Arbeit unterstützt durch NIH HD-32116, der Sandler Familie Unterstützung Foundation, The John Bowes Stem Cell Fund, MEXT, MHLW und HFSP. ZM durch die Carlos Baldoceda Foundation und UCSF Krevans Fellowship unterstützt. AA-B. hält die Heather und Melanie Muss Stiftungslehrstuhl.

<ol>
	<li>Doetsch, F., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+of+tangential+pathways+for+neuronal+migration+in+adult+mammalian+brain.">Network of tangential pathways for neuronal migration in adult mammalian brain.</a> Proc. Natl. Acad. Sci. USA. 93, 14895-14900 (1996).</li><li>Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+SVZ+stem+cells+lie+in+a+vascular+niche:+a+quantitative+analysis+of+niche+cell-cell+interactions.">Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions.</a> Cell Stem Cell. 3, 289-300 (2008).</li><li>Tavazoie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specialized+vascular+niche+for+adult+neural+stem+cells.">A specialized vascular niche for adult neural stem cells.</a> Cell Stem Cell. 3, 279-288 (2008).</li><li>Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+confer+unique+pinwheel+architecture+to+the+ventricular+surface+in+neurogenic+regions+of+the+adult+brain.">Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain.</a> Cell Stem Cell. 3, 265-278 (2008).</li><li>Mirzadeh, Z., Han, Y. G., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+organize+ependymal+planar+polarity.">Cilia organize ependymal planar polarity.</a> J Neurosci. 30, 2600-2610 (2010).</li><li>Doetsch, F., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+a+germinal+layer+in+the+adult+mammalian+brain.">Regeneration of a germinal layer in the adult mammalian brain.</a> Proc. Natl. Acad. Sci. USA. 96, 11619-11624 (1999).</li><li>Sawamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+follow+the+flow+of+cerebrospinal+fluid+in+the+adult+brain.">New neurons follow the flow of cerebrospinal fluid in the adult brain.</a> Science. 311, 629-632 (2006).</li><li>Coskun, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++neural+stem+cells+in+the+ependyma+of+mammalian+postnatal+forebrain.">CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain.</a> Proc. Natl. Acad. Sci. USA. 105, 1026-1031 (2008).</li><li>Merkle, F. T., Mirzadeh, Z., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+organization+of+neural+stem+cells+in+the+adult+brain.">Mosaic organization of neural stem cells in the adult brain.</a> Science. 317, 381-384 (2007).</li><li>Young, K. M., Fogarty, M., Kessaris, N., Richardson, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+zone+stem+cells+are+heterogeneous+with+respect+to+their+embryonic+origins+and+neurogenic+fates+in+the+adult+olfactory+bulb.">Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb.</a> J Neurosci. 27, 8286-8296 (2007).</li><li>Waclaw, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+finger+transcription+factor+Sp8+regulates+the+generation+and+diversity+of+olfactory+bulb+interneurons.">The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons.</a> Neuron. 49, 503-516 (2006).</li><li>Kohwi, M., Osumi, N., Rubenstein, J. L., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+is+required+for+making+specific+subpopulations+of+granule+and+periglomerular+neurons+in+the+olfactory+bulb.">Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb.</a> J Neurosci. 25, 6997-7003 (2005).</li><li>Kohwi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subpopulation+of+olfactory+bulb+GABAergic+interneurons+is+derived+from+Emx1-+and+Dlx5/6-expressing+progenitors.">A subpopulation of olfactory bulb GABAergic interneurons is derived from Emx1- and Dlx5/6-expressing progenitors.</a> J Neurosci. 27, 6878-6891 (2007).</li><li>Hack, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+fate+determinants+of+adult+olfactory+bulb+neurogenesis.">Neuronal fate determinants of adult olfactory bulb neurogenesis.</a> Nat Neurosci. , (2005).</li><li>Ventura, R. E., Goldman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+radial+glia+generate+olfactory+bulb+interneurons+in+the+postnatal+murine+brain.">Dorsal radial glia generate olfactory bulb interneurons in the postnatal murine brain.</a> J Neurosci. 27, 4297-4302 (2007).</li><li>Kelsch, W., Mosley, C. P., Lin, C. W., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mammalian+precursors+are+committed+to+generate+neurons+with+defined+dendritic+projection+patterns.">Distinct mammalian precursors are committed to generate neurons with defined dendritic projection patterns.</a> PLoS Biol. 5, e300 (2007).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+centrosome+inheritance+maintains+neural+progenitors.">Asymmetric centrosome inheritance maintains neural progenitors.</a> Nature. 461, 947-955 (2009).</li></ol>

