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

Gli esperimenti sugli animali sono stati approvati dal comitato etico per la sperimentazione animale nel dell&#39;Università Ben-Gurion (IL-39-08-2010). 1 del mouse Cervello estrazione di proteine <ol><li> Preparare 3 tubi da 15 ml contenenti ciascuna 5 ml di soluzione e posizionare i tubi di Ringer su ghiaccio. </li><li> Sacrifica il mouse utilizzando anestesia isoflurano (ATTENZIONE - utilizzare cappa chimica) per 10-20 secondi seguita da dislocazione cervicale e la decapitazione immediata. </li><li> Rimuovere il cervello dal cranio e sciacquare per qualche secondo con la soluzione ghiacciata di Ringer per evitare danni al tessuto cerebrale. </li><li> Utilizzando una lama chirurgica, separare il cervelletto (CR) e tronco cerebrale (BS) dalla corteccia frontale. Avanti, separare il cervelletto dal tronco encefalico e tagliare il lobo frontale (FL) dalla corteccia frontale; rimuovere il bulbo olfattivo del lobo frontale. Posizionare i 3 regioni cerebrali nelle apposite provette da 15 ml. </li><li> Omogeneizzare il tessuto in ogni tubo. Aggiungi frsoluzione ghiaccio esh freddo Ringer in modo che ogni provetta contenga lo stesso volume e centrifugare per 7 minuti a 800 x g. </li><li> Rimuovere il surnatante e aggiungere 100 ml di tampone di lisi CHAPS ad ogni provetta. Mescolare bene, facendo passare la soluzione di diverse volte (~ 10) attraverso una siringa da 1 ml con un ago da 21 G, e incubare in ghiaccio per 30 min. </li><li> Trasferire il contenuto di ogni provetta in una nuova provetta micro-centrifuga e centrifugare per 30 minuti a 13.000 x g. Trasferire il surnatante in una nuova provetta micro-centrifuga e determinare la concentrazione di proteine ​​utilizzando un metodo precedentemente stabilito. Conservare gli estratti proteici in aliquote da 10 ml a -80 ° C. </li></ol> 2 TRAP Assay Preparazione degli strumenti di reazione, Soluzioni e proteine ​​Calcoli diluizione <ol><li> Preparare numero adeguato di appositi tubi micro-centrifuga etichettati per proteine ​​diluizioni estratto e tampone di reazione telomerasi. Eseguire tutti pipettaggio nel protocollo utilizzando puntali con filtro per prevenire la contaminazione. </li&gt; <li> Scongelare le seguenti soluzioni su ghiaccio: TRAP mix di reazione 10x, TS fondo, di dNTP, estratti UPW e proteine ​​(vedi tabella di materiali per reagenti e reattivi concentrazioni). </li><li> Diluire la soluzione di estratto proteico ad una concentrazione finale di 1 mg / mL con acqua ultra-pura (UPW). </li></ol> 3 telomerasi Reazione <ol><li> Preparare la miscela di reazione telomerasi aggiungendo quanto segue a un tubo micro-centrifuga: mix 2 microlitri TRAP (10x), 1 ml TS Primer (0.1 mg), 1 microlitri dNTP (10 mm) e 15 ml UPW. Per campioni multipli, moltiplicare i volumi di cui sopra per il numero di campioni, più 1. </li><li> Aggiungere 19 ml di miscela di reazione per ciascun tubo di reazione e aggiungere 1 ml di estratto proteico diluita (1 mg proteine) a ciascuna provetta per un volume finale di 20 microlitri. </li><li> Mettere le provette di reazione in un 30 ° C bagnomaria già caldo per 45 min. </li></ol> 4. PCR Reazione <ol><li> Quindici kmn prima della fine della reazione telomerasi, scongelare le seguenti soluzioni: ACX di primer, Titanium Taq Buffer, UPW. </li><li> Preparare la miscela di reazione PCR aggiungendo quanto segue a un tubo micro-centrifuga: 2,5 microlitri Titanium Taq Buffer (10x), 1 ml ACX Primer (0.1 mg), 2 ml UPW (1 ml quando si usa IC) e 0,5 ml Titanium Taq polimerasi (50x). Per campioni multipli, moltiplicare i volumi di cui sopra per il numero di campioni più 2. </li><li> Aggiungere 6 ml di miscela PCR per ciascuna provetta PCR e aggiungere l&#39;intero volume della reazione telomerasi (20 microlitri) ad ogni provetta per un volume finale di 26 microlitri. </li><li> Inserire le provette PCR ad un termo cycler PCR ed eseguire il seguente programma: - 90 ° C - 2 min. - 94 ° C -. 30 sec * - 60 ° C -. 30 sec * - 72 ° C -. 45 sec * - 72 ° C - 2 min. Conservare i prodotti di PCR a -20 ° C. * = 34 cicli </li></ol> 5. Agarose Mini-gel Preparazione, Campioni Correre, e Gel colorazione <ol><li> Aggiungere 25 ml di tampone di elettroforesi refrigerati (TBE) e ha una ancoretta in un becher che è 2-4 volte il volume della soluzione tampone e lentamente cospargere a 1.125 g del-alta risoluzione (HR) polvere di agarosio mentre la soluzione viene agitata rapidamente . Rimuovere la barra di mescolare, coprire il becher con un involucro di plastica e forare in un piccolo foro per la ventilazione. Riscaldare il bicchiere nel forno a microonde a potenza &quot;Low&quot; per 3 minuti e poi passare al potere &quot;Alto&quot; e riscaldare la soluzione a impulsi corti con attenzione a non provocare la fuoriuscita. Si noti che quando la schiuma creata dal riscaldamento diventa grosse bolle e scompare dopo pochi secondi, l&#39;agarosio è pronto. </li><li> Cast gel ad una apparecchiatura di mini-gel orizzontale, dopo il gel si solidifica a temperatura ambiente consentono il gel per raffreddare per 20 minuti a 4 ° C prima dell&#39;uso. </li><li> Caricare ogni corsia con 25 ml di campione TRAP (20 microlitri TRAP prodotti di PCR e 5 microlitri 6x gel-loading dye) e correre alle 110 V per 3 ore a 4 ° C in camera fredda. </li><li> Preparare nucleici 3x acido macchia soluzione di lavoro con l&#39;aggiunta di 15 microlitri 10.000X macchia nucleico-acido, soluzione madre a 50 ml Doppio Acqua distillata (DDW) con 0.1 M NaCl (0,29 g). Colorare il gel per 20 minuti agitando delicatamente. </li><li> Utilizzare transilluminatore UV con 302 nm di lunghezza d&#39;onda per visualizzare le bande di DNA ladder TRAP. </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.

