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

As experiências com animais foram aprovados pelo comitê de ética em experimentação animal no Universidade Ben-Gurion (IL-39-08-2010). 1. rato Cérebro extração de proteínas <ol><li> Preparar 3 tubos de 15 ml cada, contendo 5 ml de solução e colocar tubos de Ringer no gelo. </li><li> Sacrifique o mouse usando anestesia isoflurano (ATENÇÃO - usar capuz química) por 10-20 segundos, seguido por deslocamento cervical e decapitação imediata. </li><li> Remover o cérebro do crânio e enxaguar durante alguns segundos com uma solução gelada de Ringer para evitar danos no tecido cerebral. </li><li> Usando uma lâmina cirúrgica, separar o cerebelo (CR) e tronco cerebral (BS), a partir do córtex frontal. Em seguida, separar o cerebelo a partir do tronco cerebral e cortar o lobo frontal (FL) a partir do córtex frontal; remover o bolbo olfactivo do lobo frontal. Coloque as três regiões do cérebro nas designadas tubos de 15 ml. </li><li> Homogeneizar o tecido em cada tubo. Adicionar fresh solução gelo frio de Ringer assim cada tubo contenha o mesmo volume e centrifuga-se durante 7 min a 800 x g. </li><li> Remover o sobrenadante e adicionar 100 mL de tampão de lise de CHAPS a cada tubo. Misture bem, fazendo passar a solução várias vezes (~ 10) através de uma seringa de 1 ml com uma agulha G 21, e incubar em gelo durante 30 min. </li><li> Transferir o conteúdo de cada tubo para um novo tubo de micro-centrífuga e centrifuga-se durante 30 min a 13.000 x g. Transferir o sobrenadante para um novo tubo de micro-centrífuga e determinar a concentração de proteína utilizando um método previamente estabelecido. Guardar os extractos de proteína em alíquotas de 10 ul a -80 ° C. </li></ol> 2. TRAP Ensaio Preparação de Instrumentos, Soluções de reacção, e cálculos de diluição de proteína <ol><li> Prepare o número apropriado de tubos etiquetados de micro-centrifugação de diluições de extracto de proteína e tampão de reacção da telomerase. Execute todas pipetagem no protocolo usando pontas com filtro para evitar a contaminação. </li&gt; <li> Descongele as seguintes soluções no gelo: TRAP mistura de reação 10x, TS primer, dNTP, extratos UPW e proteínas (ver tabela de materiais de reagentes e reagentes concentrações). </li><li> Dilui-se a solução de extracto de proteína para uma concentração final de 1 ug / mL utilizando água ultra-pura (PFS). </li></ol> 3. telomerase Reaction <ol><li> Prepare mistura de reação telomerase, adicionando o seguinte para um tubo de micro-centrífuga: mix 2 l TRAP (10x), 1 ml TS primário (0,1 g), uma dNTPs ul (10 mm) e 15 mL UPW. Por várias amostras, multiplique volumes acima referidos pelo número de amostras, mais 1. </li><li> Adicionar 19 ul da mistura de reacção para cada tubo de reacção e adicionar 1 ul do extracto de proteína diluída (1 ug de proteínas) a cada tubo para um volume final de 20 ul. </li><li> Colocar os tubos de ensaio num banho de água pré-aquecida a 30 ° C durante 45 min. </li></ol> 4. PCR Reação <ol><li> Quinze min antes do final da reação telomerase, descongelar as seguintes soluções: primers ACX, Titanium Taq buffer, UPW. </li><li> Prepare mistura da PCR, adicionando o seguinte para um tubo de micro-centrífuga: 2.5 l Titanium Taq Buffer (10x) 1 ml ACX primário (0,1 g), 2 l UPW (1 ml quando IC é usado) e 0,5 mL Titanium Taq Polimerase (50x). Por várias amostras, multiplique volumes acima referidos pelo número de amostras mais 2. </li><li> Adicionar 6 ul de mistura de PCR, cada tubo de PCR e adicionar todo o volume da reacção da telomerase (20 ul) a cada tubo para um volume final de 26 ul. </li><li> Inserir os tubos de PCR para um termociclador PCR e executar o seguinte programa: - 90 ° C - 2 min. - 94 ° C -. 30 seg * - 60 ° C - 30. Seg * - 72 ° C - 45. Sec * - 72 ° C - 2 min. O armazenamento de produtos de PCR a -20 ° C. * = 34 ciclos </li></ol> 5. Agarose Mini-gel Preparação, Amostras Correr, e Gel Coloração <ol><li> Adicionar 25 ml de tampão de electroforese refrigerados (TBE) e uma barra de agitação para um copo que é 2-4 vezes o volume de solução tampão e, lentamente, por aspersão, em 1,125 g da alta resolução (HR) de agarose em pó, enquanto a solução é rapidamente agitada . Remova a barra de agitação, cobrir o copo com um filme plástico e furar em um pequeno buraco para ventilação. Aqueça a taça no microondas em potência &quot;Low&quot; por 3 min e depois mudar para poder &quot;High&quot; e aquecer a solução em pulsos curtos, com cuidado para não causar derramamento. Note-se que quando a espuma criada pelo aquecimento torna-se grandes bolhas e desaparece após alguns segundos, a agarose é preparado. </li><li> Lançar o gel a um aparelho de mini-gel horizontal, depois de o gel solidifica à temperatura ambiente para permitir que o gel relaxar durante 20 min a 4 ° C antes da utilização. </li><li> Coloque cada pista com 25 mL de amostra de TRAP (20 l TRAP produtos de PCR e 5 de corante gel-loading ul 6x) e executado em 110 V durante 3 horas numa câmara fria a 4 ° C. </li><li> Prepare 3x mancha de ácido nucleico solução de trabalho adicionando 15 ul 10.000x mancha de ácido nucleico-, solução de estoque a 50 ml de água duplamente destilada (ADD), com 0,1 M de NaCl (0,29 g). Corar o gel durante 20 minutos com agitação suave. </li><li> Use fonte UV com 302 nm de comprimento de onda para ver as bandas de DNA escada 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.

