This work was partially supported by B.G. Negev technology and Linda Powers's contribution.

Les expérimentations animales ont été approuvés par le comité d&#39;éthique pour l&#39;expérimentation animale dans l&#39;Université Ben Gourion (IL-39-08-2010). Extraction de la protéine 1 de cerveau de souris <ol><li> Préparer 3 tubes de 15 ml contenant chacun 5 ml de solution et placer les tubes Ringer sur la glace. </li><li> Sacrifier la souris en utilisant l&#39;isoflurane (ATTENTION - utiliser hotte chimique) pendant 10-20 sec suivie par dislocation cervicale et décapitation immédiate. </li><li> Retirez le cerveau du crâne et de rincer pendant quelques secondes avec la solution de Ringer glacée pour éviter d&#39;endommager les tissus du cerveau. </li><li> L&#39;utilisation d&#39;une lame chirurgicale, séparer le cervelet (CR) et le tronc cérébral (BS) à partir du cortex frontal. Ensuite, séparer le cervelet du tronc cérébral et couper le lobe frontal (FL) du cortex frontal; supprimer le bulbe olfactif du lobe frontal. Placez les régions 3 du cerveau dans les désignés tubes de 15 ml. </li><li> Homogénéiser le tissu dans chaque tube. Ajouter frsolution glace chair froide Ringer sorte que chaque tube contient le même volume et centrifuger pendant 7 minutes à 800 x g. </li><li> Eliminer le surnageant et ajouter 100 ul de tampon de lyse CHAPS à chaque tube. Bien mélanger, en faisant passer plusieurs fois la solution (~ 10) à travers une seringue de 1 ml avec une aiguille 21 G, et laisser incuber sur de la glace pendant 30 min. </li><li> Transférer le contenu de chaque tube dans un nouveau tube de micro-centrifugeuse et centrifuger pendant 30 min à 13 000 x g. Transférer le surnageant dans un nouveau tube de micro-centrifugeuse et déterminer la concentration de protéine en utilisant un procédé précédemment établi. Conserver les extraits de protéines dans des aliquotes de 10 pi à -80 ° C. </li></ol> 2. TRAP Essai Préparation des instruments, des solutions de réaction, et de protéines calculs de dilution <ol><li> Préparer nombre approprié de tubes de micro-centrifugation de protéines marquées pour des dilutions de l&#39;extrait et du tampon de réaction de télomérase. Effectuer toutes pipetage dans le protocole à l&#39;aide des conseils de filtrage pour éviter la contamination. </li&gt; <li> Décongeler les solutions suivantes sur glace: TRAP mélange réactionnel 10x, TS primaire, dNTP, extraits UPW et de protéines (voir tableau des matériaux pour les réactifs et réactifs concentrations). </li><li> Diluer la solution de l&#39;extrait de protéine à une concentration finale de 1 ug / ul avec de l&#39;eau ultra-pure (UPW). </li></ol> 3. télomérase Réaction <ol><li> Préparer la télomérase mélange réactionnel en ajoutant ce qui suit à un tube de micro-centrifugeuse: 2 pl TRAP mélange (10x), 1 pl TS primaire (0,1 ug), 1 dNTP pi (10 mm) et 15 pi UPW. Pour de multiples échantillons, multiplier les volumes mentionnés ci-dessus par le nombre d&#39;échantillons, plus 1. </li><li> Ajouter 19 ul du mélange réactionnel dans chaque tube de réaction et ajouter 1 pl de l&#39;extrait de protéine diluée (1 mg de protéines) à chaque tube pour un volume final de 20 ul. </li><li> Placer les tubes de réaction dans un bain d&#39;eau préchauffé à 30 ° C pendant 45 min. </li></ol> 4 La réaction PCR <ol><li> Quinze kmn avant la fin de la réaction de la télomérase, dégeler les solutions suivantes: ACX amorces, Titanium Taq tampons, UPW. </li><li> Préparer mélange réactionnel de PCR en ajoutant ce qui suit à un tube de micro-centrifugeuse: 2,5 pi titane Taq Buffer (10x), 1 pl ACX primaire (0,1 ug), 2 pl UPW (1 pi quand IC est utilisé) et 0,5 ul titane Taq polymérase (50x). Pour de multiples échantillons, multiplier les volumes mentionnés ci-dessus par le nombre d&#39;échantillons plus 2. </li><li> Ajouter 6 ul du mélange PCR dans chaque tube PCR et ajouter la totalité du volume de la réaction télomérase (20 pi) dans chaque tube pour un volume final de 26 ul. </li><li> Insérez les tubes PCR à un thermo cycleur PCR et exécuter le programme suivant: - 90 ° C - 2 min. - 94 ° C -. 30 sec * - 60 ° C -. 30 sec * - 72 ° C -. 45 sec * - 72 ° C - 2 min. Magasin de produits PCR à -20 ° C. * = 34 cycles </li></ol> 5 Agarose Mini-gel Préparation, échantillons Courir, et Gel coloration <ol><li> Ajouter 25 ml de tampon d&#39;électrophorèse frais (TBE) et une barre d&#39;agitation dans un bécher qui est 2-4 fois le volume de la solution tampon et saupoudrer lentement 1,125 g de la haute résolution (HR) de poudre d&#39;agarose alors que la solution est rapidement agité . Retirez la barre d&#39;agitation, couvrir le bécher avec un film plastique et percer dans un petit trou pour la ventilation. Chauffer le bécher dans le micro-ondes à puissance &quot;Low&quot; pendant 3 minutes, puis passer à la puissance &quot;High&quot; et chauffer la solution à impulsions courtes avec soin de ne pas causer de renverser. Notez que lorsque la mousse créée par chauffage devient grosses bulles et disparaît après quelques secondes, l&#39;agarose est prêt. </li><li> Couler le gel dans un appareil mini-gel horizontal, après que le gel se solidifie à température ambiante, le gel permet de refroidir pendant 20 min à 4 ° C avant utilisation. </li><li> Chargez chaque voie avec 25 ul d&#39;échantillon de TRAP (produits de PCR TRAP 20 pi et 5 pi 6x colorant gel-chargement) et de fonctionner à 110 V pendant 3 heures dans un 4 ° C chambre froide. </li><li> Préparer nucléiques 3x tache d&#39;acide solution de travail en ajoutant 15 pl 10.000 fois acide nucléique tache, solution stock à 50 ml Double Eau distillée (DDW) avec 0,1 M de NaCl (0,29 g). Colorer le gel pendant 20 min en remuant doucement. </li><li> Utilisez transilluminateur UV avec 302 nm de longueur d&#39;onde pour afficher les bandes d&#39;ADN TRAP échelle. </li></ol>

