Un grand merci à Elaine Pranski et Carolyn Suwyn d&#39;assistance technique excellente et à Harry LeVine III, M. Paul Murphy, et se prépare pour des conversations importantes Marla. Le financement a été fourni par RR-00165, PO1AG026423, P50AG025688, AG030539, la Fondation Woodruff et le Comité de recherche de l&#39;Université Emory.

Partie 1: Préparation des homogénats de tissus clarifié <ol><li> Dounce homogénéiser non fixée tissu cortical en 4 fois le volume du tampon glacé (0,1 M tampon phosphate salin [Ca + + - et Mg + + libre] plus un cocktail 2x inhibiteur de la protéase [Santa Cruz]), avec environ 30 coups, même un pilon (c&#39;est à dire ajouter des tampons 400μl à 100mg de tissu). </li><li> Homogénats Spin pendant 5 minutes à 3000 g (4 ° C). Retirez délicatement surnageants clarifiés et stocker des aliquotes de ces extraits à -80 ° C jusqu&#39;à utilisation. </li><li> Déterminer la concentration en protéines totales (pg / pl) pour homogénats clarifiés à l&#39;aide d&#39;un acide bicinchoninique (BCA) dosage, selon les instructions du fabricant (ThermoFisher). </li></ol> Partie 2: Préparation des échantillons SDS-PAGE <ol><li> Avec un puits 10, 10-20% de gel tricine (Invitrogen), le volume maximal qui peut être chargé par puits est ~ 25 pi. Le volume total comprend 2x tampon d&#39;échantillon SDS et 10x agent réducteur, de sorte que le volume maximal d&#39;homogénat clarifié qui peuvent être chargés par puits est 10 ul. Clarification de 20% (p / v) homogénats corticaux doit avoir une concentration totale en protéines supérieure à 5 pg / pl, permettant de 50μg de protéines totales par puits. Échantillons contenant moins de protéines 5mg/ml nécessitera de chargement moins de protéines totales par puits. Toutefois, les taux de protéines élevé sont optimales pour la détection de Aß monomérique et agrégée (protéines totales 50-60μg est optimal). Toujours charger la même quantité de protéines totales par puits. </li><li> Pour chaque gel, la charge d&#39;au moins un bien avec 10 ul d&#39;un marqueur de poids moléculaire telles que SeeBlue Plus2 (Invitrogen). Comme contrôle positif, course de 10 100 ng de synthèse Aß40 ou Aß42 peptide (dilué dans 1xPBS) dans un autre puits. </li><li> Préparer des mélanges de réaction sur la glace, en utilisant fraîchement décongelé, homogénats clarifiés. Tubes vortex pendant 5-10 secondes, de la chaleur dans un bain à sec à 100 ° C pendant 5 minutes, puis rapidement tourner tous les échantillons pour enlever la condensation dans le bouchon. </li></ol> Partie 3: L&#39;électrophorèse sur gel SDS-PAGE <ol><li> Charger les échantillons au vortex sur un gel tricine 10-20% dans l&#39;XCell Sure Lock mini-cellule Gelbox, en utilisant Tricine tampon SDS courir, conformément aux instructions du fabricant (Invitrogen), </li><li> Exécuter gel à une tension constante de 125V pendant environ 90 minutes. Laissez échantillons courir jusqu&#39;à la bande de marqueur 4KDa est d&#39;environ 1 cm du bas du gel. </li></ol> Partie 4: Protéines Transfert à partir de gels d&#39;Membranes <ol><li> Retirez délicatement les gels de boîtier en plastique et d&#39;assembler les sandwichs le transfert à l&#39;intérieur du module XCell II Blot, conformément aux instructions du fabricant (Invitrogen). Pré-humide buvard plaquettes et des membranes de nitrocellulose 0,2 um avec du Tris-Glycine transfert de mémoire tampon (20% de méthanol), et éliminer les bulles d&#39;coussinets buvard ou papier filtre / membrane sandwich. </li><li> Dans le Gelbox XCell, remplir la chambre intérieure avec un tampon de transfert et de remplir la chambre extérieure avec DIH 2 O (exposition méthanol peut s&#39;user l&#39;gelbox en plastique au cours du temps). </li><li> Exécuter le transfert pendant 2-3 heures à une intensité constante de 25mA. </li><li> Lorsque le transfert est terminé, déconstruire le sandwich et le lieu des gels en DIH 2 O et les membranes dans 1xPBS, à la fois dans le carré des boîtes de Pétri en plastique (ou un autre récipient approprié). </li><li> Pour visualiser l&#39;efficacité du transfert de protéines, les gels peuvent être colorés avec Simply