Análise de actividade da telomerase no cérebro do rato através do ensaio TRAP é constituído por quatro etapas principais: 1) extracção de proteínas a partir de regiões específicas do tecido cerebral 2) TS alongamento do iniciador pela telomerase - reacção da telomerase 3) amplificação dos produtos de reacção da telomerase por RCP 4 ) separação dos produtos de PCR em gel de agarose, utilizando alta resolução. Este ensaio TRAP modificado aborda duas grandes dificuldades que...

A telomerase é uma ribonucleoproteína composto por transcriptase inversa da telomerase (TERT), a subunidade catalítica de telomerase e um componente de RNA (TERC). O papel canônica da telomerase é preservar o comprimento adequado dos telômeros pela adição de sequências repetitivas (TTAGGG) para as extremidades teloméricas, portanto, promover a integridade genômica e proliferação celular 1. Papéis adicionais foram atribuídos a TERT em células, ou seja, que confere resistência à apoptose induzida por vários agentes prejudiciais 2, protege as células estaminais mesenquimais humanas a partir de estresse oxidativo 3, e desempenha um papel pró-sobrevivência em neurónios completamente diferenciados pela sua associação com o ARN positivo TIA1 grânulos 4. TERT é expresso e activo, principalmente em células somáticas de se dividir e, na maioria dos tipos de cancro, enquanto que os níveis de expressão e de actividade da enzima estão fortemente regulada em células que não se dividem 5,6. Estudos têm demonstrado que, na rcérebro Odent, a atividade da telomerase torna-se indetectável por dia pós-natal 10, enquanto TERT mRNA é mantida em níveis mais baixos para a idade adulta 7. Outros estudos demonstraram a actividade da telomerase na zona sub-ventricular adulto, o bolbo olfactivo, o hipocampo e no cerebelo e córtex adulto 8. Recentemente, demonstrou que novos compostos que aumentam a expressão e a actividade de telomerase no cérebro e espinal cabos exerceu efeitos neuroprotectores em N-metil-D-aspartato (NMDA) - ratinhos injectados, atrasou o aparecimento e progressão da doença de esclerose lateral amiotrófica em SOD1 transgénico ratinhos e aumentou a sobrevivência de neurónios motores na espinal medula de ratinhos estas 9. Para compreender totalmente a função pró-sobrevivência de telomerase em neurónios e no cérebro, o que é essencial para desenvolver um ensaio simples e relativamente sensível rápido para a detecção da actividade da telomerase em várias regiões do cérebro. O telomérica rprotocolo de amplificação epita (TRAP) é um ensaio de sensibilidade conhecida a combinação de actividade de telomerase e canónico a reacção em cadeia da polimerase (PCR). Neste ensaio, a telomerase adiciona TTAGGG repete a um substrato de telomerase (TS iniciador) oligonucleótido, seguido de amplificação de PCR, que gera 6 pares de bases (pb) escada de DNA usando o TS inversa iniciador -. ACX 32 P dCTP marcado, são utilizados para detectar a pequena quantidade de produtos de PCR. A escada de ADN foi resolvido por electroforese em gel de sequenciação de poliacrilamida (PAGE). Tanto a intensidade das bandas de DNA e o comprimento da escada de ADN reflectem a quantidade de moléculas de enzimas activas e sua processabilidade, respectivamente, 10. Este ensaio tradicional detém algumas das maiores dificuldades: O perigo de exposição a substâncias radioactivas, grandes e difíceis de lidar com o seqüenciamento eo consumo de tempo do gel de corrida e exposição do filme do produto radioativo (durante a noite, em ambos os casos). </p&gt; Este protocolo apresenta um ensaio de agarose não radioactivo melhorada mini-gel à base. O DNA 6 bp ladder é resolvido usando 4,5% de alta resolução em gel de agarose ea detecção é realizada com mancha de ácidos nucleicos altamente sensível. A combinação de alta resolução utilizando mini-gel de agarose e coloração de ácido nucleico sensível oferece fácil de manusear ensaio, elimina o risco de exposição à radioactividade e encurtar significativamente a duração total do ensaio de 3 dias a várias 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

