Gostaríamos de agradecer a BK agosto da Universidade de Wisconsin-Madison Facility Microscópio Eletrônico para a microscopia eletrônica. Este trabalho foi financiado pelo NIH concede R01-DA026067 e P30-HD03352 (a JSM).

1. Preparações 1-4 <ol><li> Prepare 500 mL de tampão de meio de gradiente (GM), misturando 50 mL de uma pasta de sacarose 2,5 M, 2,5 mL de uma 1M Tris-HCl, pH 7,5 valores, e 0,1 mL de um 0,5 m EDTA, pH 8,0 estoque com mili- Q água para volume. Filtro de esterilizar a solução de alíquota, e armazenar congelados. </li><li> Prepare um estoque de 1000x tetrodotoxina (TTX) fazendo uma solução de 1 mM em mili-Q H 2 O. Alíquotas de TTX podem ser armazenadas a -20 ° C. </li><li> Preparar o tampão de Estimulação 10x, fazendo uma solução de 100 mM Tris (pH 7,5), 5 mM Na 2 HPO 4, 4 mM KH 2 PO 4, 40 mM NaHCO 3, e 800 mM NaCl em água milli-Q. Estoque pode ser armazenado a 4 ° C. </li><li> Pré-cool centrífugas a 4 ° C. </li><li> Prepare uma solução estoque de Percoll isosmótica (SIP), adicionando 9 volumes de Percoll (18 mL) para 1 volume de 2,5 M de sacarose (2 mL) em água milli-Q e misture bem. </li><li> Preparar a 3, 10, 15, e camadas gradiente de 23%, adicionando os respectivos montantes de SIP para a GM de buffer e cada mistura: </li></ol><img alt="Tabela 0" src="/files/ftp_upload/3196/3196table0.jpg" /><ol start="7"><li> Despeje camadas gradiente por pipetagem de 2 ml cada um dos 23, 15, 10, e 3% em soluções de Percoll isosmótica os tubos de centrifugação Beckman com tampas usando um P1000 Gilson Pipetman. A interface entre as camadas devem ser claramente distinguíveis, sem mistura das camadas. Armazenar os gradientes de temperatura de 4 ° C durante pelo menos 20 min antes do uso. Os gradientes podem ser armazenadas por até 24 horas, embora use mesmo dia é recomendado. [Pessoal de laboratório inexperientes podem praticar gradientes preparando adicionando corante azul para as soluções isomotic Percoll para a visualização das interfaces da camada.] </li></ol> 2. Dissecção do mouse uma <ol><li> Garantir que todos os criação de mouse e eutanásia procedimentos são realizados de acordo com as diretrizes do NIH e da Universidade. Euthanize filhotes (idade P13 a P21) por asfixia dióxido de carbono ou por um método aprovado no protocolo animal. </li><li> Pin o mouse para uma placa de dissecção e pulverizar a parte de trás do pescoço e da cabeça com etanol. Usando uma tesoura afiada corte através da medula espinhal na base do crânio, retire a pele da parte superior do crânio, corte no crânio lateralmente entre o osso eo osso parietal interparietal ou entre o cérebro e as regiões do córtex. Em seguida, corte no crânio da base para o nariz (ao longo da sutura sagital), remova cuidadosamente o osso parietal macia puxando cada hemisfério para o lado e remover o córtex com uma espátula. Inserir a espátula no cérebro acima do cerebelo e, em seguida, retire o córtex e colocá-lo na gelada tampão GM. Proceder de imediato para a etapa de homogeneização. Não deixe que os córtices sentar na gelada tampão por muito tempo como este irá afectar negativamente a formação de SNs. </li></ol> 3. Preparação de homogenato e SNs 04/01 <ol><li> Enxágüe os córtices no gelada tampão GM e transferência de dois córtices para um homogeneizador de vidro Dounce contendo 5 mL de tampão GM frio. </li><li> Homogeneizados suavemente os córtices com golpes 10/05 do pilão solto (pilão &quot;A&quot;), seguido por acidentes vasculares cerebrais 10/05 do pilão apertado (pilão &quot;B&quot;). Strokes deve ser rápido, mas lento o suficiente para não causar bolhas de ar. A imagem do homogenato após homogeneização está no Esquema 1. O número de acidentes vasculares cerebrais irá variar consoante a homogeneizador individuais e pilões de acompanhamento. Quando terminar os gradientes deve dar uma banda SN forte, se não, ajustar o número de acidentes vasculares cerebrais. </li><li> Transferir o homogeneizado para a 15 mL cônica e centrifugar a 1000xg por 10 min a 4 ° C em um rotor de caçamba móvel (Allegra 6KR centrífuga) para