<ol>
	<li>Urquidi, V., Tarin, D., Goodison, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+telomerase+in+cell+senescence+and+oncogenesis.">Role of telomerase in cell senescence and oncogenesis.</a> Annu Rev Med. 51, 65-79 (2000).</li><li>Cong, Y., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actions+of+human+telomerase+beyond+telomeres.">Actions of human telomerase beyond telomeres.</a> Cell Res. 18, 725-732 (2008).</li><li>Tichon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+protection+by+novel+telomerase+activators+in+mesenchymal+stem+cells+derived+from+healthy+and+diseased+individuals.">Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.</a> Curr.Mol. Med. 13, 1010-1022 (2013).</li><li>Lannilli, F., Zalfa, F., Gartner, A., Bagni, C., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+TERT+associates+to+RNA+granules+in+fully+mature+neurons:+Role+in+the+translational+control+of+the+cell+cycle+inhibitor+p15INK4B.">Cytoplasmic TERT associates to RNA granules in fully mature neurons: Role in the translational control of the cell cycle inhibitor p15INK4B.</a> PloS one. 8, e66602 (2013).</li><li>De Jesus, B. B., Blasco, 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=Potential+of+telomerase+activation+in+extending+health+span+and+logevity.">Potential of telomerase activation in extending health span and logevity.</a> Curr opinion in Cell Biology. 24, 1-5 (2012).</li><li>Blackburn, E., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase:+An+RNP+enzyme+synthesizes+DNA.">Telomerase: An RNP enzyme synthesizes DNA.</a> Cold Spring Harb Perspect Biol. 3, 1-9 (2011).</li><li>Klapper, W., Shin, T., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+telomerase+activity+and+TERT+expression+during+brain+development+in+mice.">Differential regulation of telomerase activity and TERT expression during brain development in mice.</a> J. Neurosci. Res. 64, 252-260 (2001).</li><li>Caporaso, G. L., Lim, D. A., Alvarez-Buylla, A., Chao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+activity+in+the+subventricular+zone+of+adult+mice.">Telomerase activity in the subventricular zone of adult mice.</a> Mol. Cell Neurosci. 23, 693-702 (2003).</li><li>Eitan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Telomerase-increasing+compound+in+mouse+brain+delays+the+onset+of+amyotrophic+lateral+sclerosis.">Novel Telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.</a> EMBO Mol. Med. 4, 313-329 (2012).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repat+amplification+protocol+(TRAP).">Advances in quantification and characterization of telomerase activity by the telomeric repat amplification protocol (TRAP).</a> Nuc. Acid Res. 25, 2595-2597 (1997).</li></ol>