Die meisten Studien der Neurogenese in ventrikuläre und subventrikulären Zonen auf klassischen Schnitte Techniken verlassen, um die Mikroanatomie und zellulären Zusammenhänge in diesen Regionen zu untersuchen. Hier beschreiben wir eine alternative Technik, zuerst verwendet, um das Netzwerk der wandernden Ketten von Neuroblasten in der SVZ 1 erzeugt analysieren, dann verwendet werden, um die Regeneration des SVZ Vorläufer-Population nach anti-mitotische Behandlung 6, und in jüngster Zeit verwendet, um den genauen apikalen Studie Studie und basalen Zell-Zell-Interaktionen von Erwachsenen SVZ neuralen Stammzellen 2,3,4. Interessanterweise hat diese Technik ergeben, dass die neuralen Stammzellen, oder geben Sie B1-Zellen, der erwachsenen SVZ Teil eines gemischt Neuroepithel mit differenzierten nicht-teilenden Ependymzellen sind. En-face-Aufnahme nach wholemounts hat gezeigt, dass dieser gemischten Neuroepithel Windrad-Architektur, bestehend aus den apikalen Enden des Typs B1-Zellen durch große apikalen Oberflächen der Ependymzellen 4 umgeben ist. Das En-face-Analyse hat unser Verständnis von der Linie von neuralen Stammzellen in embryonalen und adulten Gehirn als bestehend aus Zellen mit apikalen Enden an den Ventrikel Oberfläche und basale Prozesse Kontaktaufnahme mit einem vaskulären Nische geklärt. Diese Ergebnisse würden beinahe unmöglich gewesen Verwendung klassischer Schnitte Techniken. Wholemounts auch die Identifizierung von neuralen Stammzellen über ihre Ventrikel-Kontakt apikale Prozess. Als weitere spezifische Marker für diese Stammzellen gefunden werden, wird wholemounts ein integraler Bestandteil der Identifizierung und Analyse von neuronalen Stammzellen Verhalten sein. Wholemounts der Seitenventrikel Wände bieten auch die ideale Perspektive für das Studium der planaren Polarität der Ependymzellen. Ependymzellen sind multiciliated Zellen entlang der Herzkammern, die CSF in einer koordinierten Art und Weise anzutreiben Funktion. Mit dem wholemount Technik wird die gesamte Ependymzellen Epithel en-face ausgesetzt und kann gebeizt und umfassend von der vorderen untersucht, um posterior und dorsal nach ventral Grenzen. Darüber hinaus Ependymzellen-Flow-Assays auf akut seziert durchgeführt, live wholemounts robust zeigen die ebene polarisierte flow von Ependymzellen Cilien erzeugt. Neuere Arbeiten mit wholemount Ansätze hat zellulären Determinanten dieser Ependymzellen planaren Polarität 5 freigelegt. Interessanterweise haben wholemount Studien auch vorgeschlagen, dass Ependymzellen-generated Liquorfluss Steigungen chemorepellents, dass die Abwanderung junger Neuronen in der SVZ 7 Leitfaden stellt. Wholemount Ansätze, die zunächst identifiziert das Netzwerk der wandernden Nervenzellen Ketten sind daher weiterhin Einblicke in Mechanismen, die Kette Migration. Die Analyse der VZ und SVZ durch wholemount Bildgebung wird ein neuer Ansatz für zukünftige Studien und ein Weg, um unser Verständnis der vorhandenen Studien zu klären. Zum Beispiel, schlug einer aktuellen Studie, dass neurale Stammzellen im erwachsenen SVZ CD133 + / CD24-Zellen wurden in Kontakt mit der Herzkammer 8. Basierend auf ihrer Immunfärbung in den Abschnitten, behauptete diese Autoren, dass diese Zellen eine Subpopulation von multiciliated