This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

1 Preparação de Agudo fatias do hipocampo de ratos adultos Cérebro <ol><li> Anestesiar ratos com isoflurano. ATENÇÃO: Execute o procedimento usando um exsicador fechado, não inalar o isoflurano. Certifique-se de que o animal está completamente anestesiado. </li><li> Decapitar os ratos, isolar rapidamente o cérebro, e mergulhá-lo num precarbonated (95% de O2 / 5% 2 mistura de gás de CO) gelada solução de Gey (composição em mM: 130 de NaCl, 4,9 de KCl, 1,5 de CaCl 2 2H 2 O , 0,3 MgSO 4 2H 2 O, 11 de MgCl2 · 6H 2 O, 0,23 KH 2 PO 4, 0,8 de Na 2 HPO 4 · 7H 2 O, 5 de glucose-H2O, 25 HEPES, 22 de NaHCO3, pH 7,32) durante 30 min 1,2,10,16. </li><li> Remover o cerebelo e parte do córtex entorrinal. Separe os hemisférios corticais com um corte médio-sagital, em seguida, coloque cada hemisfério para baixo em sua superfície medial. A partir de então fazerum corte de 50-70 ° (50-70 ° transversal) ao longo da superfície dorsal de cada hemisfério 13,14. </li><li> Cole cada hemisfério com a superfície recém-cortada na plataforma de corte do sistema de corte. A plataforma deve ser coberto por solução precarbogenated de Gey gelada. </li><li> Cortar 350um 50-70 ° fatias transversais de anterior para posterior lateral usando um vibratome ajustado para minimizar a oscilação do eixo z. A formação do hipocampo, córtex entorrinal e subicular, bem como os córtices que estão localizados no hipocampo dorsolateral fará parte das fatias utilizados para experiências 13,14. </li><li> Transferir as fatias de hipocampo de uma forma de U e submerso tipo incubadora e incubar durante pelo menos 2 horas a 32 ° C com carbogenated fluido cerebroespinal artificial (ACSF contendo em mM: 110 de NaCl, 2,5 de KCl, 2,5 de CaCl2 2H 2 O, 1,5 MgSO 4 2H 2 O, 1,24 KH 2 PO 4, 10 Glicose · H 2S, 27,4 NaHCO3, pH 7,3) 1,2,10,16. </li></ol> 2. posicionamento dos eletrodos, Base de gravação, e indução de LTP <ol><li> Transferir a fatia de hipocampo de uma câmara de registo tipo submerso montado sob um microscópio. Perfundir (6-7 ml / min) com ACSF carbogenated durante pelo menos 30 min a 32 ° C. </li><li> Prepare microeletrodos capilares de vidro cheios de ACSF (resistência de ponta é de 3-5 mohms). </li><li> Coloque um microeléctrodos de vidro cheios com ACSF nas fibras Schaffer-CA1 colaterais para a estimulação e na radiatum CA1 estrato fEPSP gravação 1,2,10 (Figura 2). A distância entre os eléctrodos deve ser de cerca de 300 um. </li><li> Potenciais campo excitatórios pós-sinápticos (fEPSPs) evocar por estimulação das fibras Schaffer-colaterais com impulsos de corrente bifásica rectangulares (200 ms / polaridade) em uma gama de 3-4 V. </li><li> Realizar o teste máximo de estimulação através da medição do INPrelação ut-saída e definir a força de estimulação, como 40% dos valores máximos fEPSP-inclinação obtidos e mantê-la constante durante todo o experimento. </li><li> Comece a gravação da linha de base durante pelo menos 15 minutos após o teste de estimulação máxima. Medem-se as respostas a estímulos testar cada minuto durante todo o experimento. Realize gravações iniciais com estimulação de baixa freqüência. </li><li> Gravar a linha de base durante pelo menos 30 min. </li><li> Para indução Late-LTP aplicar de alta freqüência de 100 Hz