pellet restos celulares e núcleos. A Imagem do homogeneizado é girado no Esquema 1. </li><li> Layer 2 mL do sobrenadante por Percoll-sacarose gradiente de tampão, os tubos, e centrifugar a 32.500 xg por 5 min a 4 ° C em um rotor de ângulo fixo (Beckman J2-21 centrífuga, JA-17 rotor) o uso de adaptadores apropriados ( ver Reagentes e Equipamentos Tabela). </li><li> Pipeta e descartar a solução acima da banda SN usando um copo Pasteur pipeta. Pipeta off da banda SN na interface 15/23%, transferir para um cone e armazenar no gelo. Cada gradiente ou um córtex produzirá 0,9-1,1 mL de SN. </li><li> Ajustar a concentração de sal do SNS, adicionando um décimo volume de tampão de estimulação 10x, adicione CaCl 1000x 2 a uma concentração final de 12 nM, e adicionar 1 mM de ações TTX a uma concentração final de 1 mM para suprimir a excitação inespecífica. (Adição de CaCl 2 é opcional se ele vai interferir com aplicações a jusante SN). </li><li> Determinar a concentração de proteína do SNs usando o Micro BCA Protein Assay Kit. (O teste de proteína Bradford não é recomendado porque de inferência do Percoll.) </li><li> SNs podem ser usados ​​diretamente e rapidamente para a proteína traduçãoção estudos. Para outras aplicações SN lisado podem ser limpos ou concentrado com o Pierce SDS-PAGE Sample Kit Prep. </li></ol> 4. Resultados representativos: Quando o mouse córtex foi homogeneizado e separados em descontínuo de Percoll-sacarose gradientes (Esquema 1), 6 bandas ou frações foram (Figura 1). Foi mostrado anteriormente para o córtex cerebral de ratos 3,5 que os componentes da alta bandas de peso molecular foram quebrados e membrana de mielina e que o material continha mitocôndrias peletizada ou organelas (Tabela 1). Os 23% interface / 15% (faixa 5) continha os SNs enriquecido. A banda na interface de 23% / 15%, contendo SNs intacta, foi retirado e examinado por microscopia eletrônica (EM) conforme descrito anteriormente. 2,6 EM mostrou a presença de vesículas sinápticas intacta ea preservação de elementos pré-sináptico e pós-sinápticos (Figura 2). Isolamento de compartimentos pré e pós-sináptica é importante para o exame de sinalização e de síntese de proteína, que ocorre em ambos os compartimentos. 7-13 O gradiente de Percoll-sacarose é um purificação bruto então vimos menor contaminação, celular organela nuclear e na fração de SN, mas o separação geral foi bom. Quando examinados por Western Blot, as frações Percoll continha os marcadores de proteína com um aumento esperado na pureza sináptica na banda SN (Figura 3, Tabela 2). Marcadores sináptica e neuronal foram mantidos na faixa de SN enriquecido (faixa 5), ​​mas a abundância de outros marcadores foi significativamente reduzida. Em particular, a presença de GFAP, um marcador de células neoplásicas e astrócitos de origem glial, e Lamin-B1, um marcador nuclear, foram insignificantes, enquanto marcadores estruturais e sinápticas foram mantidos ou melhorados. Além disso, houve retenção de marcadores de organelas, como as mitocôndrias e Golgi, que desempenham um papel na síntese protéica. Rendimentos típicos para a banda SN foram 250-500 ng de proteína por mL, conforme determinado pelo BCA ensaio de proteína. A atividade do SNS foi determinada pela quantidade de síntese protéica de novo presente como monitorados por [35S]-Met incorporação (Figura 4). Basal 2 atividade translacional aumentou 1,56 vezes quando SNs foram estimulados com 50 mM mM glicina glutamate/10 (Glu), ou 5,12 vezes quando ativada com o inibidor juglone Pin1. Inibição Pin1 é conhecido por resultar em aumento da síntese protéica. 2 Para confirmar que o aumento do [35S]-Met incorporação era devido a síntese de proteínas de novo, nós adicionamos 40 anisomicina mM, um inibidor da síntese de proteínas, e viu uma acentuada diminuição na incorporação na presença e ausência de Glu, quando comparado aos níveis basais. Também, mediante a adição de 2% Triton-X 100, o que perturba a integridade dos SNs, tradução basal foi reduzida em 75%. 