Ependymzellen wurden. Doch in unserer Studie mit dem wholemount Ansatz, der eine umfassendere Sicht auf die gesamte Ependymzellen Epithel gibt, fanden wir, dass alle Ependymzellen ausdrückliche CD24 und die einzige Herzkammer-Kontakt-Zellen, die CD133 wurden + / CD24-waren eine Teilmenge des Typs B1 Zellen 4. Darüber hinaus verspricht die wholemount Technik, um auch in Zukunft Studien, die kürzlich beschriebenen Mosaik Organisation von neuronalen Stammzellen im erwachsenen Gehirn 9 Nützliche. Mehrere Studien haben gezeigt, dass neurale Stammzellen im erwachsenen Gehirn keine homogene Bevölkerung, sind aber regional festgelegt und produzieren normalerweise nur bestimmte Subtypen der Riechkolben Interneuronen. Diese Studien haben vorgeschlagen, dass verschiedene Subpopulationen von neuralen Stammzellen können entweder durch die Expression spezifischer Transkriptionsfaktoren 10,11,12,13,14 und / oder durch ihre regionale Lokalisation entlang der dorsal-ventral und anterior-posterior Ausdehnungen der unterschieden werden Seitenwand 9,15,16. Als weitere molekulare Marker der regional festgelegten Subpopulationen von adulten neuralen Stammzellen identifiziert sind, sollte wholemount Bildgebung bieten einen umfassenden Blick auf die parcelation dieser unterschiedlichen Vorläuferzellen Domänen entlang der Ventrikelwand. Die wholemount Dissektion und bildgebende Verfahren hier vorgestellten kann auch verwendet werden, um die Ventrikelwände in den Embryo zu analysieren. Die Zerlegung des embryonalen lateralen Wand durchgeführt wird, Schritt-für-Schritt, in der gleichen Weise. Es gibt nur geringe Unterschiede in der Schwierigkeit, die embryonale Ventrikel sind relativ größer machen die Präparation einfacher, aber das Gewebe weicher macht Manipulationen erschwert. Insbesondere kann eine ähnliche Darstellung des lateralen Ventrikel in Embry verwendet werdenos zu sezieren die kortikale Wand des Ventrikels auf kortikale Neurogenese zu studieren. Jüngsten Untersuchungen zufolge asymmetrische Zentrosom Erbe pflegt radialen Gliazellen an der ventrikulären Oberfläche während kortikale Neurogenese 17. En-face Bildgebung von radialen Gliazellen apikalen Oberflächen bieten Einblicke in die Zentrosomen in diesen teilende Zellen sind asymmetrisch vererbt. Wie bei den meisten Techniken, insbesondere solche, bei denen eine präzise Fingerfertigkeit erfordert Meisterschaft zu praktizieren. Es gibt jedoch einige Elemente in der Sektion, die Schlüssel zu einer besseren Ergebnisse sind: 1) Beleuchtung Anpassung der Beleuchtung der Probe, um Schatten zu schaffen liefert wertvolle Kontrast bei der Präparation von Gewebe, die sonst relativ homogen, 2) mit der Zange wie zwei Insektennadeln der Zange in dieser Technik sind nie verwendet werden, um Prise zusammen oder holen Sie sich Gewebe, sind aber als wendiger Stifte, die ständig nachjustiert, um das Gewebe zu stabilisieren können beim Schneiden, 3) ein ausgewogenes Verhältnis von sanften Einfahren und Schneiden der Messer sollte nicht nur verwendet werden, um geschnitten, sondern auch auf sanfte Rückzug liefern, um die medialen und lateralen Wände trennen, nicht vergessen, dass die Mehrheit dieser Sektion ist eigentlich durch sanfte Einziehen nur mit unterbrochenem Schnitt durchgeführt werden.

