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

Los experimentos con animales fueron aprobados por el comité de ética de la experimentación con animales en la Universidad Ben-Gurion (IL-39-08-2010). 1. ratón Cerebro Proteína Extracción <ol><li> Preparar 3 tubos de 15 ml cada una que contienen tubos de 5 ml de solución de Ringer y colocar en hielo. </li><li> Sacrificar el ratón usando anestesia isoflurano (PRECAUCIÓN - Utilizar campana química) durante 10-20 segundos, seguido por dislocación cervical y decapitación inmediata. </li><li> Retire el cerebro del cráneo y enjuague durante unos segundos con una solución helada de Ringer para evitar daños en el tejido cerebral. </li><li> Usando una cuchilla quirúrgica, separar el cerebelo (CR) y el tronco cerebral (BS) a partir de la corteza frontal. A continuación, separar el cerebelo de cortar el tronco cerebral y el lóbulo frontal (FL) desde la corteza frontal; quitar el bulbo olfatorio del lóbulo frontal. Coloque las regiones 3 cerebrales en los designados tubos de 15 ml. </li><li> Homogeneizar el tejido en cada tubo. Añadir frsolución de Ringer frío hielo carne de manera que cada tubo contiene el mismo volumen y centrifugar durante 7 min a 800 x g. </li><li> Eliminar el sobrenadante y añadir 100 l de tampón de lisis CHAPS a cada tubo. Mezclar bien, haciendo pasar la solución varias veces (~ 10) a través de una jeringa de 1 ml con una aguja de calibre 21, y se incuba en hielo durante 30 min. </li><li> Transferir el contenido de cada tubo a un tubo nuevo micro-centrífuga y centrifugar durante 30 minutos a 13.000 x g. Transferir el sobrenadante a un nuevo tubo de microcentrífuga y determinar la concentración de proteína usando un método previamente establecido. Guardar los extractos de proteína en alícuotas de 10 l a -80 ° C. </li></ol> 2. TRAP ensayo Preparación de los instrumentos, soluciones de reacción, y cálculos de dilución de proteínas <ol><li> Preparar número apropiado de tubos de microcentrífuga etiquetados para diluciones de extracto de proteína y tampón de reacción de la telomerasa. Realizar todas pipeteo en el protocolo utilizando puntas con filtro para evitar la contaminación. </li&gt; <li> Descongele las siguientes soluciones en el hielo: mezcla TRAP reacción 10x, primer TS, dNTPs, extractos de agua ultrapura y proteínas (véase la tabla de materiales para reactivos y reactivos concentraciones). </li><li> Diluir la solución del extracto de proteína a una concentración final de 1 g / l usando agua ultra-pura (agua ultrapura). </li></ol> 3. telomerasa Reacción <ol><li> Prepare la mezcla de reacción telomerasa añadiendo lo siguiente a un tubo de micro-centrífuga: mezcla 2 l TRAP (10x), 1 l TS imprimación (0,1 mg), 1 mu l dNTPs (10 mM) y 15 l de agua ultrapura. Para múltiples muestras, multiplicar los volúmenes mencionados por el número de muestras, más 1. </li><li> Añadir 19 l de la mezcla de reacción a cada tubo de reacción y añadir 1 l de extracto de proteína diluida (1 mg proteínas) a cada tubo para un volumen final de 20 l. </li><li> Colocar los tubos de reacción en un baño de agua pre-calentada 30 ° C durante 45 min. </li></ol> 4. PCR Reacción <ol><li> Quince millasn antes de la final de la reacción de la telomerasa, descongelar las siguientes soluciones: imprimación ACX, Titanium Taq Buffer, agua ultrapura. </li><li> Prepare la mezcla de reacción de PCR añadiendo lo siguiente a un tubo de micro-centrífuga: 2.5 l Titanium Taq Buffer (10x), 1 l imprimación ACX (0,1 mg), 2 l de agua ultrapura (1 l cuando se utiliza IC) y 0,5 l Titanium Taq Polimerasa (50x). Para múltiples muestras, multiplicar los volúmenes mencionados por el número de muestras más 2. </li><li> Añadir 6 l de la mezcla de PCR a cada tubo de PCR y añadir todo el volumen de la reacción telomerasa (20 l) a cada tubo para un volumen final de 26 l. </li><li> Inserte los tubos de PCR a un termo termociclador PCR y ejecutar el siguiente programa: - 90 ° C - 2 min. - 94 ° C - 30 seg. * - 60 ° C - 30 seg. * - 72 ° C -. 45 segundos * - 72 ° C - 2 min. Guarde los productos de PCR a -20 ° C. * = 34 ciclos </li></ol> 5. agarosa Mini-gel Preparación, Ejecución de muestras, y Gel de tinción <ol><li> Añadir 25 ml de tampón de electroforesis enfriada (TBE) y una barra de agitación a un vaso de precipitados que es 2-4 veces el volumen de la solución tampón y espolvorear lentamente en 1.125 g de la alta resolución (HR) de polvo de agarosa, mientras que la solución se agitó rápidamente . Retire la barra de agitación, cubrir el vaso con un envoltorio de plástico y perfore en un pequeño agujero para ventilación. Calentar el vaso en el microondas a potencia &quot;baja&quot; durante 3 minutos y luego cambiar a energía &quot;Alto&quot; y calentar la solución en pulsos cortos con cuidado de no causar derrames. Tenga en cuenta que cuando la espuma creada por calentamiento se convierte en burbujas grandes y desaparece después de unos segundos, la agarosa está listo. </li><li> El gel fundido a un aparato de mini-gel horizontal, después de que el gel se solidifica a temperatura ambiente permiten que el gel se enfríe durante 20 min a 4 ° C antes de su uso. </li><li> Cargue cada carril con 25 l de muestra TRAP (20 l TRAP productos de PCR y 5 l 6x tinte de carga de gel) y correr a las 110 V durante 3 horas en un cuarto frío 4 ° C. </li><li> Preparar 3x colorante de ácidos nucleicos solución de trabajo mediante la adición de 15 l 10,000x de ácido nucleico mancha, solución madre de 50 ml de agua bidestilada (DDW) con NaCl 0,1 M (0,29 g). Manchar el gel durante 20 min con agitación suave. </li><li> Utilice transiluminador UV con longitud de onda de 302 nm para ver las bandas de ADN escalera 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.