A telomerase, uma ribonucleoprotein, é responsável por manter o comprimento dos telômeros e, portanto, promover a integridade genômica, proliferação e longevidade. Além disso, a telomerase protege a mitocôndria do estresse oxidativo e confere resistência à apoptose, sugerindo sua possível importância para a sobrevivência dos não-mitótico, células altamente ativos, tais como os neurônios. Nós já demonstraram a capacidade dos novos ativadores de telomerase para aumentar a atividade da telomerase e expressão nas diferentes regiões do cérebro do rato e para proteger as células neurônios motores do stress oxidativo. Estes resultados vêm reforçar a noção de que a telomerase está envolvida na protecção dos neurónios de várias lesões. Para sublinhar o papel da telomerase no cérebro, nós aqui comparar a atividade da telomerase em masculino e feminino cérebro do rato e sua dependência da idade. Ensaio TRAP é um método padrão para a detecção de actividade da telomerase em várias linhas celulares ou tecidos. Aqui demonstramos a Analysis da actividade da telomerase em três regiões do cérebro do rato por não-desnaturante CHAPS extracção de proteína utilizando um tampão de lise seguido por uma modificação do ensaio TRAP padrão. Neste ensaio, em 2 passos, a telomerase endógeno alonga um substrato da telomerase específico (TS iniciador) adicionando TTAGGG 6 repetições bp (reacção da telomerase). Os produtos de reacção da telomerase são amplificados por meio de reacção de PCR a criação de uma escada de DNA de incrementos de 6 bp. A análise do segmento de ADN é feita por electroforese em gel de agarose de alta resolução de 4,5%, seguido por coloração com mancha de ácido nucleico altamente sensível. Em comparação com o ensaio TRAP tradicionais que utilizam 32 marcado com P radioactivo de dCTP do para a detecção do ADN e de electroforese em gel de poliacrilamida para resolver a escada de DNA, este protocolo oferece um tempo de não-tóxico poupança ensaio TRAP para a avaliação da actividade de telomerase no cérebro do rato, o que demonstra a capacidade de detectar diferenças na telomerasatividade e nos diversos região do mouse cérebro feminino e masculino.

Extratos de proteínas de células inteiras foram preparados a partir do lobo frontal (FL), tronco cerebral (BS) e cerebelo (CR), obtido a partir de 3 CD-1 do sexo feminino e 3 CD-1 camundongos machos com as idades de 1 e 3 meses de idade para a ensaio TRAP modificado e 3 ratinhos CD-1 do sexo masculino com a idade de 2 meses de idade para o ensaio TRAP radioactivos. A actividade da telomerase foi detectada em 1 mg de extracto de proteínas usando o ensaio TRAP modificado e extrair 2 ug de proteínas para o ensaio TRAP ...

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.

A atividade da telomerase nas diversas regiões de cérebro de camundongo: A telomerase não-radioativo Repetir 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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Neuroscience

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

14.5K visualizações.  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.

Vídeo: 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.

Crescer a partir de células-tronco neurais convencionais e não convencionais Regiões do cérebro adulto de roedores

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

Neurociência

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.

Purificação de embarcações do cérebro do rato

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 resolução quantitativa Synaptic Proteome Profiling de rato regiões cerebrais Depois Auditivo Discriminação Aprendizagem

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.

Análise do elevado-throughput de comportamento Locomotor no ensaio de ilha 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 zinco sináptica histoquímica para revelar as diferentes regiões e lâminas no cérebro em desenvolvimento e 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.

Localização do Locus Coeruleus no cérebro do rato

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.

Quantificando a ubiquitina subcelular-atividade de proteasome no cérebro de 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.

Coleção de regiões cerebrais de roedores congelados para análises a jusante

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.

Dissecção e isolamento da glia murina de múltiplas regiões do sistema nervoso 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 aguda do rato para investigar atividade espontânea da rede hipocampal

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