Analisi delle attività della telomerasi nel cervello di topo mediante il dosaggio TRAP composto da 4 fasi principali: 1) proteine ​​estrazione dalle regioni specifiche del tessuto cerebrale 2) TS allungamento fondo da telomerasi - reazione telomerasi 3) amplificazione dei prodotti di reazione telomerasi mediante PCR 4 ) separazione dei prodotti PCR utilizzando alta risoluzione gel di agarosio. Questo saggio TRAP modificato affronta due principali difficoltà che si innalzano dal test tr...

La telomerasi è un ribonucleoproteina composto di telomerasi trascrittasi inversa (TERT), la subunità catalitica della telomerasi, e una componente RNA (TERC). Il ruolo canonico di telomerasi è quello di preservare la corretta lunghezza dei telomeri con l&#39;aggiunta di sequenze ripetitive (TTAGGG) alle estremità telomeriche, promuovendo quindi l&#39;integrità genomica, e la proliferazione delle cellule 1. Ruoli supplementari sono stati attribuiti al TERT nelle cellule, vale a dire che conferisce resistenza all&#39;apoptosi indotta da vari agenti dannosi 2, protegge le cellule staminali mesenchimali umane da stress ossidativo 3, e svolge un ruolo pro-sopravvivenza nei neuroni completamente differenziati dalla sua associazione di TIA1 RNA positivo granuli 4. TERT è espresso e attivo soprattutto nelle cellule somatiche dividendo e nella maggior parte dei tipi di tumori, mentre i livelli di espressione e l&#39;attività enzimatica sono strettamente regolati in cellule non-dividendo 5,6. Studi hanno dimostrato che nel rcervello Odent, l&#39;attività della telomerasi diventa inosservabile da giorno postnatale 10, mentre TERT mRNA viene mantenuta a livelli inferiori in età adulta 7. Altri studi hanno dimostrato l&#39;attività della telomerasi nella zona sub-ventricolare adulto, il bulbo olfattivo, l&#39;ippocampo, e nel cervelletto e nella corteccia adulta 8. Abbiamo recentemente dimostrato che nuovi composti che aumentano l&#39;espressione e l&#39;attività della telomerasi nelle corde cerebrali e spinali hanno esercitato effetti neuroprotettivi in ​​N-metil-D-aspartato (NMDA) - topi iniettati, ritardato l&#39;insorgenza e la progressione della malattia sclerosi laterale amiotrofica in SOD1 transgenici topi e ha aumentato la sopravvivenza dei neuroni motori nel midollo spinale di questi topi 9. Per comprendere appieno il ruolo pro-sopravvivenza della telomerasi nei neuroni e nel cervello, è necessario sviluppare un test semplice e relativamente veloce sensibile per la rilevazione dell&#39;attività della telomerasi nelle varie regioni del cervello. Il telomerica rprotocollo di amplificazione EPEAT (TRAP) è un noto test sensibile che unisce l&#39;attività canonica della telomerasi e la reazione a catena della polimerasi (PCR). In questo saggio, la telomerasi aggiunge TTAGGG ripete ad un substrato telomerasi (TS primer) oligonucleotide, seguita da amplificazione PCR che genera 6 coppie di basi (bp) scala del DNA utilizzando il TS inverso fondo -. ACX 32 P etichettato dCTP di sono utilizzati per rilevare la bassa quantità di prodotti di PCR. La scala di DNA viene risolto mediante elettroforesi su gel di poliacrilamide sequenziamento (PAG). Sia l&#39;intensità delle bande di DNA e la lunghezza del DNA ladder riflettono la quantità di molecole di enzima attivo e la loro processività rispettivamente 10. Questo test tradizionale contiene alcune importanti difficoltà: Il pericolo di esposizione a sostanze radioattive, grandi e difficili da gestire sequenziamento PAGE e il consumo di tempo del gel corsa e l&#39;esposizione pellicola del prodotto radioattivo (pernottamento in entrambi i casi). </p&gt; Questo protocollo offre un miglioramento non radioattivo agarosio saggio basato mini-gel. Il DNA 6 bp ladder viene risolto utilizzando 4,5% ad alta risoluzione su gel di agarosio e l&#39;individuazione ha luogo con colorazione molto sensibile acido nucleico. La combinazione di alta risoluzione usando agarosio mini-gel e macchia sensibile acido nucleico offre maneggevole dosaggio, elimina il rischio di esposizione alla radioattività e abbreviare notevolmente la durata complessiva dosaggio da 3 giorni a diverse ore.

