Les travaux pris en charge par NIH HD-32116, la famille Sandler Soutenir la Fondation, le Fonds John Bowes de cellules souches, le MEXT, le MHLW et HFSP. ZM soutenu par la Fondation Carlos Baldoceda et UCSF Krevans Bourse. AA-B. Heather détient la présidence et Melanie Muss Doté.

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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. 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La plupart des études de la neurogenèse dans les zones ventriculaires et sous-ventriculaire se sont appuyés sur des techniques classiques de sectionnement d&#39;examiner les relations microanatomie et cellulaires dans ces régions. Nous décrivons ici une technique alternative, d&#39;abord utilisé pour analyser le réseau de chaînes migratoires des neuroblastes générés dans le SVZ 1, puis utilisées pour étudier la régénération de la population progénitrices SVZ suivantes antimitotiques traitement de 6, et plus récemment utilisée pour étudier les apicale précise basale et interactions cellule-cellule des cellules souches neurales adultes SVZ 2,3,4. Fait intéressant, cette technique a révélé que les cellules souches neurales, ou des cellules de type B1, de l&#39;adulte SVZ font partie d&#39;un neuroépithélium mélangé avec différenciées ne se divisent cellules épendymaires. En face d&#39;imagerie utilisant des échantillons entiers a montré que ce mélange a une architecture neuroépithélium moulinet composé des terminaisons apicale des cellules de type B1 entouré par de grandes surfaces apicales des cellules épendymaires 4. Cette analyse en face a clarifié notre compréhension de la lignée de cellules souches neurales dans le cerveau embryonnaire et adulte comme étant composé de cellules avec des terminaisons à la surface apicale du ventricule et des processus basale contactant une niche vasculaire. Ces résultats aurait été presque impossible en utilisant les techniques classiques de sectionnement. Échantillons entiers aussi faciliter l&#39;identification des cellules souches neurales via leur ventricule contactant processus apical. Comme marqueurs plus spécifiques de ces cellules souches sont trouvées, des échantillons entiers seront une partie intégrante de l&#39;identification et l&#39;analyse de comportement des cellules souches neurales. Échantillons entiers des murs ventricule latéral fournissent également la perspective idéale pour étudier la polarité planaire des cellules épendymaires. Cellules épendymaires sont les cellules tapissant les ventricules multiciliated cette fonction pour propulser le LCR de façon coordonnée. Avec la technique wholemount, l&#39;épithélium épendymaire entière est exposée en face et peut être teinté et étudiées en détail de son avant vers l&#39;arrière et dorsale des frontières ventrale. Par ailleurs, des tests de débit effectués sur épendymaires aiguë disséqué, échantillons entiers vivent robuste montrer le flux planaire polarisée générés par les cils épendymaires. Des travaux récents utilisant des approches wholemount a découvert déterminants cellulaires de cette polarité planaire épendymaires 5. Fait intéressant, les études wholemount ont également suggéré que épendymaires-généré des flux de LCR établit des gradients de chemorepellents qui guident la migration des jeunes neurones dans le SVZ 7. Des approches Wholemount qu&#39;il a identifié le réseau de chaînes de neurones migratoires sont donc continuer à fournir des indications sur les mécanismes de régulation de la migration en chaîne. L&#39;analyse de la VZ et SVZ par wholemount imagerie ajoute une nouvelle approche à la fois pour les études futures et une façon de clarifier notre compréhension des études existantes. Par exemple, une étude récente suggère que les cellules souches neurales dans le SVZ adulte ont été CD133 + / CD24-cellules en contact avec les 8 ventricule. Basé sur leur immunomarquage dans les sections, ces auteurs ont prétendu que ces cellules ont été une sous-population de cellules épendymaires multiciliated. Cependant, dans notre étude en utilisant l&#39;approche wholemount, ce qui donne une vue plus complète de l&#39;épithélium toute épendymaires, nous avons constaté que toutes les cellules épendymaires exprimer CD24 et le seul ventricule contact de cellules qui ont été CD133 + / CD24-ont un sous-ensemble de l&#39;type B1 cellules 4. Par ailleurs, la technique wholemount promet d&#39;être utile dans de futures études examinant l&#39;organisation de mosaïque récemment décrit des cellules souches neurales dans le cerveau adulte 9. Plusieurs études ont montré que les cellules souches neurales dans le cerveau adulte ne sont pas une population homogène, mais sont spécifiées régionalement et, normalement, ne produisent que des sous-types spécifiques d&#39;interneurones bulbe olfactif. Ces études ont proposé que différentes sous-populations de cellules souches neurales peuvent être distingués soit par l&#39;expression des facteurs de transcription spécifiques 10,11,12,13,14 et / ou par leur localisation régionale le long de la dorsale des degrés-ventral et antéro-postérieure de la paroi latérale 9,15,16. Comme plusieurs marqueurs moléculaires de la sous-populations régionalement spécifié de cellules souches neurales adultes sont identifiés, l&#39;imagerie wholemount devrait fournir une vue complète de l&#39;parcelation de ces domaines progénitrices différents le long de la paroi ventriculaire. La dissection wholemount et des techniques d&#39;imagerie présentées ici peuvent également être utilisés pour analyser les parois ventriculaires chez l&#39;embryon. La dissection de la paroi latérale est réalisée embryonnaire, étape par étape, de la même manière. Il ya seulement de légères différences dans le niveau de difficulté; les ventricules embryonnaires sont relativement plus faire la dissection plus facile, mais le tissu est doux rendant la manipulation plus difficile. En particulier, une exposition similaire du ventricule latéral peut être utilisé dans EmbryOS pour disséquer la paroi corticale du ventricule pour étudier la neurogenèse corticale. Des données récentes suggèrent que l&#39;héritage asymétriques centrosome maintient glie radiaire à la surface ventriculaire pendant la neurogenèse corticale 17. En face de l&#39;imagerie radiale gliales surfaces apicales peuvent fournir des indications sur la manière dont les centrosomes au sein de ces cellules qui se divisent de façon asymétrique sont héréditaires. Comme avec la plupart des techniques, en particulier celles impliquant la dextérité précise, la maîtrise exige la pratique. Il ya, cependant, quelques éléments dans la dissection qui sont essentiels à de meilleurs résultats: 1) l&#39;éclairage ajuster l&#39;éclairage de l&#39;échantillon pour créer des ombres donne un contraste précieuse lors de la dissection du tissu qui est par ailleurs relativement homogène, 2) à l&#39;aide de la pince, comme deux broches d&#39;insectes de la pince dans cette technique sont jamais utilisés pour pincer ensemble ou ramasser des tissus, mais ils sont utilisés comme des épingles maniable qui peut être constamment réajustés afin de stabiliser le tissu tout en réduisant, 3) un équilibre de rétractation doux et la coupe du couteau ne doit pas seulement être utilisé pour couper, mais aussi de fournir la rétraction douce pour séparer les murs médial et latéral, en rappelant que la majorité de cette dissection est effectivement réalisé par le biais de rétraction douce avec seulement la coupe intermittente.