2 Além disso, ao examinar as bandas (B4-B6), que mostrou o enriquecimento marcador mais sináptica, O SNS ou Banda 5 teve a maior atividade de síntese protéica de novo (Figura 5). Assim, o descontínuo de Percoll-sacarose gradientes rapidamente produzir um altamente ativa e enriquecido banda SN que pode ser usado para estudar a tradução proteica. O procedimento de gradiente descontínuo de Percoll-sacarose é mais vantajoso do que outros métodos, porque dano mecânico é causado pelas condições mais duras. Quando comparado com o método de filtração, em que apurou homogeneizado cortical é passado através de uma série de filtros que diminuem em tamanho, 3,14-15 o método Percoll formas SNs mais com maior atividade. Como visto na Figura 6, quando uma alíquota de SNs preparados pelo método de filtração foi passada ao longo de um gradiente de Percoll-sacarose há SNs muito menos na interface 15/23% e uma maior quantidade de membranas quebrado. Quando de novo a síntese de proteínas foi medida através S35 met-incorporação, o SNS preparado pelo método de filtração eram muito menos ativa (Figura 7). Para maximizar a atividade translacional usamos ratos que estão entre a idade P14 e P18. SN pode ser preparado a partir de camundongos adultos, mas observamos um aumento significativo de atividade translacional com SN preparados a partir de ratinhos jovens (Figura 8). <img alt="Tabela 1" src="/files/ftp_upload/3196/3196table1.jpg" /> Tabela 1. Constituintes das bandas do Percoll descontínuo gradiente de fracionamento do córtex do rato homogeneizado. 3,5 <img alt="Tabela 2" src="/files/ftp_upload/3196/3196table2.jpg" /> Tabela 2. Resumo da análise de parafuso ocidental de Percoll descontínuo de sacarose-constituintes banda degradê. Concentrações de proteína relativa para cada uma das frações isoladas banda usando um Percol descontínual de sacarose-gradiente foi determinada por Western Blot. Sem proteína presente (-), 0 ≥ 0,2 (+), 0,2 ≥ 0,8 (++), ou&gt; 0,8 vezes (+++) = maior proteína presente no sobrenadante representante (S, centrifugado córtex homogenato), de n 3. <img alt="Esquema 1" src="/files/ftp_upload/3196/3196scheme1.jpg" /> Esquema 1: Esquema de uma preparação SN utilizando um gradiente de densidade descontínua Percoll-sacarose córtex cerebral de camundongos foram dissecados, homogeneizado, e centrifugação a baixa velocidade para remover não-homogeneizado de tecido, células, organelas todo, como núcleos e membranas.. O gradiente descontínuo deve ter camadas bem definidas. A imagem mostra um exemplo de inserção das camadas, que têm sido tingido de azul apenas para fins ilustrativos, a fim de visualizar as camadas. O sobrenadante é camadas em cima de um gradiente de Percoll-sacarose descontínuo e centrifugado a velocidade média. A banda SN é então recuperado do gradiente e está pronto para usar. <img alt="Figura 1" src="/files/ftp_upload/3196/3196fig1.jpg" /> Figura 1. Fracionamento homogeneizado em um gradiente Percoll-sacarose descontínua. Córtex do rato foi homogeneizado e separados em um gradiente Percoll-sacarose descontínua dando origem a 6 bandas ou frações. A purificada, ativa SNs (*) foram encontrados na faixa 5. <img alt="Figura 2" src="/files/ftp_upload/3196/3196fig2.jpg" /> Figura 2. Preparações SN dar uma mistura heterogênea de SNs que retêm elementos pré-sináptico e pós-sináptica. Uma micrografia eletrônica de uma preparação SN preparados a partir de camundongos C57BL / 6 (idade P13 a P21) foi realizada como descrito anteriormente SNs e shows com preservados elementos pré-sináptico e pós-sináptica. 