acids Res. 29, 87-90 (2001).</li><li>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=Zebrafish+Brain+Ventricle+Injection.">Zebrafish Brain Ventricle Injection.</a> J. Vis. Exp. (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>

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Die Verwendung dieser Technik, um manuell ablaufen ECSF vom Zebrafisch Hirnkammern nützlich sein wird für die Bestimmung der Voraussetzung für ECSF während der Entwicklung. Darüber hinaus wird diese Technik Beschreibung des ECSF Proteinprofil Laufe der Embryonalentwicklung ermöglichen. Identifizierung verschiedener Proteine ​​während dieser Zeit ermöglicht genauere Ermittlung der Funktion des CSF und seine potenzielle Rolle während der Entwicklung des Gehirns. In Amnioten haben einige Faktoren ECSF (IGF2, F...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Eppendorf CellTram Oil </td><td> Eppendorf </td><td> 516 000.025 </td></tr><tr><td> Mineralöl </td><td> Sigma </td><td> M8410 </td></tr><tr><td> Tricaine Pulver </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Kapillarrohre </td><td> FHC Inc. </td><td> 30-30-1 </td></tr></tbody></table>

manual drainage of the zebrafish embryonic brain ventricles

Liquor (CSF) ist ein Protein reiche Flüssigkeit innerhalb der Ventrikel im Gehirn enthalten. Es ist während der frühen Wirbeltiere Embryonalentwicklung vorhanden und bleibt während des gesamten Lebens. Adult CSF wird angenommen, dass das Gehirn dämpfen, entfernen Abfallprodukte und tragen sezernierte Moleküle 1,2. Beim Erwachsenen und älteren Embryo, wird die Mehrheit der CSF durch den Plexus choroideus, eine Reihe von stark vaskularisierten sekretorischen Bereiche benachbart angeordnet, um den Hirnkammern 3-5 hergestellt. Im Zebrafisch wird der Plexus komplett bei 144 Stunden nach der Befruchtung (hpf) 6 gebildet. Zuvor sowohl Zebrafisch und andere Wirbeltierembryonen einschließlich Maus, eine signifikante Menge an embryonalen CSF (ECSF) vorhanden ist. Diese Daten und Studien chick vermuten, dass die Neuroepithel sekretorischen früh in der Entwicklung ist, und kann die Hauptquelle der ECSF vor Plexus Entwicklung 7 sein. ECSF enthält etwa dreimal mehr Protein than Erwachsenen CSF, was darauf hindeutet, dass es eine wichtige Rolle bei der Entwicklung 8,9 haben. Studien bei Huhn und Maus demonstrieren, dass sekretierten Faktoren im ECSF, Fluiddruck oder eine Kombination von diesen, die wichtig sind für die Neurogenese, Genexpression, Zellproliferation und Zellüberleben im Neuroepithel 10-20. Proteomik Analysen von Mensch, Ratte, Maus und chick ECSF haben viele Proteine, die notwendig sein CSF-Funktion identifiziert. Dazu gehören Komponenten der extrazellulären Matrix, Apolipoproteine, den osmotischen Druck regulierende Proteine ​​und Proteine ​​in Zelltod und Proliferation 21-24 beteiligt. Jedoch sind die komplexen Funktionen des ECSF weitgehend unbekannt. Wir haben ein Verfahren zum Entfernen von Zebrafisch ECSF Hirnkammern entwickelt, so dass für die Identifizierung des ECSF Komponenten und zur Analyse der ECSF Anforderung während der Entwicklung. Obwohl mehr ECSF aus anderen Wirbeltieren Systeme w gesammelt werdenith größeren Embryonen können ECSF von den frühesten Stadien der Zebrabärblingentwicklung gesammelt werden, und unter genetischen oder Umgebungsbedingungen, die zu abnormen Hirnventrikel Volumen oder Morphologie führen. Entfernen und Sammeln von ECSF ermöglicht für die massenspektrometrische Analyse, Ermittlung von ECSF Funktion, und die Wiedereinführung der ausgewählten Faktoren in die Ventrikel zu Assay ihre Funktion. So ist die Erreichbarkeit des frühen Zebrafischembryo ermöglicht eine detaillierte Analyse der ECSF Funktion während der Entwicklung.

Ein Beispiel eines durchlässigen Hirnventrikel ist in 1B-C gezeigt. Hirnkammern sind zusammengebrochen, als sie ECSF (Abbildung 1B vs C) fehlt. Wie in dorsalen Bilder (1B-C und 2A-D) die Hinterhirn Neuroepithel macht behalten ihre charakteristische Morphologie und scheint trotz fehlender ECSF wahrscheinlich durch robuste Scharnier-Punkte offen gesehen. Allerdings zeigen Seitenansichten (Abbildung 2A&#39;-D &#39;), dass die Hinterhirn V...

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

Wir präsentieren eine Methode zur Liquor (CSF) zu sammeln und ein System, das CSF fehlt im embryonalen Zebrafischgehirn ventrikuläre System zu erstellen. Dies ermöglicht eine weitere Untersuchung der CSF Zusammensetzung und seine Forderung während der embryonalen Entwicklung des Gehirns.

Manuelle Drainage der Zebrafisch Embryonale Hirnkammern

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 Aufrufe.  Massachusetts Institute of Technology. Wir präsentieren eine Methode zur Liquor (CSF) zu sammeln und ein System, das CSF fehlt im embryonalen Zebrafischgehirn ventrikuläre System zu erstellen. Dies ermöglicht eine weitere Untersuchung der CSF Zusammensetzung und seine Forderung während der embryonalen Entwicklung des Gehirns.

Serie: 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.

Ein Assay zur Durchlässigkeit des Zebrafisch-Embryos Neuroepithel

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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Neurowissenschaften

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.

Mikromanipulation der Genexpression in der Erotik-Zebrafischgehirn Mit cerebroventricular Mikroinjektion von Morpholino Oligonukleotiden

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-Aufnahmen aus embryonalen Zebrafisch-Mauthner-Zellen

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.

Eine umfassende Protokoll für die manuelle Segmentierung der medialen Temporallappen Structures

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.

Isolierung und Kultivierung von Endothelzellen aus dem embryonalen Vorderhirn

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.

Elektrophysiologische Aufnahme im Gehirn von erwachsenen Zebrafisch Intact

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.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

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.

Bedingte Genetische transsynaptische Tracing in der embryonalen Maus-Gehirn

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.

Live-Imaging des Ependymale Cilia in der Seitenventrikel des Maus-Gehirn-

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.