tetanização que consiste em três trens de estímulo 1 seg a 100 Hz com um intervalo de 5 min intertrain. Para aumentar o campo de estimulação dentro região CA1 5 uM bicuculina pode ser lavada em 2 min antes de tetanização e deve ser lavada imediatamente após a última tetanização. </li></ol> 3 Coleção de fatias após a indução da LTP e snap Congelamento <ol><li> Parar LTP gravação 2 min ou 30 min depois de três trens de estimulação tetânica induzindo Late-LTP. </li><li&gt; Retirar os eléctrodos e rapidamente transferir a fatia em uma plataforma metálica precold colocada em gelo seco. Repita o procedimento para uma fatia de controle mantidos em incubadora em forma de U. CUIDADO: Use luvas ao trabalhar com gelo seco. </li><li> Recolhe cada fatia num tubo de 1,5 ml e armazenar a -80 ° C. </li></ol> 4 Isolamento de fracções nucleares enriquecidos a partir da região CA1 do hipocampo <ol><li> Tome 1,5 ml Eppendorf tubos com as fatias congeladas entre -80 ° C e mantê-los no gelo. </li><li> Adicionar 0,5 ml de tampão contendo a protease fresca fria TBS (PI) e inibidores de fosfatase (PS), para dentro do tubo. Incubar por 2-3 min e transferir para um microscópio estereoscópico. CUIDADO: Use luvas de proteção e um jaleco, enquanto trabalhava com PI e PS. </li><li> Dissecar região pyramidale CA1 do hipocampo estrato usando duas agulhas. Use uma agulha para a realização da fatia, ao microscópio estereoscópico ea segunda agulha para cortar a área de CA1. </li><li> Recolher o CA dissecadaUma região de 5 fatias (para cada grupo) para um novo tubo de 1,5 ml contendo 50 ul de tampão de lise (1x tampão de lise hipotónico contendo em mM: 10 HEPES, 1,5 de MgCl2, 10 KCl, pH 7,9, com IP e PS). Note-se que esta quantidade de material vai ser suficiente para executar 2-3 immunoblots. </li><li> Homogeneizar o tecido coletado por pipetagem up cuidadoso e para baixo. Usar uma pipeta de 200 uL. Incubar o ligado em gelo durante 5-7 minutos para permitir que as células inchar. </li><li> Tome 2 mL do lisado, cair sobre uma lâmina de microscópio, e visualizar o inchaço das células em um microscópio de campo claro. Com inchaço adequada das células dos núcleos aparecem como rodada estruturas intactas. </li><li> Recolha 20 ul de amostra para um novo tubo de 1,5 mL como &quot;fracção de homogenato. Adicionar 8 mL de tampão de desnaturação da amostra 4x SDS. </li><li> Centrifugar os lisados ​​restantes em 11.000 rpm durante 1 min. </li><li> Recolher cuidadosamente o sobrenadante a partir do topo (&#39;fracção citosólica&#39;), transfer para um novo tubo e adicionar 20 ul de tampão de amostra 4x. </li><li> Ressuspender o sedimento em 60 mL de tampão hipotónico e adicionar 20 ul de tampão de amostra 4x. Esta fracção é referido como uma &quot;fracção enriquecida nuclear&quot;. </li><li> Homogeneizado, citosólica, e fracções enriquecidas nucleares podem ser armazenadas a -20 ° C ou -80 ° C para posterior immunoblotting. </li></ol> 5. Immunoblotting semiquantitativa de proteínas nucleares Synapto <ol><li> Descongelar as amostras e ferve-las durante 5 min a 95 ° C. </li><li> Tirar 5 ul de cada fracção e determinar a concentração de proteína pelo teste de amido preto ou o teste de BCA. </li><li> Carga igual quantidade de amostras de proteína a partir de homogeneizado, fracções enriquecidas