2 , 6 pontos setas vermelhas para pós-sinápticos densidades. Barra de escala é 1 mícron. <img alt="Figura 3" src="/files/ftp_upload/3196/3196fig3.jpg" /> Figura 3. Análises de Western blot de preparações SN mostraram que os marcadores sináptica e neuronal foram mantidas no SNs purificada (Band 5), mas a abundância de outros marcadores foi significativamente reduzida. Immunoblots do sobrenadante (S, centrifugado homogeneizado cortical) e Bandas 1-6 de uma descontínua Percoll-sacarose gradiente foram sondados para β-actina (controle, estruturais de carga), GFAP (astrocitária), GP73 (Golgi), HSC70 (citoplasmática e nuclear), Lamin-B1 (nuclear), Prohibitin (mitocondrial), PSD95 (densidade pós-sináptica ), SNAP25 (sináptica), sinaptofisina (synaptic) e β3-tubulina (neurônio-específica). As bandas foram visualizadas utilizando uma tempestade 860 Phosphorimager, representante de n = 3. <img alt="Figura 4" src="/files/ftp_upload/3196/3196fig4.jpg" /> Figura 4. SNs são ativos e apresentam a síntese de proteínas de novo através de [35S]-Met incorporação. SNs foram pré-equilibradas a 37 ° C por 10 min. Em seguida, o SNs foram pré-tratados ou não tratada por 15 min com 40 mM anisomicina ou 1 mM juglone a 37 ° C seguido por adição de [35S]-Met, 20 l expresso Etiqueta Pro Mix S35 Tag Fácil, mais ou menos Glu a 37 ° C por 30 min. As amostras foram preparadas usando o Pierce SDS-PAGE Sample Kit Prep, executado em um gel de poliacrilamida 15%, secos e quantificado através de um Molecular Dynamics Tempestade 860 phosphorimager, representante de n = 3, ± SEM. <img alt="Figura 5" src="/files/ftp_upload/3196/3196fig5.jpg" /> Figura 5. Banda de 5 a partir do gradiente de Percoll-sacarose descontínua continha os mais altos níveis de síntese protéica de novo. Bandas 4, 5 e 6 e do sobrenadante (S, centrifugado homogeneizado cortical) foram pré-equilibradas a 37 ° C por 10 min. Então, [35S]-Met, expresso Mix Etiqueta Pro S35 Tag Fácil, foi adicionado, mais ou menos Glu a 37 ° C por 30 min. As amostras foram preparadas usando o Pierce SDS-PAGE Sample Kit Prep, executado em um gel de poliacrilamida 15%, secos e quantificado através de um Molecular Dynamics Tempestade 860 phosphorimager, representante de n = 3, ± SEM. <img alt="Figura 6" src="/files/ftp_upload/3196/3196fig6.jpg" /> Figura 6. SNs preparados a partir de um método de filtração contêm membranas mais quebrado e SNs menos do que toda SNs preparado a partir do método do gradiente descontínuo de Percoll-sacarose. SNs foram preparadas pela passagem de córtex homogeneizada através de uma série de filtros com a diminuição da dimensão dos poros, como descrito anteriormente. 3,14-15 alíquota Um dos SNs preparados pelo método de filtração foi centrifugado em um gradiente de Percoll-sacarose descontínuo e em comparação com um montante equivalente de material usando o método do gradiente descontínuo de Percoll-sacarose. <img alt="Figura 7" src="/files/ftp_upload/3196/3196fig7.jpg" /> Figura 7. SNs preparados a partir de um método de filtragem de novo contêm menos proteínas do que atividade de síntese SNs preparado from o método do gradiente descontínuo de Percoll-sacarose. SNs foram preparadas pela passagem de córtex homogeneizada através de uma série de filtros com a diminuição da dimensão dos poros, como descrito anteriormente, 3,14-15 e pelos métodos Percoll descontínuo de sacarose-gradiente. SNs de ambas as preparações foram pré-equilibradas a 37 ° C por 10 min. Então, [35S]-Met, expresso Mix Etiqueta Pro S35 Tag Fácil, foi adicionado, mais ou menos Glu a 37 ° C por 30 min. As amostras foram preparadas usando o Pierce SDS-PAGE Sample Kit Prep, executado em um gel de poliacrilamida 15%, secos e quantificado através de um Molecular Dynamics Tempestade 860 phosphorimager, representante de n = 3, ± SEM. <img alt="Figura 8" src="/files/ftp_upload/3196/3196fig8.jpg" /> Figura 8. SNs de ratos jovens (P14) têm maior atividade translacional do que os camundongos mais velhos (P85). SNs foram preparados a partir de diferentes ratos idosos usando o método do gradiente descontínuo de Percoll-sacarose, preequilibrated a 37 ° C por 10 min, tratada ou não com Glu na presença de [35S]-Met (Express Etiqueta Pro Mix S35 Tag Fácil) por 30 min, snap congelados, e depois limpou com o Kit Prep Pierce SDS-PAGE da amostra. Os SNs foram executados em um 12% de gel de poliacrilamida SDS, secas e fotografada em uma tempestade Molecular Dynamics 860 phosphorimager representante do n = 3, ± SEM. SNs de P14 camundongos foram pelo menos 3 vezes mais ativo do que P85 camundongos.