Análisis de la actividad de la telomerasa en el cerebro del ratón a través de la trampa de ensayo consiste en 4 pasos principales: 1) proteínas de extracción a partir de regiones específicas del tejido cerebral 2) TS de elongación de iniciador de telomerasa por - reacción de la telomerasa 3) amplificación de los productos de reacción de telomerasa mediante PCR 4 ) separación de los productos de PCR usando gel de agarosa de alta resolución. Este ensayo TRAP modificado aborda dos g...

La telomerasa es una ribonucleoproteína compuesta de la telomerasa transcriptasa inversa (terc), la subunidad catalítica de la telomerasa, y un componente de ARN (TERC). El papel canónica de la telomerasa es preservar la longitud adecuada de los telómeros mediante la adición de secuencias repetitivas (TTAGGG) a los extremos teloméricas, por lo tanto, la promoción de la integridad genómica, y la proliferación celular 1. Funciones adicionales se atribuyeron a de TERT en células, es decir, que confiere resistencia a la inducida por diversos agente que daña el 2 apoptosis, protege a las células madre mesenquimatosas humanas del estrés oxidativo 3, y desempeña un papel pro-supervivencia en neuronas completamente diferenciadas por su asociación a TIA1 ARN positivo gránulos 4. De TERT se expresa y se activa principalmente en las células somáticas de dividirse y en la mayoría de tipos de cáncer, mientras que los niveles de expresión y actividad de la enzima están estrechamente regulado en las células que no se dividen 5,6. Los estudios han demostrado que en el rOdent cerebro, actividad de la telomerasa se ​​convierte en indetectable por día postnatal 10, mientras que el ARNm de TERT se mantiene a niveles más bajos en la edad adulta 7. Otros estudios demostraron actividad de la telomerasa en la zona sub-ventricular adulto, el bulbo olfatorio, el hipocampo, y en el cerebelo y la corteza adultos 8. Recientemente hemos demostrado que nuevos compuestos que aumentan la expresión y la actividad de la telomerasa en las cuerdas cerebro y la médula ejercen efectos neuroprotectores en N-metil-D-aspartato (NMDA) - ratones inyectados, retrasaron el inicio y la progresión de la enfermedad esclerosis lateral amiotrófica en transgénicos SOD1 ratones y aumentó la supervivencia de las neuronas motoras en la médula espinal de estos ratones 9. Para entender completamente la función pro-supervivencia de la telomerasa en las neuronas y en el cerebro, es esencial para desarrollar un ensayo rápido simple y relativamente sensible para la detección de la actividad de la telomerasa en las diversas regiones del cerebro. El telomérica rprotocolo de amplificación EPEAT (TRAP) es un ensayo sensible conocida la combinación de la actividad de la telomerasa y canónica de la reacción en cadena de la polimerasa (PCR). En este ensayo, la telomerasa añade TTAGGG repite a un sustrato de telomerasa (TS imprimación) oligonucleótido, seguido de la amplificación por PCR que genera 6 pares de bases (pb) escalera de ADN utilizando el TS cebador inverso -. ACX marcado con 32P dCTP de se utilizan para detectar la baja cantidad de productos de PCR. La escalera de ADN se resolvió por electroforesis en gel de poliacrilamida de secuenciación (PAGE). Tanto la intensidad de las bandas de ADN y la longitud de la escalera de ADN reflejan la cantidad de moléculas de enzimas activas y su procesividad respectivamente 10. Este ensayo tradicional tiene algunas dificultades importantes: El peligro de la exposición a sustancias radiactivas, grandes y difíciles de manejar PÁGINA secuenciación y el consumo de tiempo de la carrera en gel y exposición de la película del producto radiactivo (durante la noche en ambos casos). </p&gt; Este protocolo ofrece un ensayo de agarosa no radiactivo mejora mini-gel a base. La escalera del ADN 6 pb se resuelve usando gel de agarosa de alta resolución de 4,5% y la detección se logra utilizando colorante de ácidos nucleicos altamente sensibles. La combinación de alta resolución de agarosa usando mini-gel y tinción de ácido nucleico sensible ofrece fácil de manejar ensayo, elimina los peligros de la exposición a la radiactividad y se reducirá considerablemente la duración total del ensayo de 3 días a varias horas.

