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

Tierversuche wurden von der Ethikkommission für Tierversuche in der Ben-Gurion-Universität (IL-39-08-2010) zugelassen. 1. Maus Gehirn Proteinextraktion <ol><li> Bereiten Sie 3 Rohre von je 15 ml 5 ml Ringer-Lösung und Ort Röhrchen auf Eis enthält. </li><li> Opfern Sie die Maus mit Isofluran-Narkose (Achtung - chemische Haube) für 10-20 Sekunden, gefolgt von Genickbruch und sofortige Enthauptung. </li><li> Entfernen das Gehirn aus dem Schädel und gründlich für einige Sekunden mit eiskaltem Ringer-Lösung, um Schäden am Hirngewebe zu verhindern. </li><li> Verwendung einer chirurgischen Klinge, trennen das Cerebellum (CR) und Hirnstamm (BS) von dem frontalen Kortex. Als nächstes trennen das Kleinhirn aus dem Hirnstamm und schneiden den Frontallappen (FL) aus dem frontalen Kortex; entfernen Sie den Riechkolben aus der Frontallappen. Legen Sie die 3 Hirnregionen in den dafür vorgesehenen 15-ml-Röhrchen. </li><li> Homogenisieren des Gewebes in jedes Röhrchen. Fr hinzufügenesh eiskalt Ringer-Lösung, so dass jeder Röhre enthält das gleiche Volumen und Zentrifuge für 7 min bei 800 x g. </li><li> Entfernen des Überstandes und Zugabe von 100 ul Lysepuffer CHAPS in jedes Röhrchen. Gut mischen, indem die Lösung mehrmals (~ 10) durch ein 1-ml-Spritze mit einer 21 G-Nadel, und Inkubation auf Eis für 30 min. </li><li> Wird der Inhalt jedes Röhrchens in eine neue Mikrozentrifugenröhrchen und Zentrifuge für 30 min bei 13.000 x g. Den Überstand in ein neues Mikrozentrifugenröhrchen und bestimmt die Proteinkonzentration unter Verwendung eines vorher festgelegten Verfahrens. Speichern die Proteinextrakte in Aliquots von 10 ul bei 80 ° C aufweist. </li></ol> 2. TRAP-Assay Vorbereitung der Instrumente, Reaktionslösungen und Protein Verdünnung Berechnungen <ol><li> Bereiten Sie entsprechende Anzahl von markierten Mikrozentrifugenröhrchen für Proteinextrakt Verdünnungen und Telomerase-Reaktionspuffer. Führen Sie alle Pipettieren im Protokoll mit Filterspitzen, um eine Verunreinigung zu verhindern. </li&gt; <li> Tauen die folgenden Lösungen auf Eis: TRAP-Reaktionsmischung 10x, TS-Primer, dNTPs, UPW und Proteinextrakte (siehe Tabelle der Werkstoffe für Reagenzien und Reagenzien Konzentrationen). </li><li> Verdünne die Lösung der Proteinextrakt in einer Endkonzentration von 1 ug / ul mit ultrareinem Wasser (UPW). </li></ol> 3. Telomerase-Reaktion <ol><li> Bereiten Telomerase Reaktionsmischung durch Hinzufügen der folgenden, um einen Mikrozentrifugenröhrchen: 2 ul TRAP-Mix (10x), 1 ul TS-Primer (0,1 ug), 1 ul dNTPs (10 mM) und 15 ul UPW. Für mehrere Proben, multiplizieren vorgenannten Volumina der Anzahl von Proben, plus 1. </li><li> Fügen 19 ul der Reaktionsmischung in jedes Reaktionsgefäß und mit 1 ul der verdünnten Proteinextrakt (1 ug Proteine) zu jedem Röhrchen für ein Endvolumen von 20 ul. </li><li> Zeigen die Reaktionsrohre in einem 30 ° C vorgewärmten Wasserbad für 45 