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+neuronal+excitability,+synaptic+plasticity,+and+learning+in+aged+Kvbeta1.1+knockout+mice.">Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice.</a> Curr. Biol. 14, 1907-1915 (2004).</li><li>Norris, C. M., Halpain, S., Foster, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+Age-Related+Alterations+in+Synaptic+Plasticity+by+Blockade+of+L-Type+Ca21+Channels.">Reversal of Age-Related Alterations in Synaptic Plasticity by Blockade of L-Type Ca21 Channels.</a> J. Neurosci. 18, 3171-3179 (1998).</li><li>Sallert, M., Malkki, H., Segerstr&#228;lea, M., Tairaa, T., Lauri, S. 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C., Constantine-Patton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor-mediated+control+of+proteins+synthesis+at+developing+synapses.">NMDA receptor-mediated control of proteins synthesis at developing synapses.</a> Nat. Neurosci. 3, 211-216 (2000).</li><li>Villasana, L. E., Klann, E., Tejada-Simon, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+synaptoneurosomes+and+postsynaptic+densities+from+adult+mouse+hippocampus.">Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus.</a> J. Neurosci. Methods. 158, 30-36 (2006).</li><li>Weiler, I. J., Greenough, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptors+trigger+postsynaptic+protein+synthesis.">Metabotropic glutamate receptors trigger postsynaptic protein synthesis.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 7168-7171 (1993).</li><li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+nerve+endings+from+brain:+An+electron+microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation.">The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation.</a> J. Anat. 96, 79-88 (1962).</li><li>Gylys, K. H., Fein, J. A., Yang, F., Cole, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+Presynaptic+and+Postsynaptic+Markers+by+Size-Based+Gating+Analysis+of+Synaptosome+Preparations+From+Rat+and+Human+Cortex.">Enrichment of Presynaptic and Postsynaptic Markers by Size-Based Gating Analysis of Synaptosome Preparations From Rat and Human Cortex.</a> Cytometry A. 60, 90-96 (2004).</li><li>Haj&#243;s, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+preparation+of+synaptosomal+fractions+in+high+purity.">An improved method for the preparation of synaptosomal fractions in high purity.</a> Brain Res. 93, 485-489 (1975).</li><li>Bai, F., Witzmann, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptosome+Proteomics.">Synaptosome Proteomics.</a> Subcell. Biochem. 43, 77-98 (2007).</li><li>Rao, A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+protein+constituents+of+postsynaptic+membrane+specializations+are+locally+synthesized:+analysis+of+proteins+synthesized+within+synaptosomes.">Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes.</a> J Neurosci. 11, 2881-2895 (1991).</li><li>Pertoft, H., Laurent, T. C., Catsimpoolas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isopycnic+separation+of+cells+and+cell+organelles+by+centrifugation+and+modified+colloidal+silica+gradients.">Isopycnic separation of cells and cell organelles by centrifugation and modified colloidal silica gradients.</a> Methods of Cell Separation. , 25-65 (1977).</li><li>Ramarao, C. S., Acharya, S. R., Krishnan, K. S., Kenkare, U. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+affinity+uptake+of+L-glutamate+and+&amp;gamma;-aminobutyric+acid+in+Drosophila+melanogaster.">High affinity uptake of L-glutamate and &amp;gamma;-aminobutyric acid in Drosophila melanogaster.</a> J. Biosci. 11, 119-135 (1987).</li><li>Munteanu, L. S., Dinu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+granulocytes+from+whole+human+blood+by+centrifugation.+Practical+hints.">Fractionation of granulocytes from whole human blood by centrifugation. Practical hints.</a> Romanian J. Biophys. 14, 53-58 (2004).</li><li>Vlasselaer, P., Van Palathumpat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+composition,+kit+and+method.">Cell separation composition, kit and method.</a> US patent. , (1997).</li></ol>