citoplasmáticos e nucleares em SDS-PAGE. As amostras de controle e fatias LTP deve ser colocado no mesmo gel para comparação direta. </li><li> Executar um procedimento de western-blot padrão (transferência molhado é recomendado). </li><li> Sondar as membranas com tque o anticorpo de escolha. Posteriormente as mesmas manchas (ou mesmas amostras são executados em paralelo) pode ser sondado com uma citoplasmática marcador - enolase2 neurônio específico (NSE2) e marcador nuclear - NeuN e um anticorpo actina como controle de carga. </li></ol> Análise de Dados 6. <ol><li> A eficiência de indução da LTP pode ser analisada pelo software Clampfit. A inclinação média de gravações de base foi comparado com as pistas após tetanização usando two-way ANOVA, p &lt;0,05 foi considerado significativamente diferente. Os valores de declive fEPSP foram representados em diagramas como a média ± SEM </li><li> Para a quantificação de immunoblots, digitalizar o filme de auto-radiografia e analisar a densidade integrada de bandas de proteínas por ImageJ ou bandas de fluorescência com um sistema de Licor. Valores de bandas imunorreactivas deve ser normalizada para o controle de carregamento e absorvente. Para a comparação dos grupos controle e LTP um nonparametrical Mann-Whitney U-teste pode ser usado. </li></ol><table border="0"cellpadding = &quot;0&quot; cellspacing = &quot;0&quot;&gt; <tbody><tr><td> Nome do buffer </td><td> Reagente </td><td> Concentração (mM) </td><td> Valor </td><td> Comentários / Descrição </td></tr><tr><td> Solução de Gey (pH: 7,3 ~ 7,4) </td><td> NaCl </td><td> 130 </td><td> 7,6 g </td><td> filtração estéril </td></tr><tr><td> 1000 ml </td><td> KCl </td><td> 4.9 </td><td> 0,37 g </td><td></td></tr><tr><td></td><td> CaCl 2 2H 2 O </td><td> 1.5 </td><td> 0,22 g </td><td></td></tr><tr><td></td><td> MgSO 4 2H 2 O </td><td> 0,3 </td><td> 0,0739 g </td><td></td></tr><tr><td></td><td> MgCl2 · 6H 2 O </td><td> 11 </td><td> 2,24 g </td><td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 0,23 </td><td> 0,0313 g </td><td></td></tr><tr><td></td><td> Na 2 HPO 4 · 7H 2 O </td><td> 0,8 </td><td> 0,2145 g </td><td></td></tr><tr><td></td><td> Glicose · H2O </td><td> 5 </td><td> 0,9909 g </td><td></td></tr><tr><td></td><td> HEPES </td><td> 25 </td><td> 5,96 g </td><td></td></tr><tr><td></td><td> NaHCO3 </td><td> 22 </td><td> 1,85 g </td><td></td></tr><tr><td> ACSF (pH: 7,3 ~ 7,4) </td><td> NaCl </td><td> 110 </td><td> 6,428 g </td><td> filtração estéril </td></tr><tr><td> 1000 ml </td><td> KCl </td><td> 2,5 </td><td> 0,1865 g </td><td></td></tr><tr><td></td><td> CaCl 2 2H 2 O </td><td> 2,5 </td><td> 0,368 g </td><td></td></tr><tr><td></td><td> MgSO 4 2H 2 O </td><td> 1.5 </td><td> 0.370 g </td&gt; <td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 1,24 </td><td> 0,169 g </td><td></td></tr><tr><td></td><td> Glicose · H2O </td><td> 10 </td><td> 1,9817 g </td><td></td></tr><tr><td></td><td> NaHCO3 </td><td> 27.4 </td><td> 2,3 g </td><td></td></tr><tr><td> 1x tampão hipotónico (pH: 7.9) </td><td> HEPES </td><td> 10 </td><td> 0,23 g </td><td></td></tr><tr><td> 100 ml </td><td> MgCl2 · 6H 2 O </td><td> 1.5 </td><td> 0,0304 g </td><td></td></tr><tr><td></td><td> KCl </td><td> 10 </td><td> 0,07455 g </td><td></td></tr></tbody></table> Tabela 1 Os tampões.