The authors declare that they have no competing financial interests.

Analyse de l&#39;activité de la télomérase dans le cerveau de la souris par le test TRAP est constitué de quatre étapes principales: 1) l&#39;extraction des protéines à partir de régions spécifiques du tissu cérébral 2) TS élongation d&#39;amorce par la télomérase - réaction télomérase 3) l&#39;amplification des produits de la réaction de télomérase par PCR 4 ) séparation des produits de PCR en utilisant un gel d&#39;agarose de haute résolution. Ce test TRAP modifié r?...

La télomérase est une ribonucléoprotéine composé de télomérase transcriptase inverse (TERT), la sous-unité catalytique de la télomérase, et un composant d&#39;ARN (TERC). Le rôle canonique de la télomérase est de préserver la bonne longueur des télomères en ajoutant des séquences répétitives (TTAGGG) aux extrémités télomériques, favorisant ainsi l&#39;intégrité du génome, et une prolifération cellulaire. Autres rôles ont été attribués à TERT dans les cellules, c&#39;est à dire qu&#39;il confère une résistance à l&#39;apoptose induite par divers agent endommageant 2, protège les cellules souches mésenchymateuses humaines du stress oxydatif 3, et joue un rôle pro-survie dans les neurones complètement différenciées par son association à TIA1 ARN positif 4 granules. TERT et est exprimé principalement actif dans les cellules somatiques et de division dans la plupart des types de cancers, alors que les niveaux d&#39;expression et l&#39;activité enzymatique sont étroitement régulés dans les cellules ne se divisant pas 5,6. Des études ont montré que dans le rcerveau Odent, l&#39;activité de la télomérase devient indétectable par jour postnatal 10, tandis que TERT ARNm est maintenue à des niveaux inférieurs à l&#39;âge adulte 7. D&#39;autres études ont démontré l&#39;activité télomérase à l&#39;intérieur de la zone sous-ventriculaire adulte, le bulbe olfactif, l&#39;hippocampe et dans le cervelet et le cortex adulte 8. Nous avons récemment démontré que les nouveaux composés qui augmentent l&#39;expression et l&#39;activité de la télomérase dans le cerveau et la moelle épinière des cordons exercent des effets neuroprotecteurs de la N-méthyl-D-aspartate (NMDA) - souris injectées, ont retardé l&#39;apparition et la progression de la maladie de sclérose latérale amyotrophique dans SOD1 transgénique souris et augmente la survie des motoneurones dans la moelle épinière de ces souris 9. Pour bien comprendre le rôle pro-survie de la télomérase dans les neurones et dans le cerveau, il est essentiel de développer un test rapide simple et relativement sensible pour la détection de l&#39;activité de la télomérase dans les différentes régions du cerveau. Le télomérique rprotocole d&#39;amplification épéter (TRAP) est un essai sensible bien connu combinant l&#39;activité de la télomérase canonique et la réaction en chaîne par polymérase (PCR). Dans cet essai, la télomérase ajoute TTAGGG répète sur un substrat de la télomérase (TS amorce) oligonucléotide, suivie d&#39;une amplification PCR qui génère des 6 paires de bases (pb) échelle d&#39;ADN à l&#39;aide du TS amorce inverse -. ACX marqué au 32P dCTP son sont utilisés pour détecter le faible quantité de produits de PCR. L&#39;échelle d&#39;ADN est résolu par électrophorèse sur gel de séquençage de Polyacrylamide (PAGE). Tant l&#39;intensité des bandes d&#39;ADN et la longueur de l&#39;échelle d&#39;ADN reflètent la quantité de molécules d&#39;enzymes actives et leur aptitude à la maturation, respectivement 10. Cette analyse traditionnelle détient des difficultés majeures: Le danger de l&#39;exposition à des substances radioactives, de grandes et difficiles à gérer séquençage PAGE et la consommation de temps de gel fonctionnement et l&#39;exposition du film du produit radioactif (nuit dans les deux cas). </p&gt; Ce protocole offre un test d&#39;agarose non radioactif amélioration mini-gel à base. Le pb échelle ADN 6 est résolu en utilisant un gel à 4,5% d&#39;agarose à haute résolution et la détection est réalisée en utilisant très sensible aux taches d&#39;acide nucléique. La combinaison de l&#39;utilisation à haute résolution d&#39;agarose des mini-gel et sensible aux taches d&#39;acide nucléique propose dosage facile à manipuler, permet d&#39;éliminer le risque d&#39;exposition à la radioactivité et de raccourcir considérablement la durée totale de l&#39;essai de 3 jours à plusieurs heures.