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A preparação de gradiente descontínuo de Percoll-sacarose aqui descrito é um método rápido e confiável para isolar SNs ativo, que pode ser usado em uma variedade de experimentos de transmissão sináptica. Este método do gradiente, que é baseado no método desenvolvido por Dunkley et al., 3,4 é um procedimento de fracionamento subcelular cérebro que isola pré e pós-sináptica da membrana derivada vesículas que estão associados com o outro. Sem purificação adicional esses SNs são compatíveis...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Perfurar </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Pescador </td><td> C79-500 </td><td></td></tr><tr><td> Gás CO2 </td><td> Airgas (UW-MDS) </td><td> CD 50 </td><td></td></tr><tr><td> EDTA </td><td> RPI </td><td> E57020 </td><td></td></tr><tr><td> EtOH </td><td> Pescador </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Pescador </td><td> A142-212 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Pescador </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS PAGE-Sample Prep Kit </td><td> Perfurar </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Pescador </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Pescador </td><td> S381-500 </td><td></td></tr><tr><td> Sacarose </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> Tris Base de Dados </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxina </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Expressar Etiqueta Pro Mix S35 Tag Fácil </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Ferramentas de dissecção </td><td></td><td></td><td></td></tr><tr><td> Dounce homogeneizador, 7 mL (vem com dois pilões de vidro &quot;A&quot; e &quot;B&quot;) </td><td> Wheaton </td><td></td><td></td></tr><tr><td> P1000 Gilson Pipetman </td><td> Gilson </td><td> F123602 </td><td></td></tr><tr><td> Allegra Centrifugar 6KR </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH Rotor 3.8, rotor de caçamba móvel </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Centrífuga Beckman J2-21 </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tubos com tampas </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Branco adaptadores murada </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Azul adaptadores murada </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, rotor de ângulo fixo </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabela de anticorpos utilizados para western blots: <table border="1"><tbody><tr><th> Nome de anticorpos </th><th> Companhia </th><th> Número de catálogo </th><th> Espécies hospedeiras </th><th> Fator de diluição </th></tr><tr><td> β-Actina </td><td> Sigma </td><td> A5441 </td><td> mouse </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> coelho </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> cabra </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> coelho </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> mouse </td><td> 5 mg / mL </td></tr><tr><td> SNAP25 </td><td> Abcam </td><td> ab5666-100 </td><td> coelho </td><td> 1:2000 </td></tr><tr><td> Sinaptofisina </td><td> Millipore </td><td> MAB368 </td><td> mouse </td><td> 1:500 </td></tr><tr><td> β-3-tubulina </td><td> Santa Cruz </td><td> sc-80016 </td><td> mouse </td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes (SNS) são obtidos após homogeneização e fracionamento de rato córtex cerebral. Eles são selados vesículas ou terminais isolados que romper com os terminais do axônio quando o tecido cortical é homogeneizado. Os SNs reter características pré e pós-sináptico, o que os torna útil no estudo da transmissão sináptica. Conservem as máquinas moleculares utilizados na sinalização neuronal e são capazes de captação, armazenamento e liberação de neurotransmissores. A produção e isolamento de ativos SNs pode ser problemático mídias usando como Ficoll, que podem ser citotóxicos e centrifugação prolongada devido a alta densidade, e filtração e métodos de centrifugação, que pode resultar em baixa atividade, devido a danos mecânicos do SNS. No entanto, o uso de gradientes descontínuos de Percoll-sacarose de densidade para isolar SNs fornece um método rápido para produzir bons rendimentos de SNs traducionalmente ativo. O método do gradiente Percoll-sacarose é rápido e gentil como ele emprega condições isotônica, tem spins centrifugação menos e mais curtos e evita que as etapas de centrifugação pellet SNs e causar danos mecânicos.