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The authors have nothing to disclose.

Os passos descritos no protocolo acima fornecer orientações como preparar hipocampo aguda fatias de ratos jovens ou adultos, induzir e registro LTP, dissecar rapidamente área estimulada de fatia, e preparar fração enriquecida nuclear para estudar a atividade dinâmica de proteínas dependentes. Esta abordagem resulta da combinação de vários métodos diferentes utilizados independentemente um do outro. Nós otimizamos um fluxo de trabalho e fornecer detalhes suficientes para o iniciante a criar as suas próprias ...

Synaptic N-metil-D-aspartato-receptores (NMDAR) desempenham um papel crucial na plasticidade sináptica e a sobrevivência das células de sinalização enquanto a activação de NMDAR extrasynaptic pode provocar neurodegeneração e morte celular. Essas mudanças dependem de expressão rigidamente controlado gene dependente / atividade regulamentada e, portanto, requerem uma comunicação constante entre sinapses ativadas ou dendritos e do núcleo 7. O MAP-quinases ERK1 / 2 são efectores a jusante de sinalização de NMDAR sinápticas e são envolvidos na expressão de genes induzidos por NMDAR-activação, enquanto que a sinalização através de NMDAR extrasynaptic não tem ou tem um efeito inibidor sobre a ERK1 / 2 atividade 8,11. Há um número de proteínas que têm sido mostrados para transporte entre dendritos distais e do núcleo. Muitas destas proteínas conter um sinal de localização nuclear e são activamente transportados ao longo de microtúbulos em uma maneira dependente da dineína e-importin 6,9 para o núcleo. Interestingly, alguns destes mensageiros trânsito apenas para o núcleo, em resposta a estímulos específicos sinápticos. Por exemplo, o transporte retrógrado de elemento de resposta cíclica AMP ligação às proteínas 2 (CREB2) é induzida por LTD química, mas não LTP 12. Localizada estimulação sináptica dependente de NMDAR impulsiona coactivador de transcrição regulada de CREB (CRTC1) para o núcleo, um processo de translocação, que está envolvida na plasticidade do hipocampo a longo prazo 4. Foi demonstrado recentemente que o mensageiro proteína Jacob transloca para o núcleo após tanto, a activação sináptica e extrasynaptic NMDAR e regula CREB gene dependente de transcrição 5. A origem sináptica ou extrasynaptic do sinal é codificado em uma modificação pós-tradução de Jacob. Actividade sináptica induz ERK1 / 2 dependente da fosforilação de serina-Jacob numa cruciais na posição 180 (pJacobS 180), que é um requisito necessário para a subsequente translocação para o núcleo, em cultura primária do hipocampo. Além disso, in neurônios CA1 de fatias de hipocampo agudas pJacobS 180 transloca para o núcleo após Schaffer garantia LTP mas não LTD 1,10. PS180 Jacob leva a um aumento da expressão de genes relacionados com a plasticidade e a expressão do gene realimenta a função sináptica. Em nítido contraste, Jacob que transloca para o núcleo após a ativação NMDAR extrasynaptic não é fosforilada na Ser180 e pode estar associada a diferentes complexos proteína no núcleo causando &#39;CREB desligado&#39; e uma retração de contatos sinápticos 10. A maioria dos estudos publicados sobre a importação nuclear de synapto-nuclear mensageiro proteína ter sido feito em culturas primárias de neurônios dissociados. Por isso, seria interessante ver se esses resultados podem ser reproduzidos em fisiologicamente mais relevante condições usando fatias de hipocampo onde a conectividade e função neuronal são muito mais bem preservado. Aqui apresentamos um protocolo otimizado para avaliação LTP-dependent translocação nuclear de mensageiros proteicos por imunotransferência. Este método também é adequado para a análise da actividade de fosforilação de proteínas dependentes de uma fracção nuclear em bruto. Especificamente, o protocolo atual envolve a preparação de fatias aguda CA1 do hipocampo, indução e gravação de LTP. Em seguida, a região CA1 é microscopicamente dissecados para isolar a região estimulada. Nós combinamos e modificou o protocolo para o isolamento nuclear fornecido pela CellLytic nuclear Extraction Kit com alterações introduzidas pela Zhao e seus colegas 17. O procedimento optimizado inclui a lise das regiões CA1 dissecados em tampão hipotónico permitindo inchaço celular e a libertação de núcleos. A lise celular e morfologia de núcleos pode ser determinado por exame microscópico. Enriquecimento nuclear é conseguida por um passo de centrifugação curta. Immunoblotting análise com anticorpos contra NeuN e NSE2, marcadores específicos de fracções nucleares ou citosólicas, indica que esta abordagem pode ser usada como um rápidoe protocolo reprodutível para isolar essas frações subcelulares e estudar modificações pós-tradução muito instáveis, como a fosforilação de proteínas. Além disso, este método é vantajoso para pequenas amostras de tecido derivadas de regiões CA1 dissecados de fatias do hipocampo e pode ser usado em combinação com imuno-histoquímica de fatias do hipocampo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Equipment</td>