<table border="0" cellpadding="0" cellspacing="0" width="1102">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name of Material/ Equipment</td>
			<td style="width:83px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:628px;">Comments/Description</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:251px;">TRAP Mix (X10)</td>
			<td style="width:83px;"></td>
			<td style="width:143px;"></td>
			<td style="width:628px;">Made manually, mix the following in UPW: 630mM KCl, 200mM Tris-HCl pH8.2, 10mM EDTA, 15mM MgCl2, 1mg/ml BSA, 0.5% TWEEN (purchased from sigma). Aliquots are freezed&#160; in -20 C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:251px;">Ringer&#39;s solution</td>
			<td style="width:83px;"></td>
			<td style="width:143px;"></td>
			<td style="width:628px;">Made manually, mix the following in DDW: 124mM NaCl, 3mM KCl, 1.25mM NaH2PO4*H2O, 2mM MgSO4, 26mM NaHCO3, 1.802 g/L D-(+)-glucose anhydrous.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Isoflurane</td>
			<td style="width:83px;">Minrad INC.</td>
			<td style="width:143px;">NDC 60307-110-10</td>
			<td style="width:628px;">Handle with care in a chemical hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">dNTP&#39;s 10 mM</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">D7295</td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">TS primer (5&#39;-AATCCGTCGAGCAGAGTT-3&#39;)</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;"></td>
			<td style="width:628px;">DNA oligos orderd in DRY format and UPW added according to requierd concentration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">ACX primer (5&#39;-GCGCGGCTTACCCTTACCCTTACCCTAACC-3&#39;)</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;"></td>
			<td style="width:628px;">DNA oligos orderd in DRY format and UPW added according to requierd concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Titanium Taq Polymerase/Buffer</td>
			<td style="width:83px;">Clontech</td>
			<td style="width:143px;">S1792/S1793</td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">High Resolution agarose</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">A4718</td>
			<td style="width:628px;">Any high quality high-resolution agarose may be sutible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mini-gel apparatus&#160;</td>
			<td style="width:83px;">Bio-Rad</td>
			<td style="width:143px;">170-4466</td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Nucleic-acid Stain (GEL-RED)</td>
			<td style="width:83px;">Biotium</td>
			<td style="width:143px;">41003</td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">IC primer (5&#39;-ATCGCTTCTCGGCCTTTT-3&#39;)</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;"></td>
			<td style="width:628px;">DNA oligos orderd in DRY format and UPW added according to requierd concentration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">CHAPS lysis buffer</td>
			<td style="width:83px;">Cell Signaling Technology</td>
			<td style="width:143px;">9852S</td>
			<td>Add PMSF (protease inhibitor) to a final concentration of 1mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Ultra Pure Water (UPW)</td>
			<td style="width:83px;">Biological Industries</td>
			<td style="width:143px;">01-866-1B</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:251px;">CD-1 mice</td>
			<td style="width:83px;">HARLAN Laboratories INC.</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay

La télomérase, une ribonucléoprotéique, est responsable du maintien de la longueur des télomères et donc la promotion de l&#39;intégrité génomique, la prolifération et la durée de vie. En outre, la télomérase protège les mitochondries de stress oxydatif et confère une résistance à l&#39;apoptose, ce qui suggère son importance possible pour le survivant de non-mitotique des cellules hautement actifs tels que les neurones. Nous avons démontré précédemment la possibilité de nouveaux activateurs de la télomérase pour augmenter l&#39;activité de la télomérase et d&#39;expression dans les diverses régions du cerveau de la souris et pour protéger les cellules des neurones moteurs du stress oxydatif. Ces résultats renforcent l&#39;idée que la télomérase est impliqué dans la protection des neurones à partir de diverses lésions. Pour souligner le rôle de la télomérase dans le cerveau, nous comparons ici l&#39;activité de la télomérase dans le cerveau mâle et femelle souris et sa dépendance à l&#39;âge. test TRAP est un procédé standard pour la détection de l&#39;activité de la télomérase dans différents tissus ou lignées cellulaires. Ici, nous démontrons l&#39;analysis de l&#39;activité télomérase dans les trois régions du cerveau de souris non dénaturant par extraction de protéines à l&#39;aide de CHAPS tampon de lyse suivi d&#39;une modification de la méthode TRAP standard. Dans cet essai, deux étapes, la télomérase endogène s&#39;allonge un substrat spécifique de la télomérase (TS d&#39;amorce) en ajoutant 6 répétitions TTAGGG pb (réaction de télomérase). Les produits de la réaction de télomérase sont amplifiés par réaction de PCR créer une échelle d&#39;ADN de six incréments de pb. L&#39;analyse de l&#39;échelle de l&#39;ADN est effectué par 4,5% en haute résolution électrophorèse sur gel d&#39;agarose suivie par une coloration avec très sensible aux taches d&#39;acide nucléique. Par rapport à la méthode TRAP traditionnels qui utilisent marqué au 32P radioactif dCTP de la détection de l&#39;ADN et de polyacrylamide électrophorèse sur gel de résolution de l&#39;échelle de l&#39;ADN, ce protocole offre un gain de test TRAP pour évaluer l&#39;activité de la télomérase dans le cerveau de la souris, ce qui démontre la capacité de temps de non-toxique détecter des différences dans telomerasl&#39;activité dans les différents e région du cerveau de la souris mâle et femelle.

Des extraits protéiques de cellules entières ont été préparés à partir du lobe frontal (FL), le tronc cérébral (BS) et le cervelet (CR), dérivé de la femelle et 3 souris CD-1 mâles de 3 1 CD aux âges de 1 et 3 mois pour l&#39; test TRAP modifié et 3 souris CD-1 mâles à l&#39;âge de 2 mois pour le test TRAP radioactifs. L&#39;activité de télomérase a été détectée dans une ug de protéines extrait en utilisant le test TRAP à jour et 2 mg de protéines pour extraire le test TRAP radioactif. Détect...

Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Activité de la télomérase dans les différentes régions du cerveau de souris: la télomérase non-radioactifs Répétition protocole d&#39;amplification (TRAP) Dosage

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

telomerase-activity-various-regions-mouse-brain-non-radioactive

Research

JoVE Journal

Neuroscience

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

14.5K vues.  Ben-Gurion University of the Negev. Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Vidéo: Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Grandir cellules souches neurales de régions classiques et non conventionnelles du cerveau adulte rongeurs

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

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Neurosciences

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Purification de souris vaisseaux cérébraux

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

Résolution quantitative Haute Synaptic Proteome Profiling des souris régions cérébrales Après Auditory Discrimination apprentissage

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Analyse de haut-débit du comportement locomoteur dans le dosage de l’île de drosophile

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Utilisation de Zinc synaptique histochimie pour révéler les différentes régions et les limbes dans le cerveau en développement et Adult

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

Localisation du Locus Coeruleus dans le cerveau de souris

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

Quantification de l'activité subcellulaire ubiquitine-protéasome dans le cerveau des rongeurs

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Collection de régions de cerveau de rongeur congelés pour des analyses en aval

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissection et isolement de la glie murine de plusieurs régions du système nerveux central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Le cerveau aigu de souris tranche pour étudier l’activité spontanée de réseau hippocampal

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...