min. </li></ol> 4. PCR-Reaktion <ol><li> Fünfzehn min vor dem Ende der Telomerase-Reaktion, tauen die folgenden Lösungen: ACX Grundierung, Titanium Taq-Puffer, UPW. </li><li> Bereiten PCR-Reaktionsmischung durch Zugabe Folgendes in einer Mikrozentrifugenröhrchen: 2,5 ul Titanium Taq-Puffer (10x), 1 ul ACX-Primer (0,1 ug), 2 ul UPW (1 ul, wenn IC verwendet wird) und 0,5 ul Titanium Taq-Polymerase (50x). Für mehrere Proben, multiplizieren vorgenannten Volumina der Anzahl von Proben plus 2. </li><li> Sie 6 ul des PCR-Gemisches zu jeder PCR-Röhrchen und fügen das gesamte Volumen des Telomerase-Reaktion (20 ul) zu jedem Röhrchen für ein Endvolumen von 26 ul. </li><li> Setzen Sie die PCR-Röhrchen zu einem PCR-Thermocycler und führen Sie das folgende Programm: - 90 ° C - 2 min. - 94 ° C - 30 Sek. * - 60 ° C - 30 sec. * - 72 ° C -. 45 s * - 72 ° C - 2 min. Speicher PCR-Produkte bei -20 ° C. * = 34 Zyklen </li></ol> 5. Agarose Mini-gel Vorbereitung, Proben Laufen, und Gelfärbung <ol><li> 25 ml gekühltes Elektrophorese-Puffer (TBE) und einen Rührstab in ein Becherglas, das 2-4 mal das Volumen des Pufferlösung ist und langsam in 1,125 g des hochauflösenden (HR) Agarose-Pulver bestreuen, während die Lösung schnell gerührt . Entfernen Sie den Rührstab, decken Sie den Becher mit einer Kunststofffolie und Pierce in einem kleinen Loch für die Belüftung. Heizen Sie den Becher in der Mikrowelle auf &quot;Low&quot; Macht für 3 min und wechseln Sie dann auf &quot;Hoch&quot; Macht und erwärmen die Lösung in kurzen Pulsen mit Sorgfalt Verschütten nicht verursachen. Beachten Sie, dass, wenn der Schaum erzeugt wird durch Erhitzen großen Blasen und verschwindet nach einigen Sekunden ist die Agarose bereit. </li><li> Guss das Gel auf einer horizontalen Mini Gel Vorrichtung, nachdem die Gel erstarrt bei Raumtemperatur zu ermöglichen, um das Gel für 20 min bei 4 ° C zu kühlen, bevor Gebrauch. </li><li> Laden Sie jeder Spur mit 25 ul TRAP Probe (20 ul TRAP PCR-Produkte und 5 ul 6x Gel-Ladefarbstoff) und 11 laufen0 V für 3 Stunden in einer 4 ° C kalten Raum. </li><li> Bereiten Nukleinsäurefarbstoff 3x Arbeitslösung durch Zugabe von 15 ul 10.000-Nukleinsäure-Färbung, Stammlösung zu 50 ml doppelt destilliertem Wasser (DDW) mit 0,1 M NaCl (0,29 g). Färben Sie das Gel für 20 min unter leichtem Schütteln. </li><li> Verwenden UV-Transilluminator mit 302 nm Wellenlänge, die Falle Leiter DNA-Banden zu sehen. </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 von Telomerase-Aktivität in Gehirn der Maus über die TRAP-Assay besteht aus 4 Schritten: 1) Extraktion von Proteinen spezifische Bereiche des Hirngewebes 2) TS Primerverlängerung durch Telomerase - Telomerasereaktion 3) Amplifikation der Telomerase-Reaktionsprodukte durch PCR 4 ) Auftrennung der PCR-Produkte mit hoher Auflösung Agarosegel. Diese modifizierte TRAP-Assay befasst sich mit zwei Hauptschwierigkeiten, die sich aus der traditionellen Assay steigen: die Verwendung von ra...