		</tr>
		<tr>
			<td>CED 1401 AD/DA converter</td>
			<td>Cambridge Electronics Design, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/data_acquisition_system">Axon Digidata acquisition system</a></td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10.0 Data acquisition and analysis software</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica TCS SP2&#65292;Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Standard microelectrode puller</td>
			<td>Sutter&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/stereomicroscope">Stereomicroscope</a></td>
			<td>Leica</td>
			<td>Leica S4E, Germany</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica VT1000S, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated pulse stimulator</td>
			<td>Model2100, A-M systmes, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, HERAEUS, FRSCO17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>JULABO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field Microscope</td>
			<td>NIKON ECLIPSE TS100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Electron Corporation, HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann, SM-5, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting chamber and electric power supplier</td>
			<td>Hoefer Scientific Instruments, San Francisco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julabo, F12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, Heraeus, FRESCO 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged type Recording Chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shape and submerged type incubator&#160;</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic culture dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Reagents</td>
		</tr>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.3957.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.6781.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.02382.0500</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgSO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>5886.05</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>AppliChem</td>
			<td>CAS-NO: 7791-18-6; EC-NO:2320946</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MERCK</td>
			<td>12034.025</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.06574.1000</td>
			<td>extra pure</td>
		</tr>
		<tr>
			<td>Glucose&#183;H2O</td>
			<td>ROTH</td>
			<td>Art.-Nr.6887.1</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ROTH</td>
			<td>Art.-Nr.9105.4</td>
			<td>PUFFERAN, &#8805;99.5%, p.a.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>MERCK</td>
			<td>1.06329.1000</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>Protease inhibitor Coctail</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphostop</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isofluran</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Antibodies</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Species</td>
		</tr>
		<tr>
			<td>pJac-s180</td>
			<td>Biogenes/ purified</td>
			<td></td>
			<td>rabbit (dil. 1:100)</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Milipore</td>
			<td>MAB377</td>
			<td>mouse (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>NSE</td>
			<td>Cell Signaling</td>
			<td>D20H2</td>
			<td>rabbit (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>beta-actin</td>
			<td>Sigma</td>
			<td>A-5441</td>
			<td>mouse (dil. 1:5,000)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td>Species</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>DAKO</td>
			<td></td>
			<td>goat anti - mouse (1:5,000)</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>NEB</td>
			<td></td>
			<td>goat anti - rabbit (1:5,000)</td>
		</tr>
	</tbody>
</table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