Blue SafeStain (Invitrogen), et les membranes avec du Ponceau S tache rouge (Aldrich Sigma), à la fois selon les instructions du fabricant. Cette coloration ne pas interférer avec immunoblot. Si la coloration révèle la protéine de transfert inefficaces, de modifier le temps de transfert à l&#39;étape 3. </li></ol> Partie 5. Antigène-épitope de récupération &amp; immunoblot <ol><li> Récupération d&#39;antigène est une étape importante en révélant les épitopes Aßi sur la membrane pour la liaison des anticorps au cours immunoblot. Toute bain vapeur, micro-ondes, ou de l&#39;eau qui maintient une température constante de 100 ° C suffisent. Pour la récupération d&#39;antigène dans un bateau à vapeur, la chaleur de la membrane d&#39;étanchéité dans une pochette en plastique robuste Kapak rempli 1xPBS, à température ambiante. Posez le plat dans un sachet vapeur préchauffé, une fois le sachet commence à se dilater, incuber pendant 15 minutes supplémentaires. Laisser la membrane à refroidir lentement avant de le retirer de la poche, pour éviter le froissement excessif. </li><li> A température ambiante, retirez soigneusement la membrane de la poche vapeur et rincer la membrane pendant 5 minutes dans 1xPBS, suivie d&#39;un rinçage de 5 minutes dans du TBS-T (Tris-Buffered Saline, pH 8,0 avec 0,05% de Tween-20 [Sigma] ), sur un agitateur orbital. Incuber la membrane dans une solution de blocage (lait écrémé 2,5% dans du TBS-T) pour une heure, je tremblais. Sans rinçage, le transfert de la membrane d&#39;un plat ou d&#39;un sachet en plastique contenant l&#39;anticorps primaire dilué dans une solution de blocage (6E10 au 1:1000 [1μg/ml] 1:5.000 [0.2μg/ml] dilution, Covance), bulle en évitants. Incuber sur un agitateur orbital à température ambiante pendant une heure puis 24-48 heures d&#39;agitation à 4 ° C (incubation plus longue peut donner un meilleur signal). </li><li> Rincer la membrane pendant 30 minutes dans du TBS-T (un rinçage rapide suivie par rinçages x 3 minutes 10). </li><li> Incuber la membrane dans HRP-anticorps secondaire conjugué (Amersham ECL mouton anti-souris, GE Healthcare), dilué au 1:10000 en solution de blocage, pendant 90 minutes sur l&#39;agitateur à température ambiante. </li><li> Rincer la membrane pendant 30 minutes dans du TBS-T (un rinçage rapide suivie par rinçages x 3 minutes 10). </li><li> Sur le shaker, incuber la membrane fraîchement faite SuperSignal Ouest réactif électrochimioluminescence Pico (ThermoFisher) pendant 5 minutes, éponger l&#39;excès de réactif sur off non pelucheux papier filtre et placer la membrane dans une cassette de film, entre les protecteurs feuille de plastique. Blot hors supplémentaires réactif SuperSignal avec de la poussière sans Kimwipes si nécessaire. </li><li> Exposer à Kodak Biomax MR film pour des intervalles de 30 secondes jusqu&#39;à 30 minutes et se développer dans un développeur film. Après 5 minutes, les bandes monomère Aßi sera probablement saturé. </li><li> Les membranes peuvent être dépouillé (après rinçage dans du TBS-T) en agitant dans Restore Plus de décapage de tampon pendant 30 minutes à température ambiante et sondé avec des anticorps supplémentaires, si désiré. </li></ol> Partie 6: Représentant Immunoblot: <img src="/files/ftp_upload/1916/1916fig1a.jpg" alt="Figure 1a" /> <img src="/files/ftp_upload/1916/1916fig1b.jpg" alt="Figure 1b" /> <img src="/files/ftp_upload/1916/1916fig1c.jpg" alt="Figure 1c" /> Les figures 1a-c. Homogénats clarifié contenant 50μg de protéines totales de 7 sujets humains sont analysés pour la présence de Aßi multimérique et APP. Immunoblot avec des anticorps 6E10 révèle monomères Aßi, dimères, trimères, tétramères, et l&#39;APP (bande supérieure) dans tous les cas d&#39;Alzheimer, ainsi que plus abondante moléculaires multimères Aßi poids dans 2 cas après JC. Synthétique Aß40 confirme l&#39;identité des bandes plus faible poids moléculaire. AD: La maladie d&#39;Alzheimer, DLB: La démence à corps de Lewy, ND: non déments humaine (a) l&#39;exposition du film de 30 minutes, (b) l&#39;exposition du film de 5 minutes, (c) une exposition de 30 secondes du film..