Telomerase ist ein Ribonukleoprotein-Telomerase zusammen Reverse Transkriptase (TERT), der katalytischen Untereinheit der Telomerase-RNA-Komponente und eine (TERC). Die kanonische Funktion der Telomerase ist, um die richtige Länge der Telomere durch Zugabe von repetitiven Sequenzen (TTAGGG) an die telomeren Enden, damit die Förderung der genomischen Integrität und Zellproliferation 1 erhalten. Weitere Rollen wurden in TERT-Zellen zurückzuführen, dh Resistenz gegen Apoptose durch verschiedene schädigende Mittel induziert 2 schützt humanen mesenchymalen Stammzellen vor oxidativer Beanspruchung 3, und spielt eine überlebens Rolle vollständig differenzierten Neuronen durch seine Verbindung zum positiven RNA TIA1 Granulat 4. TERT exprimiert wird, und vor allem in Körperteilenden Zellen und in den meisten Krebsarten aktiv ist, während die Enzymexpression und Aktivität sind fest in nicht-teilenden Zellen 5,6 geregelt. Studien haben gezeigt, dass das ROdent Gehirn, wird die Telomerase-Aktivität nicht nachweisbar durch postnatalen Tag 10, während TERT mRNA wird auf den unteren Ebenen bis ins Erwachsenenalter 7 gehalten. Andere Studien zeigten, Telomeraseaktivität in der Erwachsenenunter ventrikulären Zone, dem Riechkolben, dem Hippocampus und im Cortex und Cerebellum Erwachsenen 8. Wir haben vor kurzem gezeigt, dass neue Verbindungen, die Telomerase-Expression und Aktivität im Gehirn und Rückenmark zu erhöhen ausgeübt neuroprotektive Effekte in N-Methyl-D-Aspartat (NMDA) - injizierten Mäusen, verzögert die Entstehung und das Fortschreiten der Krankheit Amyotrophe Lateralsklerose in SOD1 transgenen Mäuse und erhöhte das Überleben von Motoneuronen im Rückenmark von diesen Mäusen 9. Um die überlebens Rolle der Telomerase in Neuronen im Gehirn zu verstehen, ist es wichtig, ein einfaches und relativ empfindlich schnelle Methode zum Nachweis von Telomerase-Aktivität in den verschiedenen Bereichen des Gehirns zu entwickeln. Die Telomer-repeat Amplifikationsprotokoll (TRAP) ist eine bekannte empfindliche Assays Kombinieren des kanonischen Aktivität der Telomerase und der Polymerasekettenreaktion (PCR). In diesem Assay fügt Telomerase TTAGGG Wiederholungen für eine Telomerase-Substrat (TS-Primer) Oligonukleotid, gefolgt von PCR-Amplifikation mit 6 Basenpaaren (bp)-DNA-Leiter mit der TS Rückwärtsprimer erzeugt -. ACX 32 P markiertem dCTP werden verwendet, um die Erfassungs geringe Menge an PCR-Produkten. Die DNA-Leiter wird durch Sequenzierung Polyacrylamid-Gelelektrophorese (PAGE) gelöst. Sowohl die Intensität der DNA-Banden und die Länge der DNA-Leiter spiegeln die Menge an aktiven Enzymmolekülen und ihre Prozessivität bzw. 10. Das traditionelle Test hält einige größere Schwierigkeiten: Die Gefahr der Belastung durch radioaktive Stoffe, groß und schwer, Sequenzierung PAGE und der Zeitaufwand des Gels Laufen und Filmbelichtung des radioaktiven Produkt (über Nacht in beiden Fällen) zu behandeln. </p&gt; Dieses Protokoll bietet eine verbesserte nicht-radioaktiven Agarose-Gel-Mini-basierte Assays. Die DNA 6 bp-Leiter wird mit 4,5% hochauflösende Agarose-Gel aufgetrennt und die Detektion erfolgt mittels hochempfindlicher Nukleinsäure Fleck. Die Kombination von hoher Auflösung mit Agarose-Gel-Mini und empfindliche Nukleinsäurefärbung bietet einfach zu hand Assay, beseitigt die Gefahr der Radioaktivität ausgesetzt sind und die Gesamtdauer Assay 3 Tagen deutlich verkürzen, um mehrere Stunden.