the subventricular zone en-face: wholemount staining and ependymal flow

Die Wände der Seitenventrikel enthalten die größten germinal Region im erwachsenen Gehirn von Säugetieren. Die subventrikulären Zone (SVZ) in diesen Mauern ist ein umfassend untersucht Modellsystem für das Verständnis des Verhaltens von neuralen Stammzellen und die Regulation der adulten Neurogenese. Traditionell wurden diese Studien über klassische Schnitte Techniken für die histologische Analyse herangezogen. Hier präsentieren wir einen alternativen Ansatz, der wholemount Technik, die eine umfassende, en-face Angesichts dieser Keim Region bietet. Im Vergleich zu Abschnitten, zu bewahren wholemounts die komplette Zytoarchitektur und zelluläre Beziehungen innerhalb der SVZ. Dieser Ansatz hat kürzlich ergeben, dass die adulten neuralen Stammzellen, oder geben Sie B1-Zellen, Teil eines gemischt Neuroepithel mit differenzierten Ependymzellen Auskleidung der Seitenventrikel sind. Darüber hinaus hat dieser Ansatz verwendet worden, um die planare Polarisierung der Ependymzellen und der Liquor fließen sie erzeugen in den Ventrikel zu studieren. Mit den letzten Beweis dafür, dass adulte neurale Stammzellen eine heterogene Population, die regional angegeben wird, werden die wholemount Ansatz wahrscheinlich ein wesentliches Instrument für das Verständnis der Organisation und Parzellierung des Stammzell-Nische.

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Die Seitenventrikel Wände enthalten die größten germinal Region im erwachsenen Gehirn von Säugetieren. Traditionell haben die Studien auf die Neurogenese in dieser Region auf die klassische Schnitte Techniken für die histologische Analyse herangezogen. Hier präsentieren wir einen alternativen Ansatz, der wholemount Technik, die eine umfassende, en-face Angesichts dieser Keim Region bietet.

Die Subventrikularzone En-face: Wholemount Färbung und Ependymale Durchfluss

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

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Neuroscience

41.6K Aufrufe.  University of California, San Francisco - UCSF. Die Seitenventrikel Wände enthalten die größten germinal Region im erwachsenen Gehirn von Säugetieren. Traditionell haben die Studien auf die Neurogenese in dieser Region auf die klassische Schnitte Techniken für die histologische Analyse herangezogen. Hier präsentieren wir einen alternativen Ansatz, der wholemount Technik, die eine umfassende, en-face Angesichts dieser Keim Region bietet.

Serie: The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Wholemount Immunhistochemie für die Aufdeckung von komplexen Gehirn Topographie

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

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Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Vorbereitung der akuten Subventrikulärzone Slices für Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neonatal Subventrikulärzone Elektroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Zellsortierung von Neural Stamm- und Vorläuferzellen aus dem erwachsenen Maus Subventrikularzone und Live-Bildgebung ihrer Zellzyklus Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Messung des Abbaus von Hirnödem nach dem Schlaganfall, Infarktzone und Blut-Hirn-Barrierenabbau in einem einzigen Satz von Nagetier-Gehirnproben

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

Kryoschnitt-Dissektion der adulten subependymalen Zone für genaue und tiefe quantitative Proteomanalyse

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

<meta charset="utf8"></head><body>Effiziente Nukleofektion von NSCs, die aus der subventrikulären Zone der Maus isoliert wurden

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Verbesserte Nukleofektion für einen hocheffizienten Gentransfer in murinen subventrikulären Zonen abgeleiteten neuralen Stammzellkulturen

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolierung, Expansion und Nukleofektion von neuralen Stammzellen aus der adulten subventrikulären Zone der Maus

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...