<ol>
	<li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Walker, L. C., LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebral+proteopathies.">The cerebral proteopathies.</a> Neurobiol. Aging. 21, 559-561 (2000).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+amyloid,+and+Alzheimer's+disease:+a+perspective+in+honor.">Aging, amyloid, and Alzheimer's disease: a perspective in honor.</a> of Carl Cotman. Neurochem. Res. 28, 1705-1713 (2003).</li><li>Walsh, D. M., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+oligomers+-+a+decade+of+discovery.">Abeta oligomers - a decade of discovery.</a> J. Neurochem. 101, 1172-1184 (2007).</li><li>Swerdlow, P. S., Finley, D., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+immunoblot+sensitivity+by+heating+of+hydrated+filters.">Enhancement of immunoblot sensitivity by heating of hydrated filters.</a> Anal. Biochem. 156, 147-153 (1986).</li><li>Ida, N., Hartmann, T., Pantel, J., Schr&#246;der, J., Zerfass, R., F&#246;rstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. <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+hetergeneous+A4+peptides+in+human+cerebrospinal+fuid+and+blood+by+a+newly+developed+sensitive+Western+blot+assay.">Analysis of hetergeneous A4 peptides in human cerebrospinal fuid and blood by a newly developed sensitive Western blot assay.</a> J. Biol. Chem. 271, 22908-22914 (1996).</li></ol>

Malgré l&#39;importance de l&#39;agrégation Aßi dans la pathogenèse de la maladie d&#39;Alzheimer 1,4,5, peu d&#39;études ont décrit ou quantifié la distribution de divers multimères Aßi dans des échantillons de tissu cortical 2. Communément utilisé des techniques d&#39;immunohistochimie ne permettent pas la discrimination des espèces distinctes Aßi multimérique dans le tissu cortical fixe. En non fixée homogénats de tissus corticaux, multimères Aßi peuvent être séparés et év...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Protease Inhibitor Cocktail Tablets</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-29130</td>
<td>1 tablet in 25ml buffer</td>
</tr>
<tr>
<td>BCA Protein Assay kit</td>
<td>&nbsp;</td>
<td>ThermoFisher Scientific</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0002</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Sample Buffer (2X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>NuPAGE Sample Reducing Agent (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>NP0004</td>
<td>&nbsp;</td>
<tr>
<td>SeeBlue Plus2 Pre-Stained Standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5925</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10-20% Tricine Gel 1.0 mm, 10 well</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625BOX</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose membranes, 0.2 &mu;m pore size</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2000</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC3675</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>ATX Ponceau S Red staining solution</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>09276</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>Kapak heat sealable plastic sample pouches</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0181225AA</td>
<td>&nbsp;</td>
<tr>
<td>6E10 mouse monoclonal antibody to A&beta;(1-16)</td>
<td>&nbsp;</td>
<td>Covance</td>
<td>SIG-39320</td>
<td>Dilute 1:1,000 up to 1:5,000 for WB</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>ECL Mouse IgG, HRP-Linked Whole A&beta; (from sheep)</td>
<td>&nbsp;</td>
<td>GE Healthcare Life Sciences</td>
<td>NA931-1ML</td>
<td>Dilute at 1:10,000</td>
</tr>
<tr>
<td>SuperSignal West Pico Chemiluminescent Substrate</td>
<td>&nbsp;</td>
<td>ThermoFisher</td>
<td>34077</td>
<td>&nbsp;</td>
<tr>
<td>Kodak Biomax MR Film</td>
<td>&nbsp;</td>
<td>Carestream Health</td>
<td>870 1302</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