<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

Telomerase, ein ribonucleoprotein, ist für die Aufrechterhaltung der Telomerlänge und damit die Förderung der genomischen Integrität, Proliferation und Lebensdauer verantwortlich. Darüber hinaus schützt die Telomerase Mitochondrien aus oxidativem Stress und verleiht Resistenz gegenüber Apoptose, was seine mögliche Bedeutung für das Überleben der nicht-mitotischen hochaktive Zellen, wie Neuronen. Wir haben bereits gezeigt, die Fähigkeit der neuen Telomerase-Aktivatoren, um die Telomerase-Aktivität und Expression in den verschiedenen Hirnregionen der Maus zu erhöhen und die motorischen Nervenzellen Zellen vor oxidativem Stress zu schützen. Diese Ergebnisse stärken die Vorstellung, dass die Telomerase in den Schutz von Neuronen vor verschiedenen Läsionen beteiligt. Um die Rolle der Telomerase im Gehirn unterstreichen, hier vergleichen wir die Aktivität der Telomerase in männlichen und weiblichen Maus Gehirn und seine Abhängigkeit vom Alter. TRAP-Assay ist ein Standardverfahren zum Nachweis von Telomerase-Aktivität in verschiedenen Geweben oder Zelllinien. Die Analy Hier zeigen wirsis der Telomerase-Aktivität in drei Regionen des Mäusehirns durch nicht-denaturierenden Protein-Extraktion unter Verwendung von CHAPS Lysepuffer gefolgt von Modifikation der Standard-TRAP-Assay. In diesem 2-Schritt-Assay, verlängert endogenen Telomerase eine bestimmte Telomerasesubstrat (TS-Primer) durch Zugabe von 6 bp TTAGGG Wiederholungen (Telomerase-Reaktion). Die Telomerase-Reaktionsprodukte durch PCR-Reaktion die Schaffung einer DNA-Leiter mit 6 bp-Schritten amplifiziert. Die Analyse der DNA-Leiter wird durch 4,5% Agarose hochauflösende Gelelektrophorese, gefolgt von Anfärben mit hochempfindlichen Nucleinsäure-Färbung. Im Vergleich zu den traditionellen TRAP-Assay, nutzen 32 P markierten radioaktiven dCTP für DNA-Detektion und-Polyacrylamid-Gelelektrophorese für die Lösung des DNA-Leiter, bietet das Protokoll eine nicht-toxische Zeitersparnis TRAP-Assay zur Auswertung der Telomeraseaktivität im Gehirn der Maus, demonstrieren die Fähigkeit, erkennen Unterschiede in telomerasE-Aktivität in den verschiedenen weiblichen und männlichen Gehirn der Maus Region.

Gesamtzellproteinextrakte wurden aus dem Frontallappen (FL), Hirnstamm (BS) und Cerebellum (CR) hergestellt aus 3 CD-1 und 3 weibliche CD-1 männliche Mäuse im Alter von 1 und 3 Monaten für die abgeleitete TRAP-Assay modifiziert und 3 CD-1 männliche Mäuse im Alter von 2 Monate alt für die radioaktive TRAP-Assay. Telomerase-Aktivität wurde in 1 pg Proteine ​​festgestellt wurden unter Verwendung der modifizierten TRAP-Assay und 2 pg Proteine ​​zu extrahieren für die radioaktive TRAP-Assay. Nachweis von Telo...

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.

Telomerase-Aktivität in den verschiedenen Regionen der Maus Gehirn: Nicht-radioaktive Telomerase Repeat Amplification Protocol (TRAP)-Assay

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

14.6K Aufrufe.  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.

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

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

Die Neurostar TMS Device: Die Durchführung der FDA genehmigten Protokoll für die Behandlung von Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Forschung

Neurowissenschaften

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.

Wachsende neuronale Stammzellen aus konventionellen und Nonconventional Regionen der Volks Nagetier Gehirn

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

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.

Hohe Auflösung Quantitative Synaptische Proteome Profilieren von Maus Hirnregionen Nach Auditory Diskriminierung Lernen

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.

Hochdurchsatz-Analyse des Bewegungsapparates Verhaltens in der Drosophila -Insel-Assay

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.

Verwendung von synaptischen Zink Histochemie, verschiedene Regionen und Schichten im Entwicklungs- und Erwachsenen Gehirn zu offenbaren

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.

Lokalisierung von Locus Coeruleus im Gehirn Maus

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.

Quantifizierung subzellulärer Ubiquitin-Proteasom-Aktivität im Nagetierhirn

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.

Sammlung von gefrorenen Nagetier-Hirnregionen für Downstream-Analysen

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.

Dissektion und Isolierung von murinen Glia aus mehreren Regionen des zentralen Nervensystems

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.

Akute Maus Gehirn Schneiden, um spontane Hippocampal-Netzwerk-Aktivität zu untersuchen

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