<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 telomerasa, una ribonucleoproteína, es responsable de mantener la longitud de los telómeros y por lo tanto la promoción de la integridad genómica, la proliferación, y la vida útil. Además, la telomerasa protege la mitocondria de estrés oxidativo y confiere resistencia a la apoptosis, lo que sugiere su posible importancia para el sobreviviente de la no-mitótico, las células altamente activos, tales como neuronas. Hemos demostrado previamente la capacidad de los nuevos activadores de telomerasa para aumentar la actividad y la expresión de la telomerasa en las diversas regiones de cerebro de ratón y para proteger a las células neuronas motoras del estrés oxidativo. Estos resultados refuerzan la noción de que la telomerasa está implicado en la protección de las neuronas de diversas lesiones. Para subrayar el papel de la telomerasa en el cerebro, que aquí comparamos la actividad de la telomerasa en el cerebro de ratones macho y hembra y su dependencia de la edad. Ensayo TRAP es un método estándar para la detección de actividad de la telomerasa en varios tejidos o líneas celulares. Aquí se demuestra el anásis de la actividad de la telomerasa en tres regiones del cerebro de ratón por no desnaturalizante de extracción de proteínas utilizando CHAPS tampón de lisis seguido por modificación del ensayo TRAP estándar. En este ensayo de 2 pasos, la telomerasa endógena se alarga un sustrato específico de la telomerasa (cebador TS) mediante la adición de 6 repeticiones TTAGGG pb (reacción de la telomerasa). Los productos de reacción de telomerasa se amplifican por reacción PCR crear una escalera de ADN de incrementos de 6 pb. El análisis de la escalera de ADN se realizó por electroforesis en gel de agarosa de alta resolución 4,5% seguido de tinción con colorante de ácidos nucleicos altamente sensibles. En comparación con el ensayo TRAP tradicional que utilizan marcado con 32P radiactivo dCTP de para la detección de ADN y la electroforesis en gel de poliacrilamida para la resolución de la escalera del ADN, este protocolo ofrece un ahorro de ensayo TRAP para evaluar la actividad de la telomerasa en el cerebro del ratón, lo que demuestra la capacidad de tiempo no tóxica detectar diferencias en telomerasactividad de correo en los distintos región del cerebro del ratón hembra y macho.

Extractos de proteínas de células enteras se han preparado desde el lóbulo frontal (FL), el tronco cerebral (BS) y el cerebelo (CR), derivado de 3 mujeres y 3 CD-1 ratones macho-1 CD en las edades de 1 y 3 meses de edad para la ensayo TRAP modificado y 3 CD-1 ratones machos a la edad de 2 meses de edad para el ensayo TRAP radiactivo. Actividad de la telomerasa se detectó en 1 mg de extracto de proteínas usando el ensayo TRAP modificado y 2 mg extracto de proteínas para el ensayo TRAP radiactivo. La detección de a...

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.

Actividad telomerasa en las diversas regiones del cerebro de ratón: La telomerasa no radiactivo Repetir Protocolo de Amplificación (TRAP) Ensayo

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 vistas.  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.

Cultivo de células madre neurales de Regiones convencionales y no convencionales del cerebro adulto de roedores

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

Investigación

Neurociencias

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.

La purificación de los buques de cerebro de ratón

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 Resolución cuantitativa Synaptic proteoma perfiles de cerebro de ratón Regiones Después de discriminación auditiva de aprendizaje

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.

Análisis de alto rendimiento del comportamiento Locomotor en el ensayo de la isla de Drosophila

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 de Zinc sináptica histoquímica para revelar las diferentes regiones y láminas en el cerebro en desarrollo y 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.

Localización del Locus Coeruleus en el cerebro de ratón

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.

Cuantificación de la actividad ubiquitina-proteosoma subcelular en el cerebro de los roedores

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.

Colección de regiones cerebrales de roedores congelados para análisis posteriores

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.

Disección y aislamiento de la glía murina de múltiples regiones del sistema nervioso 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.

Corte cerebral de ratón agudo para investigar la actividad espontánea de la red de hipocampo

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