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

Les parois des ventricules latéraux contiennent la plus grande région germinales dans le cerveau adulte des mammifères. La zone sous-ventriculaire (SVZ) dans ces murs est un système modèle largement étudiés pour comprendre le comportement des cellules souches neurales et la régulation de la neurogenèse adulte. Traditionnellement, ces études se sont appuyés sur des techniques classiques de sectionnement pour l&#39;analyse histologique. Nous présentons ici une approche alternative, la technique wholemount, qui fournit une approche globale, en vue de face de cette région germinales. Comparé à des sections, des échantillons entiers de préserver la cytoarchitecture complète et les relations cellulaires dans le SVZ. Cette approche a récemment révélé que les cellules souches adultes de neurones, ou cellules de type B1, font partie d&#39;un neuroépithélium mélangé avec des cellules différenciées épendymaires tapissant les ventricules latéraux. En outre, cette approche a été utilisée pour étudier la polarisation planaire des cellules épendymaires et l&#39;écoulement du liquide céphalorachidien qu&#39;ils génèrent dans le ventricule. Avec des preuves récentes que cellules souches neurales adultes constituent une population hétérogène qui est régionalement spécifié, l&#39;approche wholemount sera probablement un outil essentiel pour comprendre l&#39;organisation et parcellisation de ce créneau de cellules souches.

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

Les murs du ventricule latéral contiennent la plus grande région germinales dans le cerveau adulte des mammifères. Traditionnellement, les études sur la neurogenèse dans cette région se sont appuyés sur des techniques classiques de sectionnement pour l&#39;analyse histologique. Nous présentons ici une approche alternative, la technique wholemount, qui fournit une approche globale, en vue de face de cette région germinales.

La zone sous-ventriculaire Fr-face: Coloration Wholemount et Flow épendymaire

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 vues.  University of California, San Francisco - UCSF. Les murs du ventricule latéral contiennent la plus grande région germinales dans le cerveau adulte des mammifères. Traditionnellement, les études sur la neurogenèse dans cette région se sont appuyés sur des techniques classiques de sectionnement pour l'analyse histologique. Nous présentons ici une approche alternative, la technique wholemount, qui fournit une approche globale, en vue de face de cette région germinales.

Vidéo: 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.

Immunohistochimie Wholemount de la topographie du cerveau révélatrice complexe

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.

Préparation des tranches aiguës zone sous-ventriculaire pour l&#39;imagerie calcique

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

Néonatale électroporation zone sous-ventriculaire

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.

Immunohistochimie et calcium méthodes d&#39;imagerie dans 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.

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 ...

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.

Tri cellulaire des neurones cellules souches et progénitrices de la souris adulte-ventriculaire Zone et Live-imagerie de la dynamique de leur cycle cellulaire

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.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

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.

Mesure de l’œdème cérébral post-AVC, de la zone infarctus et de la dégradation de la barrière céphalo-cérébrale dans un seul ensemble d’échantillons de cerveaux de rongeurs

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.

Dissection cryo-section de la zone sous-épendymaire adulte pour une analyse quantitative précise et approfondie du protéome

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>Nucléofection efficace de CSN isolées de la zone sous-ventriculaire murine

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>Pleins feux sur l’auteur : Nucléofection améliorée pour l’administration de gènes à haute efficacité dans des cultures de cellules souches neurales dérivées de la

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.

Isolement, expansion et nucléofection de cellules souches neurales de la zone sous-ventriculaire murine adulte

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 ...