Estudar expressão atividade da proteína dependente, translocação subcelular, ou fosforilação é essencial para compreender os mecanismos celulares subjacentes à plasticidade sináptica. A potenciação de longa duração (LTP) e depressão a longo prazo (LTD) induzida em fatias de hipocampo agudas são amplamente aceites como modelos celulares de aprendizagem e memória. Existem inúmeros estudos que utilizam abordagens de imagiologia celular ou imunohistoquímica ao vivo para visualizar a atividade dinâmica de proteínas dependentes. No entanto, estes métodos dependem da adequação de anticorpos para imunocitoquímica ou superexpressão de proteínas marcadas com fluorescência em neurônios individuais. Immunoblotting de proteínas é um método alternativo que oferece confirmação independente dos resultados. O primeiro factor limitativo na preparação de fracções subcelulares de fatias de hipocampo tetanized individuais é a baixa quantidade de material. Em segundo lugar, o procedimento de tratamento é fundamental, porque manipulações muito curtos e menores de lumprir fatias pode induzir a ativação de algumas cascatas de sinalização. Aqui, descrevemos um processo de produção optimizado, a fim de obter uma quantidade suficiente de uma fracção enriquecida nuclear de pureza suficiente da região CA1 do hipocampo agudas fatias de cérebro de rato. Como um exemplo representativo, mostra-se que a ERK1 / 2 forma fosforilada da proteína mensageira synapto nuclear Jacob activamente transloca para o núcleo mediante indução da LTP e pode ser detectada numa fracção enriquecida nuclear de neurónios CA1.

Nós já anteriormente demonstrado que a proteína mensageira synapto nuclear Jacob acumula no núcleo após a indução da LTP, mas não LTD 1. Além disso, a translocação de Jacob após estimulação sináptica requer a activação de MAPK ERK1 / 2 e a fosforilação de Jacob em Ser180 (Figura 1). Fosforilada Jacob transloca para o núcleo de uma forma dependente da importin e o estado fosforilado pode ser conservada durante períodos de tempo prolongados, por associação com o filamento...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Nós fornecer um protocolo detalhado para a indução de potenciação de longo prazo na região CA1 do hipocampo e o subsequente isolamento de fracções enriquecidas nucleares da área tetanized da fatia. Esta abordagem pode ser utilizada para determinar a actividade da importação nuclear de proteína dependente em modelos celulares de aprendizagem e memória.

Isolamento de CA1 Nucleares frações enriquecidas de fatias do hipocampo para o Estudo de dependentes de Atividade Nuclear Importação de Mensageiro Proteínas Synapto nucleares

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Research

JoVE Journal

Neuroscience

12.0K visualizações.  Leibniz Institute for Neurobiology.  Nós fornecer um protocolo detalhado para a indução de potenciação de longo prazo na região CA1 do hipocampo e o subsequente isolamento de fracções enriquecidas nucleares da área tetanized da fatia. Esta abordagem pode ser utilizada para determinar a actividade da importação nuclear de proteína dependente em modelos celulares de aprendizagem e memória. 

Vídeo: Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Preparação de fatias do hipocampo aguda de ratos e camundongos transgênicos para o Estudo das Alterações Synaptic durante o envelhecimento e amilóide Patologia

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Neurociência

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de neurônios de fatias do cérebro de roedores para estudar a transmissão sináptica e Imagem Terminais pré-sináptica

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitocôndrias associadas Membranas ER (MAMs) e glicoesfingolipídeos microdomínios enriquecidos (GEMS): Isolamento de cérebro de rato

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Emparelhados Recordings células inteiras em organotï¿½icas fatias do hipocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Investigação de Synaptic Tagging / Captura e-captura Cruz usando aguda fatias do hipocampo de roedores

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Avaliação da densidade de sinapse em fatias Hippocampal cérebro de roedor

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

Implantação de sondas de silicone crônica e gravação de lugar Hippocampal células em um aparelho de esteira enriquecido

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

Um método melhorado para coleta de líquido cefalorraquidiano de ratos anestesiados

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Preparação de fatias hipocampais humanas agudas para gravações eletrofisiológicas

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...