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Um método para preparar traducionalmente ativa, synaptoneurosomes intacta (SNs) de córtex cerebral de rato é descrito. O método utiliza um gradiente de densidade descontínua Percoll-sacarose permitindo a preparação rápida de ativos SNs.

Preparação de Synaptoneurosomes de rato Cortex usando um gradiente de densidade Percoll descontínuo Sacarose-

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Research

JoVE Journal

Neuroscience

31.2K visualizações.  University of Wisconsin. Um método para preparar traducionalmente ativa, synaptoneurosomes intacta (SNs) de córtex cerebral de rato é descrito. O método utiliza um gradiente de densidade descontínua Percoll-sacarose permitindo a preparação rápida de ativos SNs.

Vídeo: Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Imagem crônica de rato Visual Cortex usando uma preparação diluída crânio-

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Neurociência

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Remendo Recordings Grampo de neurônios da retina do rato em um Preparação Slice Dark-adaptados

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando-LacZ expressar Células de tecidos de camundongos Inner Ear usando Citometria de Fluxo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon<em&gt; In vivo</em&gt; Imagem de espinhas dendríticas no Rato Cortex Usando um desbaste-crânio Preparação

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Preparação do Synaptic Plasma Membrane e Proteínas de densidade pós-sinápticos Usando um gradiente de sacarose descontínuo

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Investigar a função do Deep Cortical e estruturas subcorticais Usando estereotáxica Eletroencefalografia: Lições da Cortex cingulado anterior

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Viver a imagem das células do córtex Cerebral primário usando um sistema de cultura 2D

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Caracterização e isolamento de Mouse Microglia primária por centrifugação gradiente de densidade

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...