Le pliage anormal et la polymérisation de la β-amyloïde (Aß) peptide est pensé pour initier la cascade dans la pathogenèse de maladies neurodégénératives maladie d&#39;Alzheimer<sup&gt; 1</sup&gt;. Aßi est surtout un 40 - peptide d&#39;acide ou de 42-amino qui est sujette à l&#39;auto-agrégation en β-feuille riche en fibrilles amyloïdes qui se trouvent dans les noyaux de plaques séniles dans le cerveau La maladie d&#39;Alzheimer. L&#39;augmentation de preuves suggère que de faible poids moléculaire, solubles multimères Aßi sont plus toxiques que l&#39;amyloïde Aß fibrillaire<sup&gt; 2</sup&gt;. L&#39;identification et la quantification des espèces de faible poids moléculaire élevé et de Aßi multimérique dans le tissu cérébral est un objectif essentiel dans la recherche la maladie d&#39;Alzheimer, et les méthodes employées peuvent également être appliquées à l&#39;identification et la caractérisation des multimères toxiques dans d&#39;autres proteopathies<sup&gt; 3</sup&gt;. Naturellement multimères Aßi peuvent être détectés par électrophorèse en gel de polyacrylamide-SDS suivie par immunoblot avec des anticorps spécifiques Aßi. Cependant, la séparation et la détection de Aß multimérique nécessite l&#39;utilisation de très concentrée homogénats corticaux et la récupération d&#39;antigène dans les petites membranes de nitrocellulose la taille des pores. Nous décrivons ici une technique pour la préparation de clarifier humaine homogénats corticaux, la séparation des protéines par SDS-PAGE, et l&#39;antigène de démas / blot avec des anticorps 6E10 à la région N-terminale du peptide Aß occidentaux. En utilisant ce protocole, nous avons constamment détecter les monomères Aßi, dimères, trimères, tétramères, et plus multimères de poids moléculaire dans le tissu cortical des humains à la pathologie d&#39;Alzheimer.

Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

SDS-PAGE/Immunoblot Detection of A&amp;#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Nous décrivons une technique pour la préparation de clarifier humaine homogénats corticaux, la séparation des protéines par SDS-PAGE, la récupération d&#39;antigène et immunoblot avec un anticorps dirigé contre le peptide Aß. En utilisant ce protocole, nous avons constamment de détecter Aßi monomère et multimère dans le tissu cortical des humains à la pathologie d&#39;Alzheimer.

Détection SDS-PAGE/Immunoblot de multimères de Aßi tissu cortical humain homogénats utilisant l&#39;antigène démas

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

sds-pageimmunoblot-detection-multimers-human-cortical-tissue

Research

JoVE Journal

Neuroscience

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

24.0K vues.  Emory University. Nous décrivons une technique pour la préparation de clarifier humaine homogénats corticaux, la séparation des protéines par SDS-PAGE, la récupération d'antigène et immunoblot avec un anticorps dirigé contre le peptide Aß. En utilisant ce protocole, nous avons constamment de détecter Aßi monomère et multimère dans le tissu cortical des humains à la pathologie d'Alzheimer.

Vidéo: SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

La cryoconservation de tissu cortical Blocs pour la génération des cultures de neurones hautement enrichi

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Recherche

Neurosciences

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Haute Sensibilité 5-hydroxyméthylcytosine détection dans le tissu cérébral des souris Balb / C

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Cohérence entre la fonction cérébrale corticale et la performance neurocognitifs dans des conditions de gravité Changé

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Imagerie fonctionnelle en calcium développement des réseaux corticaux

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantification des filamenteuses Actine (F-actine) punctas chez le rat neurones corticaux

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantification des molécules d&#39;endosome et de lysosome dans les neurones cultivés utilisant des sondes fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Visualisation des Modules corticales dans cortex mammifère aplatie

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

Modélisation statistique de la connectivité corticale à l'aide d'électroencéphalogrammes non invasifs

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...