<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

Telomerasi, un ribonucleoproteina, è responsabile del mantenimento della lunghezza dei telomeri e quindi promuovere l&#39;integrità genomica, la proliferazione, e la durata della vita. Inoltre, la telomerasi protegge i mitocondri dallo stress ossidativo e conferisce resistenza all&#39;apoptosi, suggerendo la sua possibile importanza per la sopravvivenza dei non-mitotico, cellule altamente attive come i neuroni. Abbiamo precedentemente dimostrato la capacità di attivatori telomerasi nuovi per aumentare l&#39;attività della telomerasi e di espressione nelle varie regioni del cervello del mouse e per proteggere le cellule neuroni motori dallo stress ossidativo. Questi risultati rafforzano l&#39;idea che la telomerasi è coinvolto nella protezione dei neuroni da varie lesioni. Per sottolineare il ruolo della telomerasi nel cervello, noi qui a confronto l&#39;attività della telomerasi nel cervello maschile e femminile del mouse e la sua dipendenza dell&#39;età. TRAP assay è un metodo standard per la rilevazione dell&#39;attività della telomerasi in vari tessuti o linee cellulari. Qui mostriamo il Analysis di attività della telomerasi nelle tre regioni del cervello di topo da parte di non-denaturazione estrazione delle proteine ​​usando CHAPS Lysis Buffer seguita dalla modifica del dosaggio TRAP standard. In questo test 2-step, telomerasi allunga un substrato endogeno telomerasi specifico (TS primer) con l&#39;aggiunta di TTAGGG 6 ripetizioni bp (reazione telomerasi). I prodotti di reazione telomerasi sono amplificati dalla reazione di PCR creando una scala di DNA di incrementi di 6 bp. L&#39;analisi del DNA ladder è fatto da alta risoluzione elettroforesi su gel di agarosio 4,5% seguita da colorazione con colorante altamente sensibile acido nucleico. Rispetto al tradizionale test TRAP che utilizzano 32 P etichettato radioattivo dCTP di per il rilevamento del DNA e di elettroforesi su gel di poliacrilamide per risolvere la scala del DNA, questo protocollo offre un risparmio di saggio TRAP per valutare l&#39;attività della telomerasi nel cervello di topo, dimostrando la capacità di tempo non tossico rilevare le differenze di telomerase l&#39;attività nei vari regione del cervello di topo femmina e maschio.

Estratti proteici cellulari totali sono stati preparati dal lobo frontale (FL), tronco encefalico (BS) e cervelletto (CR), derivato da 3 CD-1 femmina e 3 CD-1 topi maschi all&#39;età di 1 e 3 mesi di età per l&#39; saggio TRAP modificato e 3 CD-1 topi maschi all&#39;età di 2 mesi per il saggio TRAP radioattivo. Attività della telomerasi è stata rilevata nel 1 mg di proteine ​​estratto impiegando il saggio TRAP modificato e 2 mg di proteine ​​estrarre per il saggio TRAP radioattivo. Rilevazione di attività ...

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.

La telomerasi attività nelle varie regioni del cervello di topo: telomerasi non radioattivo Repeat Amplification Protocol (TRAP) Assay

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

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JoVE Journal

Neuroscience

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

14.5K Views.  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.

Video: 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.

Crescere cellule staminali neurali da Regioni convenzionali e non convenzionali del cervello adulto Roditore

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

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Neuroscienze

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.

Purificazione delle navi cervello di topo

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.

Alta risoluzione Quantitative Synaptic Proteome profiling di cervello di topo Regioni Dopo uditiva discriminazione Learning

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.

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila

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.

Uso di zinco sinaptica istochimica per rivelare diverse regioni e lamine nel cervello in via di sviluppo ed adulto

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.

Localizzazione del Locus Coeruleus nel cervello del topo

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.

Quantificare l'attività subcellulare ubiquitina-proteasoma nel cervello del roditore

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.

Raccolta di regioni cerebrali del roditore congelato per analisi a valle

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.

Dissezione e isolamento della glia murina da più regioni del sistema nervoso centrale

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.

Affezione cerebrale acuta del topo per indagare sull'attività spontanea della rete ippocampale

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